U.S. patent application number 13/086028 was filed with the patent office on 2011-12-29 for chinese hamster apoptosis-related genes.
This patent application is currently assigned to Agency for Science, Technology and Research. Invention is credited to Chee Furng WONG, Miranda Gek Sim Yap.
Application Number | 20110318831 13/086028 |
Document ID | / |
Family ID | 36615341 |
Filed Date | 2011-12-29 |
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United States Patent
Application |
20110318831 |
Kind Code |
A1 |
WONG; Chee Furng ; et
al. |
December 29, 2011 |
CHINESE HAMSTER APOPTOSIS-RELATED GENES
Abstract
Provided is an isolated polypeptide comprising a Cricetulus
griseus sequence capable of mediating apoptosis of a cell, the
sequence being selected from a FAIM sequence shown as SEQ ID NO: 1;
a FADD sequence shown as SEQ ID NO: 2; a PDCD6 sequence shown as
SEQ ID NO: 3; and a Requiem sequence shown as SEQ ID NO: 4.
Inventors: |
WONG; Chee Furng;
(Singapore, SG) ; Yap; Miranda Gek Sim;
(Singapore, SG) |
Assignee: |
Agency for Science, Technology and
Research
Singapore
SG
|
Family ID: |
36615341 |
Appl. No.: |
13/086028 |
Filed: |
April 13, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12946690 |
Nov 15, 2010 |
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13086028 |
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11824740 |
Jul 2, 2007 |
7846894 |
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12946690 |
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PCT/SG05/00433 |
Dec 28, 2005 |
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11824740 |
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60640333 |
Dec 30, 2004 |
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Current U.S.
Class: |
435/358 |
Current CPC
Class: |
A61P 43/00 20180101;
C07K 14/4747 20130101 |
Class at
Publication: |
435/358 |
International
Class: |
C12N 5/071 20100101
C12N005/071; C12N 5/10 20060101 C12N005/10 |
Claims
1-33. (canceled)
34. A method of modulating apoptosis of a Chinese Hamster Ovary
(CHO) cell, the method comprising modulating expression of a
cgRequiem polypeptide in the cell, wherein the cgRequiem
polypeptide comprises a sequence having at least 99% sequence
identity with SEQ ID NO: 4, thereby modulating apoptosis of the CHO
cell.
35. The method according to claim 34, wherein expression of the
cgRequiem polypeptide is down-regulated and apoptosis is
reduced.
36. The method according to claim 34, wherein the cgRequiem
polypeptide is encoded by a polynucleotide comprising a sequence
having at least 93% sequence identity to SEQ ID NO: 8; or a
sequence which is (i) complementary thereto, (ii) capable of
hybridising under stringent conditions thereto, or (iii) degenerate
thereto as a result of the genetic code.
37. The method according to claim 34, wherein the cgRequiem
polypeptide comprises SEQ ID NO: 4.
38. The method according to claim 36, wherein the polynucleotide
comprises SEQ ID NO: 8.
39. The method according to claim 35, wherein the CHO cell is
genetically engineered to down-regulate expression of the cgRequiem
polypeptide.
40. The method according to claim 35, wherein the CHO cell
comprises (i) an anti-sense construct directed against cgRequiem,
(ii) a non-functional sequence having at least 99% sequence
identity with SEQ ID NO: 4, (iii) a double-stranded (ds) RNA
corresponding to a polynucleotide encoding cgRequiem, (iv) a single
interfering RNA (siRNA) against cgRequiem or (v) a dominant
negative mutant of cgRequiem.
41. The method according to claim 40, wherein the siRNA comprises
SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 48.
42. The method according to claim 34, wherein the CHO cell
comprises a plasmid comprising SEQ ID NO: 40.
43. The method according to claim 41, wherein the sequence is
transfected, stably integrated, or transformed into the CHO
cell.
44. The method according to claim 35, further comprising expressing
a recombinant protein in the CHO cell.
45. The method according to claim 44, wherein the recombinant
protein is a heterologous protein.
46. The method according to claim 44, wherein the recombinant
protein comprises interferon gamma.
47. The method according to claim 44, wherein: (i) viability of the
cell is increased; (ii) yield of the recombinant protein is
increased; or (iii) glycosylation of the recombinant protein is
increased, compared to a method in which expression of the
cgRequiem polypeptide in a cell is not down-regulated.
48. The method according to claim 47, wherein the glycosylation is
sialylation.
49. The method according to claim 48, wherein the sialylation of
the recombinant protein is greater than 2.9 mol sialic acid/mol of
recombinant protein.
50. The method according to claim 48, wherein the sialylation of
the recombinant protein is greater than about 3.5 mol of sialic
acid/mol of recombinant protein.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 12/946,690, filed Nov. 15, 2010, which is a divisional of U.S.
application Ser. No. 11/824,740, filed Jul. 2, 2007, now U.S. Pat.
No. 7,846,894, which is a continuation-in-part of International
application no. PCT/SG2005/000433, filed Dec. 28, 2005, published
as WO 2006/071200 on Jul. 6, 2006, and claiming priority to U.S.
application No. 60/640,333, filed Dec. 30, 2004.
[0002] The foregoing applications, as well as all documents cited
in the foregoing applications ("application documents") and all
documents cited or referenced in the application documents are
incorporated herein by reference. Also, all documents cited in this
application ("herein-cited documents") and all documents cited or
referenced in herein-cited documents are incorporated herein by
reference. In addition, any manufacturer's instructions or
catalogues for any products cited or mentioned in each of the
application documents or herein-cited documents are incorporated by
reference. Documents incorporated by reference into this text or
any teachings therein can be used in the practice of this
invention. Documents incorporated by reference into this text are
not admitted to be prior art.
FIELD OF THE INVENTION
[0003] This invention relates to the fields of biotechnology and
molecular biology. The invention particularly relates to novel
genes from Chinese hamster, Cricetulus griseus, which are involved
in the mediation of apoptotic processes.
BACKGROUND OF THE INVENTION
[0004] With the completion of the human genome project, more
proteins with therapeutic potential are being discovered daily.
Many of these new biotherapeutics often require the development of
highly productive manufacturing processes to meet global demand.
One of the most commonly used cells lines for complex therapeutic
biologics production is Chinese Hamster Ovary (CHO) cells which was
originally derived from Chinese Hamster (Cricetulus griseus).
[0005] However, the genome of Cricetulus griseus is poorly
characterised, and in particular, there is lack of knowledge of
genes in that organism which control physiologically important
processes.
[0006] U.S. Pat. No. 6,562,797 describes a purified mammalian
protein designated FADD which has the ability to bind the
cytoplasmic region or domain of the Fas receptor. This document
also describes methods of regulating FAS-associated apoptosis.
However, the only sequences which are disclosed are of human
origin.
[0007] U.S. Pat. No. 6,683,168 and US Patent Application
Publication Number US 2004/0121389 describes the sequences of FAIM
sequence in a number of forms: short, long, super long and lung
cancer associated. The sequences are human and mouse sequences.
[0008] U.S. Pat. No. 6,544,523 sets out the sequence of a DNA
encoding a Fas ligand. U.S. Pat. No. 6,451,759 describes a
non-cleavable version of such a ligand.
SUMMARY OF THE INVENTION
[0009] We describe for the first time the sequences of Cricetulus
griseus FAIM, FADD, PDCD6 and Requiem.
[0010] According to a 1.sup.st aspect of the present invention, we
provide an isolated polypeptide comprising a sequence selected from
the following: (a) a cg FAIM sequence having at least 97% sequence
identity with a sequence shown in SEQ ID NO: 1; (b) a cgFADD
sequence having at least 69% sequence identity with a sequence
shown in SEQ ID NO: 2; (c) a cgPDCD6 sequence having at least 89%
sequence identity with a sequence shown in SEQ ID NO: 3; (d) a
cgRequiem sequence having at least 90% sequence identity with a
sequence shown in SEQ ID NO: 4; (e) a sequence being a fragment of
at least 15 contiguous residues of any of (a) to (d) above, which
is capable of mediating apoptosis of a cell.
[0011] There is provided, according to a 2.sup.nd aspect of the
present invention, an isolated polypeptide comprising a Cricetulus
griseus sequence capable of mediating apoptosis of a cell, the
sequence being selected from a cgFAIM sequence shown as SEQ ID NO:
1; a cgFADD sequence shown as SEQ ID NO: 2; a cgPDCD6 sequence
shown as SEQ ID NO: 3; and a cgRequiem sequence shown as SEQ ID NO:
4.
[0012] According to a 1.sup.st aspect of the present invention, we
provide an isolated polypeptide comprising a Cricetulus griseus
sequence capable of mediating apoptosis of a cell, the sequence
being selected from a cgFAIM sequence shown as SEQ ID NO: 1; a
cgFADD sequence shown as SEQ ID NO: 2; a cgPDCD6 sequence shown as
SEQ ID NO: 3; and a cgRequiem sequence shown as SEQ ID NO: 4.
[0013] There is provided, according to a 2.sup.nd aspect of the
present invention, an isolated polypeptide comprising a sequence
selected from the following: (a) a cg FAIM sequence having at least
97% sequence identity with a sequence shown in SEQ ID NO: 1; (b) a
cgFADD sequence having at least 69% sequence identity with a
sequence shown in SEQ ID NO: 2; (c) a cgPDCD6 sequence having at
least 89% sequence identity with a sequence shown in SEQ ID NO: 3;
(d) a cgRequiem sequence having at least 90% sequence identity with
a sequence shown in SEQ ID NO: 4; (e) a sequence being a fragment
of at least 15 contiguous residues of any of (a) to (d) above.
[0014] We provide, according to a 3.sup.rd aspect of the present
invention, an isolated polynucleotide comprising a sequence which
encodes a polypeptide as set out, in which the sequence is
preferably selected from the group consisting of: SEQ ID NO: 5, SEQ
ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.
[0015] As a 4.sup.th aspect of the present invention, there is
provided an isolated polynucleotide comprising a sequence selected
from the following: (a) a cgFAIM sequence which has 90% or more
sequence identity to a sequence shown as SEQ ID NO: 5; (b) a cgFADD
sequence which has 90% or more sequence identity to a sequence
shown as SEQ ID NO: 6; (c) a cgPDCD6 sequence which has 93% or more
sequence identity to a sequence shown as SEQ ID NO: 7; (d) a
cgRequiem sequence which has 89% or more sequence identity to a
sequence shown as SEQ ID NO: 8; (e) a sequence being a fragment of
at least 15 contiguous residues of any of (a) to (d) above; or a
sequence which is complementary thereto, which is capable of
hybridising under stringent conditions thereto, or which is
degenerate thereto as a result of the genetic code.
[0016] We provide, according to a 5.sup.th aspect of the present
invention, an expression sequence comprising a polynucleotide as
set out above or a portion thereof operably linked to a regulatory
sequence, the regulatory sequence capable of directing expression
of said polynucleotide.
[0017] Preferably, such an expression sequence is an expression
vector.
[0018] The present invention, in a 6.sup.th aspect, provides a
vector comprising a polynucleotide according as set out above, the
vector being capable of modulating the expression of cgFAIM,
cgFADD, cgPDCD6 or cgRequiem by a cell when exposed to the
cell.
[0019] Preferably, the vector comprises a Cricetulus griseus FAIM
sequence or a portion thereof, the vector being capable of
effecting up-regulation of cgFAIM in a cell, preferably pcDNA3.1(+)
FAIM (SEQ ID NO: 37).
[0020] Preferably, the vector comprises a Cricetulus griseus FADD
sequence or a portion thereof, the vector being capable of
effecting down-regulation of cgFADD in a cell, preferably
pcDNA3.1(+) FADD DN (SEQ ID NO: 38).
[0021] Preferably, the vector comprises a Cricetulus griseus PDCD6
sequence or a portion thereof, the vector being capable of
effecting down-regulation of cgPDCD6 in a cell, preferably
pSUPER.neo.PDCD6 siRNA (SEQ ID NO: 39).
[0022] Preferably, the vector comprises a Cricetulus griseus
Requiem sequence or a portion thereof and capable of effecting
down-regulation of cgRequiem in a cell, preferably
pSUPER.neo.Requiem siRNA (SEQ ID NO: 40).
[0023] In a 7.sup.th aspect of the present invention, there is
provided a cell comprising an expression sequence as described or a
vector as described, in which the expression sequence has
preferably been transformed into said cell.
[0024] According to an 8.sup.th aspect of the present invention, we
provide a pharmaceutical composition comprising a polypeptide as
set out, a polynucleotide as set out, an expression sequence as set
out, a vector as set out or a cell as set out, together with a
pharmaceutically acceptable carrier or diluent.
[0025] We provide, according to a 9.sup.th aspect of the invention,
a method of producing a polypeptide comprising: (a) providing an
expression sequence comprising a polynucleotide sequence as set out
and a regulatory sequence, in which the regulatory sequence is
capable of directing expression of the polypeptide from the
polynucleotide sequence, (b) allowing expression of the polypeptide
from the expression sequence under control of the regulatory
sequence, and (c) optionally purifying the polypeptide.
[0026] Preferably, the expression sequence comprises an expression
vector which is transfected into a cell, preferably a Cricetulus
griseus cell, to enable expression of the polypeptide by the
cell.
[0027] There is provided, in accordance with a 10.sup.th aspect of
the present invention, a method comprising modulating, preferably
up-regulating, the expression of a cgFAIM polypeptide having a
sequence shown as SEQ ID NO: 1 or a cgFAIM polynucleotide having a
sequence shown as SEQ ID NO: 5 in a cell, preferably a Cricetulus
griseus cell.
[0028] As an 11.sup.th aspect of the invention, we provide a method
comprising modulating, preferably down-regulating, the expression
of a cgFADD polypeptide having a sequence shown as SEQ ID NO: 2, a
cgPDCD6 polypeptide having a sequence shown as SEQ ID NO: 3 or a
cgRequiem polypeptide having a sequence shown as SEQ ID NO: 4, or a
cgFAIM polynucleotide having a sequence shown as SEQ ID NO: 6, a
cgPDCD6 polynucleotide having a sequence shown as SEQ ID NO: 7 or a
cgRequiem polynucleotide having a sequence shown as SEQ ID NO: 8,
in a cell, preferably a Cricetulus griseus cell.
[0029] Preferably, the method comprises exposing a vector as set
out to the cell, preferably transfecting the cell with the
vector.
[0030] We provide, according to a 12.sup.th aspect of the
invention, a cell, preferably a Cricetulus griseus cell, which has
been modified, preferably genetically engineered, to up-regulate
the expression of a polypeptide having a sequence shown as SEQ ID
NO: 1 or a polynucleotide having a sequence shown as SEQ ID NO: 5,
compared to a cell which has not been so modified.
[0031] According to a 13.sup.th aspect of the present invention, we
provide a cell, preferably a Cricetulus griseus cell, which has
been modified, preferably genetically engineered, to down-regulate
the expression of a polypeptide having a sequence shown as SEQ ID
NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 or a polynucleotide having a
sequence shown as SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8,
compared to a cell which has not been so modified.
[0032] There is provided, according to a 14.sup.th aspect of the
present invention, a cell line comprising a cell as described, or a
descendent thereof, preferably a Cricetulus griseus cell line.
[0033] We provide, according to a 15.sup.th aspect of the present
invention, a cell culture comprising a cell as described, or a
descendant thereof, or a cell line as described.
[0034] According to a 16.sup.th aspect of the present invention, we
provide a transgenic non-human animal comprising a cell as
described, or a descendant thereof, preferably Cricetulus
griseus.
[0035] Preferably, (i) cell viability of the cell is increased or
enhanced, preferably in which apoptosis of the cell is reduced;
(ii) protein yield, preferably recombinant expressed protein yield,
of the cell is increased or enhanced; and/or (iii) glycosylation,
preferably sialylation, of expressed protein by the cell is
increased or enhanced; compared to a cell in which expression of
the polypeptide is not so modulated.
[0036] According to a 17.sup.th aspect of the present invention, we
provide use of a method as set out, a cell as set out, a cell line
as set out, a cell culture as set out or a transgenic non-human
animal as set out, for the production of a protein, preferably a
heterologous protein, more preferably from an exogenously
introduced sequence, most preferably a recombinant protein.
[0037] We provide, according to an 18.sup.th aspect of the present
invention, a method of producing a recombinant protein, the method
comprising providing a cell as set out, transfecting the cell with
an expression vector capable of expressing the recombinant protein,
and causing expression of the recombinant protein in the cell.
[0038] According to a 19.sup.th aspect of the present invention, we
provide a polypeptide comprising a cgFADD dominant negative
sequence having SEQ ID NO: 9, or a polynucleotide capable of
encoding such a polypeptide, preferably SEQ ID NO: 10, or a
fragment, homologue, variant or derivative thereof.
[0039] As an 20.sup.th aspect of the invention, we provide a
polypeptide, preferably a recombinant protein, more preferably
interferon gamma, producable by a method according to the 17.sup.th
or 18.sup.th aspect of the invention, which polypeptide has an
increased sialylation, compared to a polypeptide producable from a
cell which is not so modified.
[0040] Preferably, the sialylation is greater than 2.9 mol sialic
acid/mol of produced polypeptide, preferably about 3.5 mol of
sialic acid/mol of produced polypeptide.
[0041] Further particular and preferred aspects of the present
invention are set out in the accompanying independent and dependent
claims. Features of the dependent claims may be combined with
features of the independent claims as appropriate, and in
combinations other than those explicitly set out in the claims.
[0042] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of chemistry,
molecular biology, microbiology, recombinant DNA and immunology,
which are within the capabilities of a person of ordinary skill in
the art. Such techniques are explained in the literature. See, for
example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989,
Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3,
Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995
and periodic supplements; Current Protocols in Molecular Biology,
ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe,
J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing:
Essential Techniques, John Wiley & Sons; J. M. Polak and James
O'D. McGee, 1990, In Situ Hybridization: Principles and Practice;
Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide
Synthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J.
E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A:
Synthesis and Physical Analysis of DNA Methods in Enzymology,
Academic Press; Using Antibodies: A Laboratory Manual: Portable
Protocol NO. I by Edward Harlow, David Lane, Ed Harlow (1999, Cold
Spring Harbor Laboratory Press, ISBN 0-87969-544-7); Antibodies: A
Laboratory Manual by Ed Harlow (Editor), David Lane (Editor) (1988,
Cold Spring Harbor Laboratory Press, ISBN 0-87969-314-2), 1855,
Lars-Inge Larsson "Immunocytochemistry: Theory and Practice", CRC
Press inc., Baca Raton, Fla., 1988, ISBN 0-8493-6078-1, John D.
Pound (ed); "Immunochemical Protocols, vol 80", in the series:
"Methods in Molecular Biology", Humana Press, Totowa, N.J., 1998,
ISBN 0-89603-493-3, Handbook of Drug Screening, edited by
Ramakrishna Seethala, Prabhavathi B. Fernandes (2001, New York,
N.Y., Marcel Dekker, ISBN 0-8247-0562-9); Lab Ref: A Handbook of
Recipes, Reagents, and Other Reference Tools for Use at the Bench,
Edited Jane Roskams and Linda Rodgers, 2002, Cold Spring Harbor
Laboratory, ISBN 0-87969-630-3; and The Merck Manual of Diagnosis
and Therapy (17th Edition, Beers, M. H., and Berkow, R, Eds, ISBN:
0911910107, John Wiley & Sons). Each of these general texts is
herein incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] The present invention will be described further, by way of
example only, with reference to preferred embodiments thereof as
illustrated in the accompanying drawings, in which:
[0044] FIGS. 1A-1D are graphs showing over-expression of FADD
Dominant Negative (FIG. 1B) and FAIM (FIG. 1A) and suppression of
Requiem (FIG. 1D) and PDCD6 (FIG. 1C) expression, in cells
transfected with relevant constructs.
[0045] FIGS. 2A-2D are graphs showing the growth kinetics of CHO
IFN-.gamma. cells over-expressing FAIM. FIG. 2A shows viable cell
density (cells/ml) versus time. FIG. 2B shows total cell density
versus time. FIG. 2C shows viability versus time. FIG. 2D shows
apoptotic cells versus time. Loss of cell culture viability is
significantly reduced with FAIM over-expression compared to control
cells (FIG. 2C) due to significant reduction in apoptotic cells
(FIG. 2D).
[0046] FIGS. 3A-3D are graphs showing the growth kinetics of CHO
IFN-.gamma. cells over-expressing FADD dominant negative. FIG. 3A
shows viable cell density (cells/ml) versus time. FIG. 3B shows
total cell density versus time. FIG. 3C shows viability versus
time. FIG. 3D shows apoptotic cells versus time. Loss of cell
culture viability is significantly reduced with FADD Dominant
Negative over-expression compared to control cells (FIG. 3C) due to
significant reduction in apoptotic cells (FIG. 3D).
[0047] FIGS. 4A-4D are graphs showing the growth kinetics of CHO
IFN-.gamma. cells with PDCD6 suppression. FIG. 4A shows viable cell
density (cells/ml) versus time, FIG. 4B shows total cell density
versus time, FIG. 4C shows viability versus time, FIG. 4D shows
apoptotic cells versus time. Loss of cell culture viability is
significantly reduced when PDCD6 is suppressed compared to control
cells (FIG. 4C) due to significant reduction in apoptotic cells
(FIG. 4D).
[0048] FIGS. 5A-5D are graphs showing the growth kinetics of CHO
IFN-.gamma. cells with Requiem suppression. FIG. 5A shows viable
cell density (cells/ml) versus time. FIG. 5B shows total cell
density versus time. FIG. 5C shows viability versus time. FIG. 5D
shows apoptotic cells versus time. Loss of cell culture viability
is significantly reduced when Requiem is suppressed compared to
control cells (FIG. 5C) due to significant reduction in apoptotic
cells (FIG. 5D).
[0049] FIGS. 6A-6E are graphs showing the activity of Caspases 2,
3, 8 and 9 in CHO cell culture. FIG. 6A shows caspase activity in
cells transfected with a control. FIG. 6B shows caspase activity in
cells over-expressing FAIM. FIG. 6C shows caspase activity in cells
over-expressing FADD Dominant Negative. FIG. 6D shows caspase
activity in cells with suppression of PDCD6. FIG. 6E shows caspase
activity in cells with suppression of Requiem. Gene targeting FAIM,
FADD Dominant Negative, PDCD6 or REQUIEM is able to either suppress
and/or delay caspases activity in culture.
[0050] FIGS. 7A-7D are graphs showing Interferon-.gamma. yields for
transfected CHO IFN-.gamma. cells. FIG. 7A shows interferon-.gamma.
activity in cells over-expressing FAIM. FIG. 7B shows
interferon-.gamma. activity in cells over-expressing FADD Dominant
Negative. FIG. 7C shows interferon-.gamma. activity in cells with
suppression of PDCD6. FIG. 7D shows interferon-.gamma. activity in
cells with suppression of REQUIEM. Significant improvement of
interferon gamma yields by up to 300% can be achieved through gene
targeting approach.
[0051] FIG. 8A shows viable cell densities of stable CHO
IFN-.gamma. clones with either Requiem or PDCD6 suppression or FADD
DN or FAIM* overexpression in fed-batch cultures. (Data presented
are the averages of two duplicate experiments).
[0052] FIG. 8B shows viable cell densities of stable CHO
IFN-.gamma. clones with either Requiem or PDCD6 suppression or FADD
DN or FAIM* overexpression in fed-batch cultures.
[0053] FIG. 9A shows interferon gamma yields of stable CHO
IFN-.gamma. clones with either Requiem or PDCD6 suppression or FADD
DN* or FAIM* overexpression in fed-batch cultures. (Data presented
are the averages of two duplicate experiments)
[0054] FIG. 9B shows interferon gamma yields of stable CHO
IFN-.gamma. clones with either Requiem or PDCD6 suppression or FADD
DN* or FAIM* overexpression in fed-batch cultures.
[0055] FIG. 10 shows sialylation of recombinant IFN-.gamma. in
stable CHO IFN-.gamma. clones with either Requiem or PDCD6
suppression or FADD DN* or FAIM* over-expression during
mid-exponential, stationary and death phase of fed-batch
cultures.
DESCRIPTION OF SEQUENCES
[0056] SEQ ID NO: 1 is the sequence of amino acid sequence of C.
griseus FAIM. SEQ ID NO: 2 is the amino acid sequence of C. griseus
FADD. SEQ ID NO: 3 is the amino acid sequence of C. griseus PDCD6.
SEQ ID NO: 4 is the amino acid sequence of C. griseus Requiem.
[0057] SEQ ID NO: 5 is the nucleic acid sequence of C. griseus
FAIM. SEQ ID NO: 6 is the nucleic acid sequence of C. griseus FADD.
SEQ ID NO: 7 is the nucleic acid sequence of C. griseus PDCD6. SEQ
ID NO: 8 is the nucleic acid sequence of C. griseus Requiem.
[0058] SEQ ID NO: 9 is the amino acid sequence of C. griseus FADD
dominant negative. SEQ ID NO: 10 is the nucleic acid sequence of C.
griseus FADD dominant negative. SEQ ID NO: 11 is the sequence of C.
griseus FADD dominant negative 5'-PCR primer. SEQ ID NO: 12 is the
sequence of C. griseus FADD dominant negative 3'-PCR primer. SEQ ID
NO: 13 is the sequence of C. griseus PDCD6 suppression vector
insert 5'. SEQ ID NO: 14 is the sequence of C. griseus PDCD6
suppression vector insert 3'. SEQ ID NO: 15 is the sequence of C.
griseus Requiem suppression vector insert 5'. SEQ ID NO: 16 is the
sequence of C. griseus Requiem suppression vector insert 3'.
[0059] SEQ ID NO: 17 is the sequence of C. griseus FAIM 5' PCR
primer. SEQ ID NO: 18 is the sequence of C. griseus FAIM 3' PCR
primer. SEQ ID NO: 19 is the sequence of C. griseus FADD 5' PCR
primer. SEQ ID NO: 20 is the sequence of C. griseus FADD 3' PCR
primer. SEQ ID NO: 21 is the sequence of C. griseus PDCD6 5' PCR
primer. SEQ ID NO: 22 is the sequence of C. griseus PDCD6 3' PCR
primer. SEQ ID NO: 23 is the sequence of C. griseus PDCD6 3'-RACE
primer. SEQ ID NO: 24 is the sequence of C. griseus Requiem 5' PCR
primer. SEQ ID NO: 25 is the sequence of C. griseus Requiem 3' PCR
primer. SEQ ID NO: 26 is the sequence of C. griseus Requiem 3'-RACE
primer.
[0060] SEQ ID NO: 27 is the sequence of C. griseus FAIM
Quantitative Real Time PCR primer 5'. SEQ ID NO: 28 is the sequence
of C. griseus FAIM Quantitative Real Time PCR primer 3'. SEQ ID NO:
29 is the sequence of C. griseus FADD Quantitative Real Time PCR
primer 5'. SEQ ID NO: 30 is the sequence of C. griseus FADD
Quantitative Real Time PCR primer 3'. SEQ ID NO: 31 is the sequence
of C. griseus PDCD6 Quantitative Real Time PCR primer 5'. SEQ ID
NO: 32 is the sequence of C. griseus PDCD6 Quantitative Real Time
PCR primer 3'. SEQ ID NO: 33 is the sequence of C. griseus Requiem
Quantitative Real Time PCR primer 5'. SEQ ID NO: 34 is the sequence
of C. griseus Requiem Quantitative Real Time PCR primer 3'. SEQ ID
NO: 35 is the sequence of .beta.-actin Quantitative Real Time PCR
primer 5'.
[0061] SEQ ID NO: 36 is the sequence of a .beta.-actin Quantitative
Real Time PCR primer 3'. SEQ ID NO: 37 is the nucleic acid sequence
of plasmid pcDNA3.1(+) FAIM. SEQ ID NO: 38 is the nucleic acid
sequence of plasmid pcDNA3.1(+) FADD DN. SEQ ID NO: 39 is the
nucleic acid sequence of plasmid pSUPER.neo.PDCD6 siRNA. SEQ ID NO:
40 is the nucleic acid sequence of plasmid pSUPER.neo.Requeim
siRNA.
[0062] The methods and compositions described here may suitably
employ any one or more of the sequences shown in the Sequence
Listing.
DETAILED DESCRIPTION
Chinese Hamster Sequences
[0063] The disclosure provides generally for certain nucleic acids,
polypeptides, as well as fragments, homologues, variants and
derivatives thereof from the Chinese hamster, Cricetulus griseus,
which are capable of modulating apoptosis in cells.
[0064] In particular, we provide for Cricetulus griseus FADD, FAIM,
PDCD6 and Requiem polypeptide and nucleic acid sequences as set out
in the Sequence Listings. In addition we provide for the use of
such genes, fragments, homologues.
[0065] Particularly preferred uses include the modification of
cells, particularly Chinese Hamster Ovary cells, for enhanced
properties, such as increased viability, increased capacity to
express proteins (particularly recombinant proteins) and increased
glycosylation, preferably sialylation, of such proteins. Such
modified cells and derivatives of these (such as colonies, clones,
cell lines, etc) are described in further detail below, and may be
used as apoptosis resistant cells for the production of recombinant
proteins.
cgFAIM, cgFADD, cgPDCD6 and cgREQUIEM Polypeptides
[0066] It will be understood that polypeptide sequences disclosed
here are not limited to the particular sequences set forth in the
sequence listing, or fragments thereof, or sequences obtained from
cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem protein, but also include
homologous sequences obtained from any source, for example related
cellular homologues, homologues from other species and variants or
derivatives thereof, provided that they have at least one of the
biological activities of cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem,
as the case may be.
[0067] This disclosure therefore encompasses variants, homologues
or derivatives of the amino acid sequences set forth in the
sequence listings, as well as variants, homologues or derivatives
of the amino acid sequences encoded by the nucleotide sequences
disclosed here. Such sequences are generally referred to as a
"cgFADD sequence", a "cgFAIM sequence", "a cgPDCD6 sequence", or a
"cgRequiem sequence", as the case may be.
[0068] Biological Activities
[0069] In highly preferred embodiments, the sequences comprise at
least one biological activity of cgFAIM, cgFADD, cgPDCD6 and/or
cgRequiem, as the case may be.
[0070] Preferably, in the case of cgFAIM, the biological activity
comprises apoptosis inhibiting activity, preferably assayed by
down-regulation of caspase activity. Thus, the cgFADD sequences
described in this document preferably are capable of inhibiting
apoptosis, specifically capable of down-regulating caspase activity
in the context of a cell.
[0071] In highly preferred embodiments, when assayed using such
methods, the cgFAIM sequences when transfected into a cell are
capable of inhibiting apoptosis by at least 10%, preferably 20%,
more preferably 30%, 40% 50%, 60%, 70%, 80%, 90% or more, compared
to a cell which has not been so transfected with the relevant
cgFAIM sequence.
[0072] In the case of cgFADD, cgPDCD6 and cgRequiem, the biological
activity preferably comprises apoptosis stimulating activity,
preferably assayed by up-regulation of caspase activity. Thus, the
cgFADD, cgPDCD6 and cgRequiem sequences described in this document
preferably are capable of up-stimulating apoptosis, specifically
capable of up-regulating caspase activity in the context of a
cell.
[0073] In highly preferred embodiments, when assayed using such
methods, the cgFADD, cgPDCD6 and cgRequiem sequences when
transfected into a cell are capable of stimulating apoptosis by at
least 10%, preferably 20%, more preferably 30%, 40% 50%, 60%, 70%,
80%, 90% or more, compared to a cell which has not been so
transfected with the relevant cgFADD, cgPDCD6 or cgRequiem
sequence.
[0074] In highly preferred embodiments, the activation or
repression of apoptosis by the cgFAIM, cgFADD, cgPDCD6 and/or
cgRequiem sequences is assayed by assaying caspase activity. Thus,
the percentage stimulation or repression of apoptosis set out above
are in highly preferred embodiments to be read as percentage
stimulation or repression of caspase activity.
[0075] Thus, apoptosis activity monitoring methods such as caspase
activity measurement assays using colorimetric or fluorometric
methods can be used to ascertain the biochemical activity of
cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem. Such methods may be
carried out in cells transfected with appropriate expression
constructs, such as by means known in the art, or using protocols
set out in the Examples, to determine whether apoptosis is affected
and/or caspase activity is up- or down-regulated.
[0076] Caspases are a large family of cysteine proteases that
mediates apoptosis (Nicholson & Thornberry 1997; Thornberry
& Littlewood 1998). Caspase-8 is an initiator caspase that act
the most upstream in receptor-mediated apoptotic pathway. Upon
activation of cell-surface receptors, caspase-8 directly or
indirectly initiates the proteolytic activities of downstream
effector caspases such as caspase-3 (Srinivasula et at 1996 and
Cohen 1997). Caspase-9, which is also another upstream caspase, is
activated via the mitochondrial release of cytochrome c to the
cytosol. Released cytochrome c binds to the apoptotic protease
activating factor, APAF-1, forming a complex that activates
procaspase-9 (Zou et at 1999 and Hu et at 1999). Active caspase-9
initiates a protease cascade that also activates caspase-3 and
other downstream caspases.
[0077] In preferred embodiments, the caspase activity that is
assayed to determine up- or down-regulation of apoptosis activity
comprises caspase-8 or caspase-9.
[0078] Methods for assaying caspase-8 and caspase-9 activity are
known in the art, and are specifically described in, for example,
Nicholson D W and Thornberry N A (1997) Caspases: killer proteases.
Trends Biochem Sci. 272: 2952-2956 and Thornberry N A and
Littlewood Y (1998) Caspases: Enemies within. Science
281:1312-1316. Any of the protocols set out in the prior art may be
used to assay caspase activity.
[0079] In preferred embodiments, however, the "Caspase Assay
Protocol" set out below is employed to assay caspase-8 and/or
caspase-9 activity.
[0080] Caspase Assay Protocol
[0081] Caspase activity can be assayed by utilizing fluorogenic
substrates specific for different caspases immobilized in the
wells. Application of cell lysates containing the active caspase to
the wells will cleave the substrate and release a fluorescent
product that can be detected using standard fluorescence plate
reader.
[0082] Specifically, BD ApoAlert.TM. Caspase assay plates
(catalogue number K2033-1, BD Biosciences Clontech, Palo Alto,
Calif., USA) uses different caspase substrates composed of short
peptides that are recognized by their respective activated
caspases. The peptides are covalently linked to the fluorogenic dye
7-amino-4-methyl coumarin (AMC). Peptide bound AMC emits in the UV
range (.lamda..sub.max=380 nm) while unbound AMC emits in the green
range (.lamda..sub.max=460 nm). This makes it possible to correlate
an increase in fluorescence intensity at 460 nm with an increase in
activity of the respective caspase in the test sample. For assaying
caspase-8 activity, the substrate used is VDVAD-AMC while an assay
for caspase-9 activity uses LEHD-AMC as its substrate.
[0083] In order to assay for caspases activity using BD
ApoAlert.TM. Caspase assay plate, cells from samples are pelleted
by centrifugation and then resuspended in 1.times. cell lysis
buffer (BD Biosciences Clontech) and incubated on ice for 10 min.
Cellular debris is then removed by centrifugation for 5 min at
4.degree. C. 50 .mu.L of 2.times. reaction buffer/DTT mix is then
added to each well of the 96-well plate that will be used. The
plate is preincubated at 37.degree. C. for 5 min. 50 .mu.L of the
appropriate cell lysate(s) is then added to the wells and incubated
at 37.degree. C. for 2 hour. A fluorescence plate reader is then
used to measure the amount of AMC released (Excitation at 380 nm,
Emission at 460 nm).
[0084] Caspase activity is defined as the absolute emission at 460
nm of a sample after subtraction from the absolute emission at 460
nm of a reference sample. The reference sample is a sample
collected at time reference zero.
[0085] Caspase activity of cells transfected with cgFAIM, cgFADD,
cgPDCD6 and/or cgRequiem expression vectors may be compared with
cells transfected with null-vectors (or untransfected) to determine
the percentage by which apoptosis is stimulated or repressed as the
case may be.
[0086] [End of "Caspase Assay Protocol"]
[0087] In preferred embodiments, when assayed using such methods,
the cgFAIM sequences when transfected into a cell are capable of
inhibiting the expression of caspase-8, or caspase-9, or both by at
least 10%, preferably 20%, more preferably 30%, 40% 50%, 60%, 70%,
80%, 90% or more, compared to a cell which has not been so
transfected with the relevant cgFAIM sequence.
[0088] In highly preferred embodiments, when assayed using such
methods, the cgFADD, cgPDCD6 and cgRequiem sequences when
transfected into a cell are capable of stimulating the expression
of caspase-8, or caspase-9, or both by at least 10%, preferably
20%, more preferably 30%, 40% 50%, 60%, 70%, 80%, 90% or more,
compared to a cell which has not been so transfected with the
relevant cgFADD, cgPDCD6 or cgRequiem sequence.
[0089] Other assays that detect apoptosis related events such as
membrane changes, DNA fragmentation and other biochemical hallmarks
of apoptosis can also be used, instead of, or in addition to, the
assays described.
[0090] Homologues
[0091] The polypeptides disclosed include homologous sequences
obtained from any source, for example related viral/bacterial
proteins, cellular homologues and synthetic peptides, as well as
variants or derivatives thereof. Thus polypeptides also include
those encoding homologues of cgFAIM, cgFADD, cgPDCD6 and/or
cgRequiem from other species including animals such as mammals
(e.g. mice, rats or rabbits), in particular rodents.
[0092] In the context of the present document, a homologous
sequence or homologue is taken to include an amino acid sequence
which is at least 60, 65, 70, 75, 80, 85, 86, 87, 88, 89 or 90%
identical, preferably at least 91, 92, 93, 94, 95, 96, 97, 98 or
99% identical at the amino acid level over at least 30, preferably
40, 50, 60, 70, 80, 90 or 100 amino acids with cgFAIM, cgFADD,
cgPDCD6 and/or cgRequiem, as the case may be, for example as shown
in the sequence listing herein. In the context of this document, a
homologous sequence is taken to include an amino acid sequence
which is at least 15, 20, 25, 30, 40, 50, 60, 65, 70, 75, 80, 85,
86, 97, 88, 89 or 90% identical, preferably at least 91, 92, 93,
94, 95, 96, 97, 98 or 99% identical at the amino acid level,
preferably over at least 15, 25, 35, 50 or 100, preferably 200,
300, 400 or 500 amino acids with the sequence of cgFAIM, cgFADD,
cgPDCD6 and/or cgRequiem. For example, a sequence may have the
stated sequence identity to cgFADD (preferably comprising a
sequence as shown in SEQ ID NO: 1), cgFAIM (preferably comprising a
sequence as shown in SEQ ID NO: 2), cgPDCD6 (preferably comprising
a sequence as shown in SEQ ID NO: 3) or cgRequiem (preferably
comprising a sequence as shown in SEQ ID NO: 4).
[0093] Although homology can also be considered in terms of
similarity (i.e. amino acid residues having similar chemical
properties/functions), in the context of the present document it is
preferred to express homology in terms of sequence identity. In
highly preferred embodiments, the sequence identity is determined
relative to the entirety of the length the relevant sequence, i.e.,
over the entire length or full length sequence of the relevant
gene, for example.
[0094] Homology comparisons can be conducted by eye, or more
usually, with the aid of readily available sequence comparison
programs. These commercially available computer programs can
calculate % homology between two or more sequences.
[0095] % homology may be calculated over contiguous sequences, i.e.
one sequence is aligned with the other sequence and each amino acid
in one sequence directly compared with the corresponding amino acid
in the other sequence, one residue at a time. This is called an
"ungapped" alignment. Typically, such ungapped alignments are
performed only over a relatively short number of residues (for
example less than 50 contiguous amino acids).
[0096] Although this is a very simple and consistent method, it
fails to take into consideration that, for example, in an otherwise
identical pair of sequences, one insertion or deletion will cause
the following amino acid residues to be put out of alignment, thus
potentially resulting in a large reduction in % homology when a
global alignment is performed. Consequently, most sequence
comparison methods are designed to produce optimal alignments that
take into consideration possible insertions and deletions without
penalising unduly the overall homology score. This is achieved by
inserting "gaps" in the sequence alignment to try to maximise local
homology.
[0097] However, these more complex methods assign "gap penalties"
to each gap that occurs in the alignment so that, for the same
number of identical amino acids, a sequence alignment with as few
gaps as possible--reflecting higher relatedness between the two
compared sequences--will achieve a higher score than one with many
gaps. "Affine gap costs" are typically used that charge a
relatively high cost for the existence of a gap and a smaller
penalty for each subsequent residue in the gap. This is the most
commonly used gap scoring system. High gap penalties will of course
produce optimised alignments with fewer gaps. Most alignment
programs allow the gap penalties to be modified. However, it is
preferred to use the default values when using such software for
sequence comparisons. For example when using the GCG Wisconsin
Bestfit package (see below) the default gap penalty for amino acid
sequences is -12 for a gap and -4 for each extension.
[0098] Calculation of maximum % homology therefore firstly requires
the production of an optimal alignment, taking into consideration
gap penalties. A suitable computer program for carrying out such an
alignment is the GCG Wisconsin Bestfit package (University of
Wisconsin, U.S.A.; Devereux et al., 1984, Nucleic Acids Research
12:387). Examples of other software than can perform sequence
comparisons include, but are not limited to, the BLAST package (see
Ausubel et al., 1999 ibid--Chapter 18), FASTA (Atschul et al.,
1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison
tools. Both BLAST and FASTA are available for offline and online
searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60).
However it is preferred to use the GCG Bestfit program.
[0099] Although the final % homology can be measured in terms of
identity, the alignment process itself is typically not based on an
all-or-nothing pair comparison. Instead, a scaled similarity score
matrix is generally used that assigns scores to each pairwise
comparison based on chemical similarity or evolutionary distance.
An example of such a matrix commonly used is the BLOSUM62
matrix--the default matrix for the BLAST suite of programs. GCG
Wisconsin programs generally use either the public default values
or a custom symbol comparison table if supplied (see user manual
for further details). It is preferred to use the public default
values for the GCG package, or in the case of other software, the
default matrix, such as BLOSUM62.
[0100] Once the software has produced an optimal alignment, it is
possible to calculate % homology, preferably % sequence identity.
The software typically does this as part of the sequence comparison
and generates a numerical result.
[0101] In preferred embodiments, sequence similarity, identity,
homology or complementarity is adjudged with respect to the entire
length of the relevant sequence used for comparison.
[0102] Variants and Derivatives
[0103] The terms "variant" or "derivative" in relation to the amino
acid sequences as described here includes any substitution of,
variation of, modification of, replacement of, deletion of or
addition of one (or more) amino acids from or to the sequence.
Preferably, the resultant amino acid sequence retains substantially
the same activity as the unmodified sequence, preferably having at
least the same activity as the cgFAIM, cgFADD, cgPDCD6 and
cgRequiem polypeptides shown in the sequence listings. Thus, the
key feature of the sequences--namely that they are capable of
modulating one or more apoptotic processes--is preferably
retained.
[0104] Polypeptides having the amino acid sequence shown in the
Examples, or fragments or homologues thereof may be modified for
use in the methods and compositions described here. Typically,
modifications are made that maintain the biological activity of the
sequence. Amino acid substitutions may be made, for example from 1,
2 or 3 to 10, 20 or 30 substitutions provided that the modified
sequence retains the biological activity of the unmodified
sequence. Amino acid substitutions may include the use of
non-naturally occurring analogues, for example to increase blood
plasma half-life of a therapeutically administered polypeptide.
[0105] Natural variants of cgFAIM, cgFADD, cgPDCD6 and cgRequiem
are likely to comprise conservative amino acid substitutions.
Conservative substitutions may be defined, for example according to
the Table below. Amino acids in the same block in the second column
and preferably in the same line in the third column may be
substituted for each other:
TABLE-US-00001 ALIPHATIC Non-polar G A P I L V Polar - uncharged C
S T M N Q Polar - charged D E K R AROMATIC H F W Y
[0106] Fragments
[0107] Polypeptides disclosed here and useful as markers also
include fragments of the above mentioned full length polypeptides
and variants thereof, including fragments of the sequences set out
in the sequence listings.
[0108] Polypeptides also include fragments of the full length
sequence of any of the cgFAIM, cgFADD, cgPDCD6 and cgRequiem
polypeptides. Preferably fragments comprise at least one epitope.
Methods of identifying epitopes are well known in the art.
Fragments will typically comprise at least 6 amino acids, more
preferably at least 10, 20, 30, 50 or 100 amino acids.
[0109] Included are fragments comprising, preferably consisting of,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125,
130, 135, 140, 145 or 150, or more residues from a cgFAIM, cgFADD,
cgPDCD6 and/or cgRequiem amino acid sequence.
[0110] Polypeptide fragments of the cgFAIM, cgFADD, cgPDCD6 and
cgRequiem proteins and allelic and species variants thereof may
contain one or more (e.g. 5, 10, 15, or 20) substitutions,
deletions or insertions, including conserved substitutions. Where
substitutions, deletion and/or insertions occur, for example in
different species, preferably less than 50%, 40% or 20% of the
amino acid residues depicted in the sequence listings are
altered.
[0111] cgFAIM, cgFADD, cgPDCD6 and cgRequiem, and their fragments,
homologues, variants and derivatives, may be made by recombinant
means. However, they may also be made by synthetic means using
techniques well known to skilled persons such as solid phase
synthesis. The proteins may also be produced as fusion proteins,
for example to aid in extraction and purification. Examples of
fusion protein partners include glutathione-S-transferase (GST),
6.times.His, GAL4 (DNA binding and/or transcriptional activation
domains) and .beta.-galactosidase. It may also be convenient to
include a proteolytic cleavage site between the fusion protein
partner and the protein sequence of interest to allow removal of
fusion protein sequences. Preferably the fusion protein will not
hinder the function of the protein of interest sequence. Proteins
may also be obtained by purification of cell extracts from animal
cells.
[0112] The cgFAIM, cgFADD, cgPDCD6 and cgRequiem polypeptides,
variants, homologues, fragments and derivatives disclosed here may
be in a substantially isolated form. It will be understood that
such polypeptides may be mixed with carriers or diluents which will
not interfere with the intended purpose of the protein and still be
regarded as substantially isolated. A cgFAIM, cgFADD, cgPDCD6
and/or cgRequiem variant, homologue, fragment or derivative may
also be in a substantially purified form, in which case it will
generally comprise the protein in a preparation in which more than
90%, e.g. 95%, 98% or 99% of the protein in the preparation is a
protein.
[0113] The cgFAIM, cgFADD, cgPDCD6 and cgRequiem polypeptides,
variants, homologues, fragments and derivatives disclosed here may
be labelled with a revealing label. The revealing label may be any
suitable label which allows the polypeptide, etc to be detected.
Suitable labels include radioisotopes, e.g. .sup.125I, enzymes,
antibodies, polynucleotides and linkers such as biotin. Labelled
polypeptides may be used in diagnostic procedures such as
immunoassays to determine the amount of a polypeptide in a sample.
Polypeptides or labelled polypeptides may also be used in
serological or cell-mediated immune assays for the detection of
immune reactivity to said polypeptides in animals and humans using
standard protocols.
[0114] cgFAIM, cgFADD, cgPDCD6 and cgRequiem polypeptides,
variants, homologues, fragments and derivatives disclosed here,
optionally labelled, my also be fixed to a solid phase, for example
the surface of an immunoassay well or dipstick. Such labelled
and/or immobilised polypeptides may be packaged into kits in a
suitable container along with suitable reagents, controls,
instructions and the like. Such polypeptides and kits may be used
in methods of detection of antibodies to the polypeptides or their
allelic or species variants by immunoassay.
[0115] Immunoassay methods are well known in the art and will
generally comprise: (a) providing a polypeptide comprising an
epitope bindable by an antibody against said protein; (b)
incubating a biological sample with said polypeptide under
conditions which allow for the formation of an antibody-antigen
complex; and (c) determining whether antibody-antigen complex
comprising said polypeptide is formed.
[0116] The cgFAIM, cgFADD, cgPDCD6 and cgRequiem polypeptides,
variants, homologues, fragments and derivatives disclosed here may
be used in in vitro or in vivo cell culture systems to study the
role of their corresponding genes and homologues thereof in cell
function, including their function in disease. For example,
truncated or modified polypeptides may be introduced into a cell to
disrupt the normal functions which occur in the cell. The
polypeptides may be introduced into the cell by in situ expression
of the polypeptide from a recombinant expression vector (see
below). The expression vector optionally carries an inducible
promoter to control the expression of the polypeptide.
[0117] The use of appropriate host cells, such as insect cells or
mammalian cells, is expected to provide for such post-translational
modifications (e.g. myristolation, glycosylation, truncation,
lapidation and tyrosine, serine or threonine phosphorylation) as
may be needed to confer optimal biological activity on recombinant
expression products. Such cell culture systems in which the cgFAIM,
cgFADD, cgPDCD6 and cgRequiem polypeptides, variants, homologues,
fragments and derivatives disclosed here are expressed may be used
in assay systems to identify candidate substances which interfere
with or enhance the functions of the polypeptides in the cell.
cgFAIM, cgFADD, cgPDCD6 and cgREQUIEM Nucleic Acids
[0118] We provide generally for a number of cgFAIM, cgFADD, cgPDCD6
and cgRequiem nucleic acids, together with fragments, homologues,
variants and derivatives thereof. These nucleic acid sequences
preferably encode the polypeptide sequences disclosed here, and
particularly in the sequence listings.
[0119] Preferably, the polynucleotides comprise cgFAIM, cgFADD,
cgPDCD6 and cgRequiem nucleic acids, preferably selected from the
group consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and
SEQ ID NO: 8 respectively.
[0120] In particular, we provide for nucleic acids or
polynucleotides which encode any of the Cricetulus griseus
polypeptides disclosed here. Thus, the terms "cgFADD sequence",
"cgFAIM sequence", "cgPDCD6 sequence" and "cgRequiem sequence"
should be construed accordingly. Preferably, however, such nucleic
acids or polynucleotides comprise any of the sequences set out as
SEQ ID NOs: 5 to 16 and SEQ ID Nos: 37, 38, 39 and 40, or a
sequence encoding any of the corresponding polypeptides, and a
fragment, homologue, variant or derivative of such a nucleic acid.
The above terms therefore preferably should be taken to refer to
these sequences.
[0121] As used here in this document, the terms "polynucleotide",
"nucleotide", and nucleic acid are intended to be synonymous with
each other. "Polynucleotide" generally refers to any
polyribonucleotide or polydeoxyribonucleotide, which may be
unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides"
include, without limitation single- and double-stranded DNA, DNA
that is a mixture of single- and double-stranded regions, single-
and double-stranded RNA, and RNA that is mixture of single- and
double-stranded regions, hybrid molecules comprising DNA and RNA
that may be single-stranded or, more typically, double-stranded or
a mixture of single- and double-stranded regions. In addition,
"polynucleotide" refers to triple-stranded regions comprising RNA
or DNA or both RNA and DNA. The term polynucleotide also includes
DNAs or RNAs containing one or more modified bases and DNAs or RNAs
with backbones modified for stability or for other reasons.
"Modified" bases include, for example, tritylated bases and unusual
bases such as inosine. A variety of modifications has been made to
DNA and RNA; thus, "polynucleotide" embraces chemically,
enzymatically or metabolically modified forms of polynucleotides as
typically found in nature, as well as the chemical forms of DNA and
RNA characteristic of viruses and cells. "Polynucleotide" also
embraces relatively short polynucleotides, often referred to as
oligonucleotides.
[0122] It will be understood by a skilled person that numerous
different polynucleotides and nucleic acids can encode the same
polypeptide as a result of the degeneracy of the genetic code. In
addition, it is to be understood that skilled persons may, using
routine techniques, make nucleotide substitutions that do not
affect the polypeptide sequence encoded by the polynucleotides
described here to reflect the codon usage of any particular host
organism in which the polypeptides are to be expressed.
[0123] Variants, Derivatives and Homologues
[0124] The polynucleotides described here may comprise DNA or RNA.
They may be single-stranded or double-stranded. They may also be
polynucleotides which include within them synthetic or modified
nucleotides. A number of different types of modification to
oligonucleotides are known in the art. These include
methylphosphonate and phosphorothioate backbones, addition of
acridine or polylysine chains at the 3' and/or 5' ends of the
molecule. For the purposes of the present document, it is to be
understood that the polynucleotides described herein may be
modified by any method available in the art. Such modifications may
be carried out in order to enhance the in vivo activity or life
span of polynucleotides.
[0125] Where the polynucleotide is double-stranded, both strands of
the duplex, either individually or in combination, are encompassed
by the methods and compositions described here. Where the
polynucleotide is single-stranded, it is to be understood that the
complementary sequence of that polynucleotide is also included.
[0126] The terms "variant", "homologue" or "derivative" in relation
to a nucleotide sequence include any substitution of, variation of,
modification of, replacement of, deletion of or addition of one (or
more) nucleotides from or to the sequence. Preferably, the
resulting sequence is capable of encoding a polypeptide which has
apoptosis mediator activity.
[0127] As indicated above, with respect to sequence identity, a
"homologue" has preferably at least 5% identity, at least 10%
identity, at least 15% identity, at least 20% identity, at least
25% identity, at least 30% identity, at least 35% identity, at
least 40% identity, at least 45% identity, at least 50% identity,
at least 55% identity, at least 60% identity, at least 65%
identity, at least 70% identity, at least 75% identity, at least
80% identity, at least 85% identity, at least 90% identity, or at
least 95% identity to the relevant sequence shown in the sequence
listings.
[0128] More preferably there is at least 95% identity, more
preferably at least 96% identity, more preferably at least 97%
identity, more preferably at least 98% identity, more preferably at
least 99% identity. Nucleotide homology comparisons may be
conducted as described above. A preferred sequence comparison
program is the GCG Wisconsin Bestfit program described above. The
default scoring matrix has a match value of 10 for each identical
nucleotide and -9 for each mismatch. The default gap creation
penalty is -50 and the default gap extension penalty is -3 for each
nucleotide.
[0129] In preferred embodiments, a cgFAIM polynucleotide has at
least 90% or more sequence identity to a sequence shown as SEQ ID
NO: 5. Preferably, the cgFAIM polynucleotide has 91% or more,
preferably 92% or more, 93% or more, 94% or more, 95% or more, 96%
or more, 97% or more, 98% or more, 99% or more or 99.5% or more
sequence identity to a sequence shown as SEQ ID NO: 5.
[0130] Similarly, in preferred embodiments, a cgFADD sequence has
at least 90% sequence identity to a sequence shown as SEQ ID NO: 6.
Preferably, the cgFADD polynucleotide has 91% or more, preferably
92% or more, 93% or more, 94% or more, 95% or more, 96% or more,
97% or more, 98% or more, 99% or more or 99.5% or more sequence
identity to a sequence shown as SEQ ID NO: 6.
[0131] In preferred embodiments, a cgPDCD6 sequence has at least
93% or more sequence identity to a sequence shown as SEQ ID NO: 7.
ably, the cgPDCD6 polynucleotide has 94% or more, 95% or more, 96%
or more, 97% or more, 98% or more, 99% or more or 99.5% or more
sequence identity to a sequence shown as SEQ ID NO: 7.
[0132] In preferred embodiments, a cgRequiem polynucleotide has at
least 90% or more sequence identity to a sequence shown as SEQ ID
NO: 5. Preferably, the cgRequiem polynucleotide has 90% or more,
preferably 91% or more, 92% or more, 93% or more, 94% or more, 95%
or more, 96% or more, 97% or more, 98% or more, 99% or more or
99.5% or more sequence identity to a sequence shown as SEQ ID NO:
8
[0133] Hybridisation
[0134] We further describe cgFAIM, cgFADD, cgPDCD6 and cgRequiem
nucleotide sequences that are capable of hybridising selectively to
any of the sequences presented herein, or any variant, fragment or
derivative thereof, or to the complement of any of the above.
Nucleotide sequences are preferably at least 15 nucleotides in
length, more preferably at least 20, 30, 40 or 50 nucleotides in
length.
[0135] The term "hybridisation" as used herein shall include "the
process by which a strand of nucleic acid joins with a
complementary strand through base pairing" as well as the process
of amplification as carried out in polymerase chain reaction
technologies.
[0136] Polynucleotides capable of selectively hybridising to the
nucleotide sequences presented herein, or to their complement, will
be generally at least 70%, preferably at least 80 or 90% and more
preferably at least 95% or 98% homologous to the corresponding
nucleotide sequences presented herein over a region of at least 20,
preferably at least 25 or 30, for instance at least 40, 60 or 100
or more contiguous nucleotides.
[0137] The term "selectively hybridisable" means that the
polynucleotide used as a probe is used under conditions where a
target polynucleotide is found to hybridize to the probe at a level
significantly above background. The background hybridization may
occur because of other polynucleotides present, for example, in the
cDNA or genomic DNA library being screened. In this event,
background implies a level of signal generated by interaction
between the probe and a non-specific DNA member of the library
which is less than 10 fold, preferably less than 100 fold as
intense as the specific interaction observed with the target DNA.
The intensity of interaction may be measured, for example, by
radiolabelling the probe, e.g. with .sup.32P.
[0138] Hybridisation conditions are based on the melting
temperature (Tm) of the nucleic acid binding complex, as taught in
Berger and Kimmel (1987, Guide to Molecular Cloning Techniques,
Methods in Enzymology, Vol 152, Academic Press, San Diego Calif.),
and confer a defined "stringency" as explained below.
[0139] Maximum stringency typically occurs at about Tm-5.degree. C.
(5.degree. C. below the Tm of the probe); high stringency at about
5.degree. C. to 10.degree. C. below Tm; intermediate stringency at
about 10.degree. C. to 20.degree. C. below Tm; and low stringency
at about 20.degree. C. to 25.degree. C. below Tm. As will be
understood by those of skill in the art, a maximum stringency
hybridisation can be used to identify or detect identical
polynucleotide sequences while an intermediate (or low) stringency
hybridisation can be used to identify or detect similar or related
polynucleotide sequences.
[0140] In a preferred aspect, we disclose nucleotide sequences that
can hybridise to a cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem nucleic
acid, or a fragment, homologue, variant or derivative thereof,
under stringent conditions (e.g. 65.degree. C. and 0.1.times.SSC
{1.times.SSC=0.15 M NaCl, 0.015 M Na.sub.3 Citrate pH 7.0}).
[0141] Where a polynucleotide is double-stranded, both strands of
the duplex, either individually or in combination, are encompassed
by the present disclosure. Where the polynucleotide is
single-stranded, it is to be understood that the complementary
sequence of that polynucleotide is also disclosed and
encompassed.
[0142] Polynucleotides which are not 100% homologous to the
sequences disclosed here but fall within the disclosure can be
obtained in a number of ways. Other variants of the sequences
described herein may be obtained for example by probing DNA
libraries made from a range of individuals, for example individuals
from different populations. In addition, other viral/bacterial, or
cellular homologues particularly cellular homologues found in
mammalian cells (e.g. rat, mouse, bovine and primate cells), may be
obtained and such homologues and fragments thereof in general will
be capable of selectively hybridising to the sequences shown in the
sequence listing herein. Such sequences may be obtained by probing
cDNA libraries made from or genomic DNA libraries from other animal
species, and probing such libraries with probes comprising all or
part of SEQ ID NO: 1 to 40 under conditions of medium to high
stringency. Similar considerations apply to obtaining species
homologues and allelic variants of cgFAIM, cgFADD, cgPDCD6 and
cgRequiem.
[0143] The polynucleotides described here may be used to produce a
primer, e.g. a PCR primer, a primer for an alternative
amplification reaction, a probe e.g. labelled with a revealing
label by conventional means using radioactive or non-radioactive
labels, or the polynucleotides may be cloned into vectors. Such
primers, probes and other fragments will be at least 15, preferably
at least 20, for example at least 25, 30 or 40 nucleotides in
length, and are also encompassed by the term polynucleotides as
used herein. Preferred fragments are less than 500, 200, 100, 50 or
20 nucleotides in length.
[0144] Polynucleotides such as a DNA polynucleotides and probes may
be produced recombinantly, synthetically, or by any means available
to those of skill in the art. They may also be cloned by standard
techniques.
[0145] In general, primers will be produced by synthetic means,
involving a step wise manufacture of the desired nucleic acid
sequence one nucleotide at a time. Techniques for accomplishing
this using automated techniques are readily available in the
art.
[0146] Longer polynucleotides will generally be produced using
recombinant means, for example using PCR (polymerase chain
reaction) cloning techniques. This will involve making a pair of
primers (e.g. of about 15 to 30 nucleotides) flanking a region of
the sequence which it is desired to clone, bringing the primers
into contact with mRNA or cDNA obtained from an animal or human
cell, performing a polymerase chain reaction under conditions which
bring about amplification of the desired region, isolating the
amplified fragment (e.g. by purifying the reaction mixture on an
agarose gel) and recovering the amplified DNA. The primers may be
designed to contain suitable restriction enzyme recognition sites
so that the amplified DNA can be cloned into a suitable cloning
vector
Uses of CG Sequences
[0147] As shown in the Examples, we have established that these
four genes are involved in the mediation of apoptosis in the
cell.
[0148] We also show that targeting of such genes by modulation of
their activity results in reduction of apoptosis and hence improved
cell viability. The genes and polypeptides and products thereof
therefore have utility in a number of fields, for example in cell
culture.
[0149] Thus, U.S. Pat. No. 6,586,206 describes the use of apoptosis
inhibitors in the production of recombinant proteins using cultured
host cells, with the effect of improved yield of the desired
protein. Accordingly, the disclosure of the sequences of Cricetulus
griseus FAIM, FADD, PDCD6 and Requiem therefore enables the
targetting of these genes in cell culture to enhance cell viability
and promote enhanced yields of recombinant protein production.
Specifically, cgFAIM, cgFADD, cgPDCD6 and cgRequiem modified cells
we describe here, preferably Cricetulus griseus cells, more
preferably Chinese Hamster Ovary cells, may be suitably employed
for production of recombinant proteins with improved yield.
cgFAIM, cgFADD, cgPDCD6 and cgREQUIEM Modified Cells
[0150] According to the methods and compositions described here,
modulation of any one or more of cgFAIM, cgFADD, cgPDCD6 and
cgRequiem in a cell improves cell viability of a population,
preferably a Cricetulus griseus population. In particular, we show
in the Examples that reduction of expression of cgFADD, cgPDCD6
and/or cgRequiem, as well as increasing expression of cgFAIM, leads
to improved cell viability.
[0151] However, it will be appreciated that methods of regulation
of any of these genes, including use of modulator entities such as
agonists and antagonists, may be employed in addition to, or as an
alternative to, modulation of polypeptide expression.
[0152] Cells in which the expression of any one or more of these
genes are modulated are referred to for convenience as "modified"
cells--although it will be appreciated that these may not be
physically modified themselves, but may be descendants of cells
which have been modified. We specifically provide for cells in
which cgFAIM expression is up-regulated, as well as for cells in
which expression of cgFADD, cgPDCD6 and/or cgRequiem, or any
combination thereof is down-regulated. Thus, it will be appreciated
that expression of one, two, three, or all four of cgFAIM, cgFADD,
cgPDCD6 and cgRequiem may be modulated in the modified cells. The
modification may be transient, or it may be permanent or long term,
depending on the mode of modification.
[0153] The modified cells may comprise mammalian cells, preferably
Cricetulus griseus cells, most preferably CHO cells. They may
comprise rodent cells, preferably mouse or rat cells. Preferably,
such modified cells comprise Cricetulus griseus cells, most
preferably CHO cells. However, they may comprise primate cells,
such as monkey cells or human cells.
[0154] The relevant cells may be modified by targeting relevant
genes by any means known in the art.
[0155] One possible approach is to express anti-sense constructs
directed against cgFADD, cgPDCD6 and/or cgRequiem, to inhibit gene
function and prevent the expression of the relevant polypeptide.
Another approach is to use non-functional variants of cgFADD,
cgPDCD6 and/or cgRequiem polypeptides that compete with the
endogenous gene product for cellular components of cell death
machinery, resulting in inhibition of function.
[0156] Alternatively, compounds identified by the assays described
above as binding to a cgFADD, cgPDCD6 and/or cgRequiem polypeptide
may be administered to cells to prevent the function of that
polypeptide. This may be performed, for example, by means of
recombinant DNA technology or by direct administration of the
compounds. Suitable antibodies directed against cgFADD, cgPDCD6
and/or cgRequiem may also be used as agents.
[0157] Alternatively, double-stranded (ds) RNA is a powerful way of
interfering with gene expression in a range of organisms that has
recently been shown to be successful in mammals (Wianny and
Zernicka-Goetz, 2000, Nat Cell Biol 2000, 2, 70-75). Double
stranded RNA corresponding to the sequence of a cgFADD, cgPDCD6
and/or cgRequiem polynucleotide can be introduced into or expressed
in cells or cell lines to enhance cell viability.
[0158] In particular, we describe modification by the use of single
interfering RNAs (siRNAs) as well as the use of dominant negative
mutants where reduction in expression is desired. We further
describe the use of vectors which enable over-expression of a
relevant sequence for increasing expression of relevant genes. The
modification may be transient, or it may be permanent. Thus, we
provide for cell lines which comprise cells with genomic and
transmittable modifications in cgFAIM, cgFADD, cgPDCD6 and/or
cgRequiem. A detailed protocol for establishing such cells lines is
set out in the Examples.
[0159] The modified cells may be provided as single cells, groups
of cells, clones, clonal lines, colonies, cell lines or tissues. We
further provide for transgenic animals whose cells comprise
down-regulated expression of cgFADD, cgPDCD6 and/or cgRequiem, or
up-regulated expression of cgFADD, or both.
[0160] Preferably, the dominant mutant comprises a cgFADD dominant
mutant comprising the sequence set out in SEQ ID NO: 9.
Alternatively, or in addition, the sequence may comprise:
TABLE-US-00002 (SEQ ID NO: 41)
FDIVCDNVGRDWKRLARQLKVSEAKIDGIEERYPRSLSEQVREALRVWKI
AEREKATVAGLVKALRACRLNLVADLVE
[0161] The dominant mutant may be encoded by a sequence set out in
SEQ ID NO:10, or alternatively,
TABLE-US-00003 (SEQ ID NO: 42)
TTTGACATTGTATGCGACAATGTGGGGAGAGATTGGAAGAGACTGGCCCG
CCAGCTGAAAGTGTCTGAGGCCAAAATTGATGGGATTGAGGAGAGGTACC
CCCGAAGCCTGAGTGAGCAGGTAAGGGAGGCTCTGAGAGTCTGGAAGATT
GCCGAGAGGGAGAAAGCCACGGTGGCTGGACTGGTAAAGGCACTTCGGGC
CTGCCGGCTGAACCTGGTGGCTGACCTGGTGGAA
[0162] Increased Cell Viability
[0163] The modified cells have several beneficial properties when
compared to cognate non-modified cells, or wild type cells, or
parental cells from which they are derived. They may have the
property of having improved cell viability. Thus, they may survive
in culture longer, in terms of time or number of generations.
[0164] Preferably, cell viability is gauged by quantitating a
viable cell density of a population of cells which have been
modified, i.e., by targeting cgFAIM, cgFADD, cgPDCD6 and cgRequiem.
Preferably, the modified cells maintain a higher cell viability,
compared to cells which have not been modified (e.g., a control
population). Cell viability is preferably measured as the
percentage of cells in the relevant cell population which are
viable.
[0165] In a preferred embodiment, cell viability is determined by a
"Trypan blue viability exclusion assay". This assay is commonly
used for cell viability determination in the field of cell culture.
A detailed protocol is set out in the Examples, but in brief: a
cell suspension is mixed with 0.4% trypan blue in phosphate
buffered solution and counted using a hemocytometer. Live cells
appear round and refractile without any blue-dye coloration while
dead cells absorb the dye and appear blue. Viability is then
expressed as a percentage of viable cells over total cells
counted.
[0166] A viable cell is defined as a cell that whose membrane
integrity is still able to prevent the absorption of trypan blue in
a trypan blue exclusion viability assay.
[0167] Preferably, the modified cells have at least 5%, preferably
10% or more, more preferably 15%, 20%, 30%, 40%, 50% or more viable
cells compared to a control population. Alternatively, or in
addition, the modified cells maintain cell viability for a longer
period of time compared to cells which have not been modified. For
example, modified cells are able to maintain a certain percentage
cell viability (e.g., 95%) for a longer period compared to control
cells.
[0168] Preferably, modified cells have extended cell viability by
at least 1 hour, more preferably at least 6 hours, most preferably
at least 12 hours or more, e.g., at least 24 hours, at least 36
hours or at least 48 hours, compared to control cells. In highly
preferred embodiments, modified cells have extended viability by at
least 24 hours before viability begins to drop below 95%, compared
to control cells.
[0169] The modified cells preferably are capable of higher viable
culture densities compared to unmodified control cells. Preferably,
the modified cells are capable of 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 100%, 150%, 200% or higher viable cell density
compared to control cells. For example, modified cells may achieve
densities as high as 9.6.times.10.sup.6 cells/ml.
[0170] The modified cells preferably display a delayed onset of
expression of an apoptosis marker, preferably caspase 2, caspase 3
or caspase 8. The modified cells may have the property of
displaying reduced apoptosis, in terms of longer time of survival
for individual cells, or the number of cells which display
apoptosis. Preferably, they have the property of being resistant to
apoptosis (see below).
[0171] Increased Protein Yield
[0172] Advantageously, the modified cells are capable of increased
protein yield, preferably increased recombinant expressed protein
yield, compared to unmodified control cells, as demonstrated in
Example 21. Preferably, modified cells are capable of 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or more higher
yield compared to control cells. More preferably, modified cells
are capable of 2.5.times., 3.times., 5.times., 10.times. or more
higher yield compared to control cells. Preferably, the recombinant
expressed protein comprises interferon gamma. We therefore provide
a method of expressing a recombinant protein, preferably a
biotherapeutic molecule, in a modified cell as described.
[0173] Preferably, the modified cells display any one or more of
their properties in batch culture, fed-batch culture or preferably
both.
[0174] Increased Glycosylation
[0175] The modified cells preferably are also capable of increased
glycosylation of expressed proteins compared to control unmodified
cells. The Examples show that the modified cells are capable of
maintaining protein glycosylation over extended cell culture time,
whether or not loss of cell culture viability has taken place. In
highly preferred embodiments, the glycosylation comprises
sialylation.
[0176] This characteristic of modified cell lines is particularly
advantageous in the manufacturing of biotherapeutics as a lower
degree of sialylation can decrease the in vivo half-life of
protein-based drugs (Varki, 1993, Biotechnol Bioeng 43:423-428;
Gramer et al., 1995, Glycobiology 3:97-130).
[0177] In preferred embodiments, the glycosylation of the expressed
protein is maintained substantially throughout one or more growth
phases of cell culture, preferably through at least part of
exponential phase (preferably at least through mid-exponential
phase), but more preferably also through the point at which maximum
viable cell density occurs, more preferably also through a point at
which cell death would occur in a parental or unmodified cell. In
such cases, the level of glycosylation is preferably maintained at
a level where it would decrease in a parental or unmodified cell.
In preferred embodiments, the glycosylation is maintained at a
level of at least 2.7, preferably at least 2.9 moles of the sugar
per mole of expressed protein.
[0178] In preferred embodiments, glycosylation of the expressed
protein by a modified cell is increased compared to a parental or
unmodified cell in a cognate point in the growth phase. In such
preferred embodiments, glycosylation may be achieved at a level of
at least 2.9, preferably at least 3, 3.1, 3.2, 3.3, 3.4 or 3.5
moles of the sugar per mole of expressed protein.
[0179] We further provide for recombinant proteins with increased
glycosylation, preferably increased sialyation, made using modified
cells as described. Such polypeptides have an increased
sialylation, compared to a polypeptide producable from a cell which
is not so modified. Preferably, the glycosylation or sialylation is
greater than 2.9 mol sialic acid/mol of produced polypeptide,
preferably about 3.5 mol of sialic acid/mol of produced
polypeptide. In highly preferred embodiments, the expressed protein
comprises interferon gamma.
[0180] We further provide methods for modifying a cell to display
any one or more of the above properties, by modulating its
expression of cgFAIM, gFADD, cgPDCD6 and/or cgRequiem.
Apoptosis
[0181] According to the invention, increase in cell viability of
cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem modified cells results
from a decrease in apoptosis in the modified cell populations. Such
modified cells may display reduced apoptosis, or be resistant to
apoptosis. The modified cells are preferably capable of maintaining
a higher viable cell density, preferably for a longer period of
time, compared to control cells. Preferably, the number of viable
cells in a modified population is higher, for example 10%, 20%,
30%, 40%, 50%, 100%, 200%, 500%, or more, compared to an ummodified
control population.
[0182] Preferably, modified cells show an extension of viability by
at least 6 hours, at least 12 hours, preferably at least 18 hours,
and most preferably at least 24 hours compared to control cells. In
highly preferred embodiments, caspase 2 and/or caspase 3 and/or
caspase 8 expression is delayed by such times compared to control
cells.
[0183] Accordingly, preferably, apoptosis in a modified cell
population is decreased by at least 10%, preferably 25% or more,
more preferably 40%, 50%, 75%, 95% or more compared to a control
population. In preferred embodiments, the percentage of apoptotic
cells in a modified cell population is decreased by such amounts.
In highly preferred embodiments, the modified cells are resistant
to apoptosis, i.e., display little or no significant apoptosis.
[0184] Methods of assaying apoptosis are known in the art, and are
described in detail below and in the Examples. A preferred method
of assaying apoptosis is set out in Example 14: Apoptosis
Assay.
[0185] An alternative assay of apoptosis involves quantitation or
measurement of levels any one or more of caspase 2, caspase 3 and
caspase 8 in the relevant cells. Thus, preferably, levels of any
one or more of these caspases is decreased in a modified cell or
population compared to one which has not been so modified, by 10%,
20%, 30%, 50%, 70%, 80%, 90% or more. Preferably, modified cells
exhibit a delay in expression of any one or more of caspase 2,
caspase 3 and caspase 8 by a period of time preferably at least 1
hour, more preferably at least 6 hours, most preferably at least 12
hours or more, e.g., at least 24 hours, at least 36 hours, at least
48 hours, at least 72 hours, at least 144 hours, at least 288
hours, or more, compared to control cells which are not modified.
Caspase levels may be assayed by any means known in the art,
including RT-PCR, RNAse protection, SDS-PAGE, immunoassays,
etc.
[0186] Cell death can occur by either of two distinct mechanisms,
necrosis or apoptosis. In addition, certain chemical compounds and
cells are said to be cytotoxic to the cell, that is, to cause its
death.
[0187] "Cytotoxicity" refers to the cell killing property of a
chemical compound (such as a food, cosmetic, or pharmaceutical) or
a mediator cell (cytotoxic T cell). In contrast to necrosis and
apoptosis, the term cytotoxicity need not necessarily indicate a
specific cellular death mechanism. For example, cell mediated
cytotoxicity (that is, cell death mediated by either cytotoxic T
lymphocytes [CTL] or natural killer [NK] cells) combines some
aspects of both necrosis and apoptosis.
[0188] "Necrosis" (also referred to as "accidental" cell death)
refers to the pathological process which occurs when cells are
exposed to a serious physical or chemical insult. Necrosis occurs
when cells are exposed to extreme variance from physiological
conditions (e.g., hypothermia, hypoxia) which may result in damage
to the plasma membrane. Under physiological conditions direct
damage to the plasma membrane is evoked by agents like complement
and lytic viruses. Necrosis begins with an impairment of the cell's
ability to maintain homeostasis, leading to an influx of water and
extracellular ions. Intracellular organelles, most notably the
mitochondria, and the entire cell swell and rupture (cell lysis).
Due to the ultimate breakdown of the plasma membrane, the
cytoplasmic contents including lysosomal enzymes are released into
the extracellular fluid. Therefore, in vivo, necrotic cell death is
often associated with extensive tissue damage resulting in an
intense inflammatory response.
[0189] "Apoptosis" ("normal" or "programmed" cell death) refers to
the physiological process by which unwanted or useless cells are
eliminated during development and other normal biological
processes. Apoptosis is a mode of cell death that occurs under
normal physiological conditions and the cell is an active
participant in its own demise ("cellular suicide"). It is most
often found during normal cell turnover and tissue homeostasis,
embryogenesis, induction and maintenance of immune tolerance,
development of the nervous system and endocrine-dependent tissue
atrophy. Cells undergoing apoptosis show characteristic
morphological and biochemical features. These features include
chromatin aggregation, nuclear and cytoplasmic condensation,
partition of cytoplasm and nucleus into membrane bound vesicles
(apoptotic bodies) which contain ribosomes, morphologically intact
mitochondria and nuclear material. In vivo, these apoptotic bodies
are rapidly recognized and phagocytized by either macrophages or
adjacent epithelial cells. Due to this efficient mechanism for the
removal of apoptotic cells in vivo no inflammatory response is
elicited. In vitro, the apoptotic bodies as well as the remaining
cell fragments ultimately swell and finally lyse. This terminal
phase of in vitro cell death has been termed "secondary
necrosis".
[0190] Table 1 summarises the various observable differences
between necrosis and apoptosis. Preferably, modified cells exhibit
a reduction in one or more of these features. Any of these
differences, alone or in combination, may be assayed in order to
determine whether cell death is occurring by apoptosis or by
necrosis.
TABLE-US-00004 TABLE 1 Differential features and significance of
necrosis and apoptosis. Necrosis Apoptosis Morphological Loss of
membrane Membrane blebbing, but no loss of features integrity
integrity Begins with swelling of Aggregation of chromatin at the
nuclear cytoplasm and membrane mitochondria Begins with shrinking
of cytoplasm and Ends with total cell lysis condensation of nucleus
No vesicle formation, Ends with fragmentation of cell into complete
lysis smaller bodies Disintegration Formation of membrane bound
vesicles (swelling) of organelles (apoptotic bodies) Mitochondria
become leaky due to pore formation involving proteins of the bcl-2
family. Biochemical Loss of regulation of ion Tightly regulated
process involving features homeostasis activation and enzymatic
steps No energy requirement Energy (ATP)-dependent (active process,
(passive process, also does not occur at 4.degree. C.) occurs at
4.degree. C.) Non-random mono- and oligonucleosomal Random
digestion of length fragmentation of DNA (Ladder DNA (smear of DNA
pattern after agarose gel electrophoresis) after agarose gel
Prelytic DNA fragmentation Release of electrophoresis) various
factors (cytochrome C, AIF) into Postlytic DNA cytoplasm by
mitochondria fragmentation (=late Activation of caspase cascade
event of death) Alterations in membrane asymmetry (i.e.,
translocation of phosphatidyl-serine from the cytoplasmic to the
extracellular side of the membrane) Physiological Affects groups of
Affects individual cells significance contiguous cells Induced by
physiological stimuli (lack of Evoked by non- growth factors,
changes in hormonal physiological environment) disturbances
Phagocytosis by adjacent cells or (complement attack, macrophages
lytic viruses, hypothermia, No inflammatory response hypoxia,
ischemica, metabolic poisons) Phagocytosis by macrophages
Significant inflammatory response
[0191] Reference is made to the following documents, which describe
apoptosis in detail, as well as various assays for measuring cell
death by apoptosis: Schwartzman, R. A. and Cidlowski, J. A. (1993).
Endocrine Rev. 14, 133; Vermes, I. and Haanan, C. (1994). Adv.
Clin. Chem. 31, 177; Berke, G. (1991). Immunol. Today 12, 396;
Krahenbuhl, O. and Tschopp, J. (1991). Immunol. Today 12, 399; Van
Furth, R. and Van Zwet, T. L. (1988). J. Immunol; Methods 108, 45.
Cohen, J. J. (1993) Apoptosis. Immunol. Today 14, 126; Savill, J.
S. et al. (1989). J. Clin. Invest. 83, 865; Wyllie, A. H. (1980).
Nature 284, 555; Leist, M. et al. (1994) Biochemica No. 3, 18-20;
Fraser, A. and Evan, G. (1996) Cell 85, 781-784; Duke, R. C.
(1983). Proc. Natl. Acad. Sci. USA 80,6361; Duke, R. C. &
Cohen, J. J. (1986). Lymphokine Res. 5, 289; Trauth, B. C. et al.
(1994) Eur. J. Cell. Biol. 63, 32, Suppl 40; Matzinger, P. (1991).
J. Immunol; Methods 145, 185; Kaeck, M. R. (1993); Anal. Biochem.
208, 393; Prigent, P. et al. (1993). J. Immunol; Methods 160, 139;
Huang, P. & Plunkett, W. (1992); Anal. Biochem. 207, 163
Bortner, C. D. et al. (1995) Trends Cell Biol. 5, 21; Gold, R. et
al. (1994); Lab. Invest. 71, 219.
[0192] Apoptosis and cell mediated cytotoxicity are characterized
by cleavage of the genomic DNA into discrete fragments prior to
membrane disintegration. Accordingly, apoptosis may be assayed by
measuring DNA fragmentation, for example, by observing the presence
of DNA ladders. DNA fragments may be assayed, for example, as
"ladders" (with the 180 by multiples as "rungs" of the ladder)
derived from populations of cells, or by quantification of histone
complexed DNA fragments via, for example, ELISA. Such an assay
relies on an one-step sandwich immunoassay to detect nucleosomes.
The procedure involves pelleting cells by centrifugation and
discarding the supernatant (which contains DNA from necrotic cells
that leaked through the membrane during incubation). Cells are
resuspended and incubated in lysis buffer. After lysis, intact
nuclei are pelleted by centrifugation. An aliquot of the
supernatant is transferred to a streptavidin-coated well of a
microtiter plate, and nucleosomes in the supernatant are bound with
two monoclonal antibodies, anti-histone (biotin-labeled) and
anti-DNA (peroxidase-conjugated). Antibody-nucleosome complexes are
bound to the microtiter plate by the streptavidin. The immobilized
antibody-histone complexes are washed three times to remove cell
components that are not immuno-reactive, and the sample is
incubated with peroxidase substrate (ABTS.RTM.). The amount of
colored product (and thus, of immobilized anti-body-histone
complexes) is then determined spectrophotometrically.
[0193] Several proteases are involved in the early stages of
apoptosis. Apoptosis may therefore also be assayed by detecting the
presence of, in addition to, or instead of, assaying the activity
of, apoptosis-induced proteases such as caspases, e.g., caspase 3.
Caspase activation can be analyzed in different ways, for example,
by an in vitro enzyme assay of, for example, cellular lysates by
capturing of the caspase and measuring proteolytic cleavage of a
suitable substrate. Furthermore, caspases may be assayed by
detection of cleavage of an in vivo caspase substrate such as PARP
(Poly-ADP-Ribose-Polymerase). Cleaved fragments of PARP may be
detected with a suitable antibody such as an anti PARP antibody.
Protease assays and DNA fragmentation assays are especially
suitable for assaying apoptosis in cell populations.
[0194] Methods for studying apoptosis in individual cells are also
available, such as ISNT and TUNEL enzymatic labeling assays. As
noted above, extensive DNA degradation is a characteristic event
which often occurs in the early stages of apoptosis. Cleavage of
the DNA yields double-stranded, low molecular weight DNA fragments
(mono- and oligonucleosomes) as well as single strand breaks
("nicks") in high molecular weight-DNA. In TUNEL, such DNA strand
breaks are detected by enzymatic labeling of the free 3'-OH termini
with suitable modified nucleotides (such as X-dUTP, X=biotin, DIG
or fluorescein). Suitable labeling enzymes include DNA polymerase
(nick translation) in ISNT ("in situ nick translation") and
terminal deoxynucleotidyl transferase (end labeling) in TUNEL
("TdT-mediated X-dUTP nick end labeling"; Huang, P. & Plunkett,
W., 1992, Anal. Biochem. 207, 163; Bortner, C. D. et al., 1995,
Trends Cell Biol. 5, 21).
[0195] Apoptosis may also be assayed by measuring membrane
alterations, including: loss of terminal sialic acid residues from
the side chains of cell surface glycoproteins, exposing new sugar
residues; emergence of surface glycoproteins that may serve as
receptors for macrophage-secreted adhesive molecules such as
thrombospondin; and loss of asymmetry in cell membrane
phospholipids, altering both the hydrophobicity and charge of the
membrane surface. In particular, the human anticoagulant annexin V
is a 35-36 kilodalton, Ca2+-dependent phospholipid-binding protein
that has a high affinity for phosphatidylserine (PS). In normal
viable cells, PS is located on the cytoplasmic surface of the cell
membrane. However, in apoptotic cells, PS is translocated from the
inner to the outer leaflet of the plasma membrane, thus exposing PS
to the external cellular environment. Annexin V may therefore be
used to detect phosphatidylserine asymmetrically exposed on the
surface of apoptotic cells (Homburg, C. H. E. et al. 1995, Blood
85, 532; Verhoven, B. et al., 1995, J. Exp. Med. 182, 1597).
Furthermore, DNA stains such as DAPI, ethidium bromide and
propidium iodide, etc may be used for differential staining to
distinguish viable and non-viable cells. Profiles of DNA content
may also be used; thus, permeabilized apoptotic cells leak low
molecular weight DNA, and detection of "sub-G 1 peaks", or "A 0"
cells (cells with lower DNA staining than that of G 1 cells) may be
detected by, for example, flow cytometry. Morphological changes
characteristic of apoptosis may also be detected in this
manner.
[0196] Detection of apoptosis-related proteins such as ced-3,
ced-4, ced-9 (Ellis, H. M. and Horvitz, H. R., 1986, Cell 44,
817-829; Yuan, J. Y. and Horvitz, H. R., 1990, Dev. Biol. 138,
33-41; Hentgartner, M. O., Ellis, R. E. and Horvitz, H. R., 1992,
Nature 356, 494-499.), Fas(CD95/Apo-1; Enari et al., 1996, Nature
380, 723-726), Bc1-2 (Baffy, G. et al., 1993, J. Biol. Chem. 268,
6511-6519; Miyashita, T. and Reed, J. C., 1993, Blood 81, 151-157;
Oltvai, Z. N., Milliman, C. L. and Korsmeyer, S. J., 1993, Cell 74,
609-619), p53 (Yonish-Rouach, E. et al., 1991, Nature 352,
345-347), etc by the use of antibodies may also be used to assay
apoptosis.
Nucleotide Vectors
[0197] The polynucleotides can be incorporated into a recombinant
replicable vector. The vector may be used to replicate the nucleic
acid in a compatible host cell. Thus in a further embodiment, we
provide a method of making polynucleotides by introducing a
polynucleotide into a replicable vector, introducing the vector
into a compatible host cell, and growing the host cell under
conditions which bring about replication of the vector. The vector
may be recovered from the host cell. Suitable host cells include
bacteria such as E. coli, yeast, mammalian cell lines and other
eukaryotic cell lines, for example insect Sf9 cells.
[0198] Preferably, a polynucleotide in a vector is operably linked
to a control sequence that is capable of providing for the
expression of the coding sequence by the host cell, i.e. the vector
is an expression vector. The term "operably linked" means that the
components described are in a relationship permitting them to
function in their intended manner. A regulatory sequence "operably
linked" to a coding sequence is ligated in such a way that
expression of the coding sequence is achieved under condition
compatible with the control sequences.
[0199] The control sequences may be modified, for example by the
addition of further transcriptional regulatory elements to make the
level of transcription directed by the control sequences more
responsive to transcriptional modulators.
[0200] Vectors may be transformed or transfected into a suitable
host cell as described below to provide for expression of a
protein. This process may comprise culturing a host cell
transformed with an expression vector as described above under
conditions to provide for expression by the vector of a coding
sequence encoding the protein, and optionally recovering the
expressed protein.
[0201] The vectors may be for example, plasmid or virus vectors
provided with an origin of replication, optionally a promoter for
the expression of the said polynucleotide and optionally a
regulator of the promoter. The vectors may contain one or more
selectable marker genes, for example an ampicillin resistance gene
in the case of a bacterial plasmid or a neomycin resistance gene
for a mammalian vector. Vectors may be used, for example, to
transfect or transform a host cell.
[0202] Control sequences operably linked to sequences encoding the
protein include promoters/enhancers and other expression regulation
signals. These control sequences may be selected to be compatible
with the host cell for which the expression vector is designed to
be used in. The term "promoter" is well-known in the art and
encompasses nucleic acid regions ranging in size and complexity
from minimal promoters to promoters including upstream elements and
enhancers.
[0203] The promoter is typically selected from promoters which are
functional in mammalian cells, although prokaryotic promoters and
promoters functional in other eukaryotic cells may be used. The
promoter is typically derived from promoter sequences of viral or
eukaryotic genes. For example, it may be a promoter derived from
the genome of a cell in which expression is to occur. With respect
to eukaryotic promoters, they may be promoters that function in a
ubiquitous manner (such as promoters of .alpha.-actin,
.beta.-actin, tubulin) or, alternatively, a tissue-specific manner
(such as promoters of the genes for pyruvate kinase). They may also
be promoters that respond to specific stimuli, for example
promoters that bind steroid hormone receptors. Viral promoters may
also be used, for example the Moloney murine leukaemia virus long
terminal repeat (MMLV LTR) promoter, the Rous sarcoma virus (RSV)
LTR promoter or the human cytomegalovirus (CMV) IE promoter.
[0204] It may also be advantageous for the promoters to be
inducible so that the levels of expression of the heterologous gene
can be regulated during the life-time of the cell. Inducible means
that the levels of expression obtained using the promoter can be
regulated.
[0205] In addition, any of these promoters may be modified by the
addition of further regulatory sequences, for example enhancer
sequences. Chimeric promoters may also be used comprising sequence
elements from two or more different promoters described above.
Expression of cgFAIM, cgFADD, cgPDCD6 and cgREQUIEM
Polypeptides
[0206] In order to express a biologically active cgFAIM, cgFADD,
cgPDCD6 and/or cgRequiem polypeptide, a Cricetulus griseus FAIM,
FADD, PDCD6 or Requiem polynucleotide sequence is brought into
association with a regulatory sequence so as to enable the
regulatory sequence to direct expression of said polynucleotide.
Expression of the polypeptide under control of the regulatory
sequence is then allowed to happen. Optionally, the polypeptide so
produced may be purified.
[0207] Preferably, the regulatory sequence is one with which the
FAIM, FADD, PDCD6 or Requiem polynucleotide sequence is not
naturally associated.
[0208] We therefore describe a method of producing polypeptide
comprising providing a cell, preferably a Cricetulus griseus cell,
in which a Cricetulus griseus FAIM, FADD, PDCD6 or Requiem
polynucleotide sequence has been brought into association with a
regulatory sequence so as to enable the regulatory sequence to
direct expression of said polynucleotide, and culturing the cell
under conditions which enable expression of the polypeptide, and
optionally purifying the polypeptide.
[0209] We further describe a method of producing a polypeptide
comprising: (a) providing an expression sequence produced by
bringing a Cricetulus griseus FAIM, FADD, PDCD6 or Requiem
polynucleotide sequence into association with a regulatory sequence
so as to enable the regulatory sequence to direct expression of
said polynucleotide; (b) allowing expression of the polypeptide
from the expression sequence under control of the regulatory
sequence, and (c) optionally purifying the polypeptide.
[0210] In particular, the nucleotide sequences encoding the
respective nucleic acid or homologues, variants, or derivatives
thereof may be inserted into appropriate expression vector, i.e., a
vector which contains the necessary elements for the transcription
and translation of the inserted coding sequence.
[0211] We also provide for a polypeptide produced by any of the
above methods.
[0212] Methods of enabling expression of cgFAIM, cgFADD, cgPDCD6
and cgRequiem polypeptides are set out below. It will be
appreciated that these methods may be suitable for use in
embodiments of the methods and compositions described here in which
up-regulation of a polypeptide is desired, e.g., up-regulation of
cgFADD in order to achieve enhanced cell viability.
[0213] One method by which to provide expressed polypeptides is by
means of an expression vector, i.e., a vector (e.g., a plasmid)
which contains a regulatable promoter, optionally with other
regulatory sequences such as enhancers, which is operably linked to
a sequence encoding a polypeptide of interest which has been cloned
into the expression vector.
[0214] Methods which are well known to those skilled in the art may
be used to construct expression vectors containing sequences
encoding cgFAIM, cgFADD, cgPDCD6 and cgRequiem and appropriate
transcriptional and translational control elements. These methods
include in vitro recombinant DNA techniques, synthetic techniques,
and in vivo genetic recombination. Such techniques are described in
Sambrook, J. et al. (1989; Molecular Cloning, A Laboratory Manual,
ch. 4, 8, and 16-17, Cold Spring Harbor Press, Plainview, N.Y.) and
Ausubel, F. M. et al. (1995 and periodic supplements; Current
Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley &
Sons, New York, N.Y.).
[0215] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding cgFAIM, cgFADD, cgPDCD6
and/or cgRequiem. These include, but are not limited to,
microorganisms such as bacteria transformed with recombinant
bacteriophage, plasmid, or cosmid DNA expression vectors; yeast
transformed with yeast expression vectors; insect cell systems
infected with virus expression vectors (e.g., baculovirus); plant
cell systems transformed with virus expression vectors (e.g.,
cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or
with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or
animal cell systems. Any suitable host cell may be employed.
[0216] The "control elements" or "regulatory sequences" are those
non-translated regions of the vector (i.e., enhancers, promoters,
and 5' and 3' untranslated regions) which interact with host
cellular proteins to carry out transcription and translation. Such
elements may vary in their strength and specificity. Depending on
the vector system and host utilized, any number of suitable
transcription and translation elements, including constitutive and
inducible promoters, may be used. For example, when cloning in
bacterial systems, inducible promoters such as the hybrid lacZ
promoter of the BLUESCRIPT phagemid (Stratagene, La Jolla, Calif.)
or PSPORT1 plasmid (GIBCO/BRL), and the like, may be used. The
baculovirus polyhedrin promoter may be used in insect cells.
Promoters or enhancers derived from the genomes of plant cells
(e.g., heat shock, RUBISCO, and storage protein genes) or from
plant viruses (e.g., viral promoters or leader sequences) may be
cloned into the vector.
[0217] In mammalian cell systems, promoters from mammalian genes or
from mammalian viruses are preferable. If it is necessary to
generate a cell line that contains multiple copies of the sequence
encoding cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem, vectors based on
SV40 or EBV may be used with an appropriate selectable marker.
[0218] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for cgFAIM, cgFADD,
cgPDCD6 and/or cgRequiem. For example, when large quantities of
cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem are needed for the
induction of antibodies, vectors which direct high level expression
of fusion proteins that are readily purified may be used. Such
vectors include, but are not limited to, multifunctional E. coli
cloning and expression vectors such as BLUESCRIPT (Stratagene), in
which the sequence encoding cgFAIM, cgFADD, cgPDCD6 and/or
cgRequiem may be ligated into the vector in frame with sequences
for the amino-terminal Met and the subsequent 7 residues of
.beta.-galactosidase so that a hybrid protein is produced, pIN
vectors (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem.
264:5503-5509), and the like. pGEX vectors (Promega, Madison, Wis.)
may also be used to express foreign polypeptides as fusion proteins
with glutathione S-transferase (GST). In general, such fusion
proteins are soluble and can easily be purified from lysed cells by
adsorption to glutathione-agarose beads followed by elution in the
presence of free glutathione. Proteins made in such systems may be
designed to include heparin, thrombin, or factor XA protease
cleavage sites so that the cloned polypeptide of interest can be
released from the GST moiety at will.
[0219] In the yeast Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters, such as alpha
factor, alcohol oxidase, and PGH, may be used. For reviews, see
Ausubel (supra) and Grant et al. (1987; Methods Enzymol.
153:516-544).
[0220] In cases where plant expression vectors are used, the
expression of sequences encoding cgFAIM, cgFADD, cgPDCD6 and/or
cgRequiem may be driven by any of a number of promoters. For
example, viral promoters such as the 35S and 19S promoters of CaMV
may be used alone or in combination with the omega leader sequence
from TMV. (Takamatsu, N. (1987) EMBO J. 6:307-311.) Alternatively,
plant promoters such as the small subunit of RUBISCO or heat shock
promoters may be used. (Coruzzi, G. et al. (1984) EMBO J.
3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and
Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.)
These constructs can be introduced into plant cells by direct DNA
transformation or pathogen-mediated transfection. Such techniques
are described in a number of generally available reviews. (See, for
example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of
Science and Technology (1992) McGraw Hill, New York, N.Y.; pp.
191-196.).
[0221] An insect system may also be used to express cgFAIM, cgFADD,
cgPDCD6 and/or cgRequiem. For example, in one such system,
Autographa californica nuclear polyhedrosis virus (AcNPV) is used
as a vector to express foreign genes in Spodoptera frugiperda cells
or in Trichoplusia larvae. The sequences encoding cgFAIM, cgFADD,
cgPDCD6 and/or cgRequiem may be cloned into a non-essential region
of the virus, such as the polyhedrin gene, and placed under control
of the polyhedrin promoter. Successful insertion of cgFAIM, cgFADD,
cgPDCD6 and/or cgRequiem will render the polyhedrin gene inactive
and produce recombinant virus lacking coat protein. The recombinant
viruses may then be used to infect, for example, S. frugiperda
cells or Trichoplusia larvae in which cgFAIM, cgFADD, cgPDCD6
and/or cgRequiem may be expressed. (Engelhard, E. K. et al. (1994)
Proc. Nat. Acad. Sci. 91:3224-3227.)
[0222] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding cgFAIM, cgFADD, cgPDCD6
and/or cgRequiem may be ligated into an adenovirus
transcription/translation complex consisting of the late promoter
and tripartite leader sequence. Insertion in a non-essential E1 or
E3 region of the viral genome may be used to obtain a viable virus
which is capable of expressing cgFAIM, cgFADD, cgPDCD6 and/or
cgRequiem in infected host cells. (Logan, J. and T. Shenk (1984)
Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcription
enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be
used to increase expression in mammalian host cells.
[0223] Thus, for example, the cgFAIM, cgFADD, cgPDCD6 and/or
cgRequiem proteins are expressed in either human embryonic kidney
293 (HEK293) cells or adherent dhfr CHO cells. To maximize receptor
expression, typically all 5' and 3' untranslated regions (UTRs) are
removed from the receptor cDNA prior to insertion into a pCDN or
pcDNA3 vector. The cells are transfected with individual receptor
cDNAs by lipofectin and selected in the presence of 400 mg/ml G418.
After 3 weeks of selection, individual clones are picked and
expanded for further analysis. HEK293 or CHO cells transfected with
the vector alone serve as negative controls. To isolate cell lines
stably expressing the individual receptors, about 24 clones are
typically selected and analyzed by Northern blot analysis. Receptor
mRNAs are generally detectable in about 50% of the G418-resistant
clones analyzed.
[0224] Human artificial chromosomes (HACs) may also be employed to
deliver larger fragments of DNA than can be contained and expressed
in a plasmid. HACs of about 6 kb to 10 Mb are constructed and
delivered via conventional delivery methods (liposomes,
polycationic amino polymers, or vesicles) for therapeutic
purposes.
[0225] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding cgFAIM, cgFADD, cgPDCD6
and/or cgRequiem. Such signals include the ATG initiation codon and
adjacent sequences. In cases where sequences encoding cgFAIM,
cgFADD, cgPDCD6 and/or cgRequiem and its initiation codon and
upstream sequences are inserted into the appropriate expression
vector, no additional transcriptional or translational control
signals may be needed. However, in cases where only coding
sequence, or a fragment thereof, is inserted, exogenous
translational control signals including the ATG initiation codon
should be provided. Furthermore, the initiation codon should be in
the correct reading frame to ensure translation of the entire
insert. Exogenous translational elements and initiation codons may
be of various origins, both natural and synthetic. The efficiency
of expression may be enhanced by the inclusion of enhancers
appropriate for the particular cell system used, such as those
described in the literature. (Scharf, D. et al. (1994) Results
Probl. Cell Differ. 20:125-162.)
[0226] In addition, a host cell strain may be chosen for its
ability to modulate expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be used to
facilitate correct insertion, folding, and/or function. Different
host cells which have specific cellular machinery and
characteristic mechanisms for post-translational activities (e.g.,
CHO, HeLa, MDCK, HEK293, and WI38), are available from the American
Type Culture Collection (ATCC, Bethesda, Md.) and may be chosen to
ensure the correct modification and processing of the foreign
protein.
[0227] For long term, high yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
capable of stably expressing cgFAIM, cgFADD, cgPDCD6 and/or
cgRequiem can be transformed using expression vectors which may
contain viral origins of replication and/or endogenous expression
elements and a selectable marker gene on the same or on a separate
vector. Following the introduction of the vector, cells may be
allowed to grow for about 1 to 2 days in enriched media before
being switched to selective media. The purpose of the selectable
marker is to confer resistance to selection, and its presence
allows growth and recovery of cells which successfully express the
introduced sequences. Resistant clones of stably transformed cells
may be proliferated using tissue culture techniques appropriate to
the cell type.
[0228] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase genes (Wigler, M. et al.
(1977) Cell 11:223-32) and adenine phosphoribosyltransferase genes
(Lowy, I. et al. (1980) Cell 22:817-23), which can be employed in
tk.sup.- or apr.sup.- cells, respectively. Also, antimetabolite,
antibiotic, or herbicide resistance can be used as the basis for
selection. For example, dhfr confers resistance to methotrexate
(Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt
confers resistance to the aminoglycosides neomycin and G-418
(Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14); and als
or pat confer resistance to chlorsulfuron and phosphinotricin
acetyltransferase, respectively (Murry, supra). Additional
selectable genes have been described, for example, trpB, which
allows cells to utilize indole in place of tryptophan, or hisD,
which allows cells to utilize histinol in place of histidine.
(Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci.
85:8047-51.) Recently, the use of visible markers has gained
popularity with such markers as anthocyanins. .beta.-glucuronidase
and its substrate GUS, and luciferase and its substrate luciferin.
These markers can be used not only to identify transformants, but
also to quantify the amount of transient or stable protein
expression attributable to a specific vector system. (Rhodes, C. A.
et al. (1995) Methods Mol. Biol. 55:121-131.)
[0229] Although the presence/absence of marker gene expression
suggests that the gene of interest is also present, the presence
and expression of the gene may need to be confirmed. For example,
if the sequence encoding cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem
is inserted within a marker gene sequence, transformed cells
containing sequences encoding cgFAIM, cgFADD, cgPDCD6 and/or
cgRequiem can be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a
sequence encoding cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem under
the control of a single promoter. Expression of the marker gene in
response to induction or selection usually indicates expression of
the tandem gene as well.
[0230] Alternatively, host cells which contain the nucleic acid
sequence encoding cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem and
express cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem may be identified
by a variety of procedures known to those of skill in the art.
These procedures include, but are not limited to, DNA-DNA or
DNA-RNA hybridizations and protein bioassay or immunoassay
techniques which include membrane, solution, or chip based
technologies for the detection and/or quantification of nucleic
acid or protein sequences.
[0231] The presence of polynucleotide sequences encoding cgFAIM,
cgFADD, cgPDCD6 and/or cgRequiem can be detected by DNA-DNA or
DNA-RNA hybridization or amplification using probes or fragments or
fragments of polynucleotides encoding cgFAIM, cgFADD, cgPDCD6
and/or cgRequiem. Nucleic acid amplification based assays involve
the use of oligonucleotides or oligomers based on the sequences
encoding cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem to detect
transformants containing DNA or RNA encoding cgFAIM, cgFADD,
cgPDCD6 and/or cgRequiem.
[0232] A variety of protocols for detecting and measuring the
expression of cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem, using
either polyclonal or monoclonal antibodies specific for the
protein, are known in the art. Examples of such techniques include
enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays
(RIAs), and fluorescence activated cell sorting (FACS). A two-site,
monoclonal-based immunoassay utilizing monoclonal antibodies
reactive to two non-interfering epitopes on cgFAIM, cgFADD, cgPDCD6
and/or cgRequiem is preferred, but a competitive binding assay may
be employed. These and other assays are well described in the art,
for example, in Hampton, R. et al. (1990; Serological Methods, a
Laboratory Manual, Section IV, APS Press, St Paul, Minn.) and in
Maddox, D. E. et al. (1983; J. Exp. Med. 158:1211-1216).
[0233] A wide variety of labels and conjugation techniques are
known by those skilled in the art and may be used in various
nucleic acid and amino acid assays. Means for producing labeled
hybridization or PCR probes for detecting sequences related to
polynucleotides encoding cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem
include oligolabeling, nick translation, end-labeling, or PCR
amplification using a labeled nucleotide. Alternatively, the
sequences encoding cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem, or any
fragments thereof, may be cloned into a vector for the production
of an mRNA probe. Such vectors are known in the art, are
commercially available, and may be used to synthesize RNA probes in
vitro by addition of an appropriate RNA polymerase such as T7, T3,
or SP6 and labeled nucleotides. These procedures may be conducted
using a variety of commercially available kits, such as those
provided by Pharmacia & Upjohn (Kalamazoo, Mich.), Promega
(Madison, Wis.), and U.S. Biochemical Corp. (Cleveland, Ohio).
Suitable reporter molecules or labels which may be used for ease of
detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic agents, as well as substrates,
cofactors, inhibitors, magnetic particles, and the like.
[0234] Host cells transformed with nucleotide sequences encoding
cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem may be cultured under
conditions suitable for the expression and recovery of the protein
from cell culture. The protein produced by a transformed cell may
be located in the cell membrane, secreted or contained
intracellularly depending on the sequence and/or the vector used.
As will be understood by those of skill in the art, expression
vectors containing polynucleotides which encode cgFAIM, cgFADD,
cgPDCD6 and/or cgRequiem may be designed to contain signal
sequences which direct secretion of cgFAIM, cgFADD, cgPDCD6 and/or
cgRequiem through a prokaryotic or eukaryotic cell membrane. Other
constructions may be used to join sequences encoding cgFAIM,
cgFADD, cgPDCD6 and/or cgRequiem to nucleotide sequences encoding a
polypeptide domain which will facilitate purification of soluble
proteins. Such purification facilitating domains include, but are
not limited to, metal chelating peptides such as
histidine-tryptophan modules that allow purification on immobilized
metals, protein A domains that allow purification on immobilized
immunoglobulin, and the domain utilized in the FLAGS
extension/affinity purification system (Immunex Corp., Seattle,
Wash.). The inclusion of cleavable linker sequences, such as those
specific for Factor XA or enterokinase (Invitrogen, San Diego,
Calif.), between the purification domain and the cgFAIM, cgFADD,
cgPDCD6 and/or cgRequiem encoding sequence may be used to
facilitate purification. One such expression vector provides for
expression of a fusion protein containing cgFAIM, cgFADD, cgPDCD6
and/or cgRequiem and a nucleic acid encoding 6 histidine residues
preceding a thioredoxin or an enterokinase cleavage site. The
histidine residues facilitate purification on immobilized metal ion
affinity chromatography (IMIAC; described in Porath, J. et al.
(1992) Prot. Exp. Purif. 3: 263-281), while the enterokinase
cleavage site provides a means for purifying cgFAIM, cgFADD,
cgPDCD6 and/or cgRequiem from the fusion protein. A discussion of
vectors which contain fusion proteins is provided in Kroll, D. J.
et al. (1993; DNA Cell Biol. 12:441-453).
[0235] Fragments of cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem, as
well as whole length polypeptides, may be produced not only by
recombinant production, but also by direct peptide synthesis using
solid-phase techniques. (Merrifield J. (1963) J. Am. Chem. Soc.
85:2149-2154.) Protein synthesis may be performed by manual
techniques or by automation. Automated synthesis may be achieved,
for example, using the Applied Biosystems 431A peptide synthesizer
(Perkin Elmer). Various fragments of cgFAIM, cgFADD, cgPDCD6 and/or
cgRequiem may be synthesized separately and then combined to
produce the full length molecule.
[0236] Other methods of expression are also known, for example, a
method known as "gene activation" may be employed to modulate
activity or expression of cgFAIM, cgFADD, cgPDCD6 and cgRequiem.
This method is described in detail in U.S. Pat. No. 5,641,670,
hereby incorporated by reference. In essence, the gene activation
method is based upon the recognition that the regulation or
activity of endogenous genes of interest in a cell can be altered
by inserting into the cell genome, at a preselected site, through
homologous recombination, a suitable DNA construct comprising: (a)
a targeting sequence; (b) a regulatory sequence; (c) an exon and
(d) an unpaired splice-donor site, wherein the targeting sequence
directs the integration of elements (a)-(d) such that the elements
(b)-(d) are operatively linked to the endogenous gene. The DNA
construct may alternatively comprise: (a) a targeting sequence, (b)
a regulatory sequence, (c) an exon, (d) a splice-donor site, (e) an
intron, and (f) a splice-acceptor site, wherein the targeting
sequence directs the integration of elements (a)-(f) such that the
elements of (b)-(f) are operatively linked to the first exon of the
endogenous gene.
[0237] The targeting sequences used are selected with reference to
the site into which the DNA is to be inserted. In both arrangements
the targeting event is used to create a new transcription unit,
which is a fusion product of sequences introduced by the targeting
DNA constructs and the endogenous cellular gene. For example, the
formation of the new transcription unit allows transcriptionally
silent genes (genes not expressed in a cell prior to transfection)
to be activated in host cells by introducing into the host cell's
genome a DNA construct as described. The expression of an
endogenous gene such as cgFAIM, cgFADD, cgPDCD6 or cgRequiem which
is expressed in a cell as obtained can be altered in that it is
increased, reduced, including eliminated, or the pattern of
regulation or induction may be changed through use of the gene
activation method.
Antibodies
[0238] Specific antagonists of cgFAIM, cgFADD, cgPDCD6 and/or
cgRequiem, which may be used to regulate the activity of these
proteins and may include antibodies against the protein(s). In
particular, antibodies capable of binding to cgFADD, cgPDCD6 and
cgRequiem, and preferably capable of inhibiting any biological
activity thereof, are suitable for use in down-regulating
expression of the relevant protein for enhancing cell
viability.
[0239] We therefore provide in particular for anti-cgFADD,
anti-cgFAIM, anti-cgPDCD6 and anti-cgRequiem antibodies, as well as
methods of producing them.
[0240] Antibodies, as used herein, refers to complete antibodies or
antibody fragments capable of binding to a selected target, and
including Fv, ScFv, Fab' and F(ab').sub.2, monoclonal and
polyclonal antibodies, engineered antibodies including chimeric,
CDR-grafted and humanised antibodies, and artificially selected
antibodies produced using phage display or alternative techniques.
Small fragments, such as Fv and ScFv, possess advantageous
properties for diagnostic and therapeutic applications on account
of their small size and consequent superior tissue
distribution.
[0241] The anti-cgFAIM, cgFADD, cgPDCD6 and cgRequiem antibodies
described here may be used for the detection of the relevant
protein, for example, within the context of a cell. Accordingly,
they may be altered antibodies comprising an effector protein such
as a label. Especially preferred are labels which allow the imaging
of the distribution of the antibody in vivo or in vitro. Such
labels may be radioactive labels or radio-opaque labels, such as
metal particles, which are readily visualisable within an embryo or
a cell mass. Moreover, they may be fluorescent labels or other
labels which are visualisable on tissue samples.
[0242] Recombinant DNA technology may be used to improve the
antibodies as described here. Thus, chimeric antibodies may be
constructed in order to decrease the immunogenicity thereof in
diagnostic or therapeutic applications. Moreover, immunogenicity
may be minimised by humanising the antibodies by CDR grafting [see
European Patent Application 0 239 400 (Winter)] and, optionally,
framework modification [EP 0 239 400].
[0243] Anti-cgFAIM, cgFADD, cgPDCD6 and cgRequiem antibodies may be
obtained from animal serum, or, in the case of monoclonal
antibodies or fragments thereof, produced in cell culture.
Recombinant DNA technology may be used to produce the antibodies
according to established procedure, in bacterial or preferably
mammalian cell culture. The selected cell culture system preferably
secretes the antibody product.
[0244] Therefore, we disclose a process for the production of an
antibody comprising culturing a host, e.g. E. coli or a mammalian
cell, which has been transformed with a hybrid vector comprising an
expression cassette comprising a promoter operably linked to a
first DNA sequence encoding a signal peptide linked in the proper
reading frame to a second DNA sequence encoding said antibody
protein, and isolating said protein.
[0245] Multiplication of hybridoma cells or mammalian host cells in
vitro is carried out in suitable culture media, which are the
customary standard culture media, for example Dulbecco's Modified
Eagle Medium (DMEM) or RPMI 1640 medium, optionally replenished by
a mammalian serum, e.g. foetal calf serum, or trace elements and
growth sustaining supplements, e.g. feeder cells such as normal
mouse peritoneal exudate cells, spleen cells, bone marrow
macrophages, 2-aminoethanol, insulin, transferrin, low density
lipoprotein, oleic acid, or the like. Multiplication of host cells
which are bacterial cells or yeast cells is likewise carried out in
suitable culture media known in the art, for example for bacteria
in medium LB, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC,
2.times.YT, or M9 Minimal Medium, and for yeast in medium YPD,
YEPD, Minimal Medium, or Complete Minimal propout Medium.
[0246] In vitro production provides relatively pure antibody
preparations and allows scale-up to give large amounts of the
desired antibodies. Techniques for bacterial cell, yeast or
mammalian cell cultivation are known in the art and include
homogeneous suspension culture, e.g. in an airlift reactor or in a
continuous stirrer reactor, or immobilised or entrapped cell
culture, e.g. in hollow fibres, microcapsules, on agarose
microbeads or ceramic cartridges.
[0247] Large quantities of the desired antibodies can also be
obtained by multiplying mammalian cells in vivo. For this purpose,
hybridoma cells producing the desired antibodies are injected into
histocompatible mammals to cause growth of antibody-producing
tumours. Optionally, the animals are primed with a hydrocarbon,
especially mineral oils such as pristane (tetramethyl-pentadecane),
prior to the injection. After one to three weeks, the antibodies
are isolated from the body fluids of those mammals. For example,
hybridoma cells obtained by fusion of suitable myeloma cells with
antibody-producing spleen cells from Balb/c mice, or transfected
cells derived from hybridoma cell line Sp2/0 that produce the
desired antibodies are injected intraperitoneally into Balb/c mice
optionally pre-treated with pristane, and, after one to two weeks,
ascitic fluid is taken from the animals.
[0248] The foregoing, and other, techniques are discussed in, for
example, Kohler and Milstein, (1975) Nature 256:495-497; U.S. Pat.
No. 4,376,110; Harlow and Lane, Antibodies: a Laboratory Manual,
(1988) Cold Spring Harbor, incorporated herein by reference.
Techniques for the preparation of recombinant antibody molecules is
described in the above references and also in, for example, EP
0623679; EP 0368684 and EP 0436597, which are incorporated herein
by reference.
[0249] The cell culture supernatants are screened for the desired
antibodies, for example by immunoblotting, by an enzyme
immunoassay, e.g. a sandwich assay or a dot-assay, or a
radioimmunoassay.
[0250] For isolation of the antibodies, the immunoglobulins in the
culture supernatants or in the ascitic fluid may be concentrated,
e.g. by precipitation with ammonium sulphate, dialysis against
hygroscopic material such as polyethylene glycol, filtration
through selective membranes, or the like. If necessary and/or
desired, the antibodies are purified by the customary
chromatography methods, for example gel filtration, ion-exchange
chromatography, chromatography over DEAE-cellulose and/or (immuno-)
affinity chromatography, e.g. affinity chromatography with cgFAIM,
cgFADD, cgPDCD6 and/or cgRequiem, or fragments thereof, or with
Protein-A.
[0251] Hybridoma cells secreting the monoclonal antibodies are also
provided. Preferred hybridoma cells are genetically stable, secrete
monoclonal antibodies of the desired specificity and can be
activated from deep-frozen cultures by thawing and recloning.
[0252] Also included is a process for the preparation of a
hybridoma cell line secreting monoclonal antibodies directed to
cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem, characterised in that a
suitable mammal, for example a Balb/c mouse, is immunised with a
one or more cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem polypeptides,
or antigenic fragments thereof; antibody-producing cells of the
immunised mammal are fused with cells of a suitable myeloma cell
line, the hybrid cells obtained in the fusion are cloned, and cell
clones secreting the desired antibodies are selected. For example
spleen cells of Balb/c mice immunised with cgFAIM, cgFADD, cgPDCD6
and/or cgRequiem are fused with cells of the myeloma cell line PAI
or the myeloma cell line Sp2/0-Ag14, the obtained hybrid cells are
screened for secretion of the desired antibodies, and positive
hybridoma cells are cloned.
[0253] Preferred is a process for the preparation of a hybridoma
cell line, characterised in that Balb/c mice are immunised by
injecting subcutaneously and/or intraperitoneally between 10 and
10.sup.7 and 10.sup.8 cells expressing cgFAIM, cgFADD, cgPDCD6
and/or cgRequiem and a suitable adjuvant several times, e.g. four
to six times, over several months, e.g. between two and four
months, and spleen cells from the immunised mice are taken two to
four days after the last injection and fused with cells of the
myeloma cell line PAI in the presence of a fusion promoter,
preferably polyethylene glycol. Preferably the myeloma cells are
fused with a three- to twentyfold excess of spleen cells from the
immunised mice in a solution containing about 30% to about 50%
polyethylene glycol of a molecular weight around 4000. After the
fusion the cells are expanded in suitable culture media as
described hereinbefore, supplemented with a selection medium, for
example HAT medium, at regular intervals in order to prevent normal
myeloma cells from overgrowing the desired hybridoma cells.
[0254] Recombinant DNAs comprising an insert coding for a heavy
chain variable domain and/or for a light chain variable domain of
antibodies directed to cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem as
described hereinbefore are also disclosed. By definition such DNAs
comprise coding single stranded DNAs, double stranded DNAs
consisting of said coding DNAs and of complementary DNAs thereto,
or these complementary (single stranded) DNAs themselves.
[0255] Furthermore, DNA encoding a heavy chain variable domain
and/or for a light chain variable domain of antibodies directed to
cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem can be enzymatically or
chemically synthesised DNA having the authentic DNA sequence coding
for a heavy chain variable domain and/or for the light chain
variable domain, or a mutant thereof. A mutant of the authentic DNA
is a DNA encoding a heavy chain variable domain and/or a light
chain variable domain of the above-mentioned antibodies in which
one or more amino acids are deleted or exchanged with one or more
other amino acids. Preferably said modification(s) are outside the
CDRs of the heavy chain variable domain and/or of the light chain
variable domain of the antibody. Such a mutant DNA is also intended
to be a silent mutant wherein one or more nucleotides are replaced
by other nucleotides with the new codons coding for the same amino
acid(s). Such a mutant sequence is also a degenerated sequence.
Degenerated sequences are degenerated within the meaning of the
genetic code in that an unlimited number of nucleotides are
replaced by other nucleotides without resulting in a change of the
amino acid sequence originally encoded. Such degenerated sequences
may be useful due to their different restriction sites and/or
frequency of particular codons which are preferred by the specific
host, particularly E. coli, to obtain an optimal expression of the
heavy chain murine variable domain and/or a light chain murine
variable domain.
[0256] The term mutant is intended to include a DNA mutant obtained
by in vitro mutagenesis of the authentic DNA according to methods
known in the art.
[0257] For the assembly of complete tetrameric immunoglobulin
molecules and the expression of chimeric antibodies, the
recombinant DNA inserts coding for heavy and light chain variable
domains are fused with the corresponding DNAs coding for heavy and
light chain constant domains, then transferred into appropriate
host cells, for example after incorporation into hybrid
vectors.
[0258] Also disclosed are recombinant DNAs comprising an insert
coding for a heavy chain murine variable domain of an antibody
directed to cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem fused to a
human constant domain g, for example .gamma.1, .gamma.2, .gamma.3
or .gamma.4, preferably .gamma.1 or .gamma.4. Likewise recombinant
DNAs comprising an insert coding for a light chain murine variable
domain of an antibody directed to cgFAIM, cgFADD, cgPDCD6 and/or
cgRequiem fused to a human constant domain .kappa. or .lamda.,
preferably .kappa. are also disclosed.
[0259] In another embodiment, we disclose recombinant DNAs coding
for a recombinant polypeptide wherein the heavy chain variable
domain and the light chain variable domain are linked by way of a
spacer group, optionally comprising a signal sequence facilitating
the processing of the antibody in the host cell and/or a DNA coding
for a peptide facilitating the purification of the antibody and/or
a cleavage site and/or a peptide spacer and/or an effector
molecule.
[0260] The DNA coding for an effector molecule is intended to be a
DNA coding for the effector molecules useful in diagnostic or
therapeutic applications. Thus, effector molecules which are toxins
or enzymes, especially enzymes capable of catalysing the activation
of prodrugs, are particularly indicated. The DNA encoding such an
effector molecule has the sequence of a naturally occurring enzyme
or toxin encoding DNA, or a mutant thereof, and can be prepared by
methods well known in the art.
Formulation and Administration
[0261] Peptides and polypeptides, such as the cgFAIM, cgFADD,
cgPDCD6 and/or cgRequiem peptides and polypeptides, nucleic acids
and polynucleotides and agonists and antagonist peptides or small
molecules, may be formulated in combination with a suitable
pharmaceutical carrier. Such formulations comprise a
therapeutically effective amount of the polypeptide or compound,
and a pharmaceutically acceptable carrier or excipient. Such
carriers include but are not limited to, saline, buffered saline,
dextrose, water, glycerol, ethanol, and combinations thereof.
Formulation should suit the mode of administration, and is well
within the skill of the art. We further describe pharmaceutical
packs and kits comprising one or more containers filled with one or
more of the ingredients of the aforementioned compositions.
[0262] Polypeptides and other compounds may be employed alone or in
conjunction with other compounds, such as therapeutic
compounds.
[0263] Preferred forms of systemic administration of the
pharmaceutical compositions include injection, typically by
intravenous injection. Other injection routes, such as
subcutaneous, intramuscular, or intraperitoneal, can be used.
Alternative means for systemic administration include transmucosal
and transdermal administration using penetrants such as bile salts
or fusidic acids or other detergents. In addition, if properly
formulated in enteric or encapsulated formulations, oral
administration may also be possible. Administration of these
compounds may also be topical and/or localize, in the form of
salves, pastes, gels and the like.
[0264] The dosage range required depends on the choice of peptide,
the route of administration, the nature of the formulation, the
nature of the subject's condition, and the judgment of the
attending practitioner. Suitable dosages, however, are in the range
of 0.1-100 .mu.g/kg of subject. Wide variations in the needed
dosage, however, are to be expected in view of the variety of
compounds available and the differing efficiencies of various
routes of administration. For example, oral administration would be
expected to require higher dosages than administration by
intravenous injection. Variations in these dosage levels can be
adjusted using standard empirical routines for optimization, as is
well understood in the art.
[0265] Polypeptides used in treatment can also be generated
endogenously in the subject, in treatment modalities often referred
to as "gene therapy" as described above. Thus, for example, cells
from a subject may be engineered with a polynucleotide, such as a
DNA or RNA, to encode a polypeptide ex vivo, and for example, by
the use of a retroviral plasmid vector. The cells are then
introduced into the subject.
Pharmaceutical Compositions
[0266] We also provide a pharmaceutical composition comprising
administering a therapeutically effective amount of the
polypeptide, polynucleotide, peptide, vector or antibody (such as a
cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem polypeptide, etc) and
optionally a pharmaceutically acceptable carrier, diluent or
excipients (including combinations thereof).
[0267] The pharmaceutical compositions may be for human or animal
usage in human and veterinary medicine and will typically comprise
any one or more of a pharmaceutically acceptable diluent, carrier,
or excipient. Acceptable carriers or diluents for therapeutic use
are well known in the pharmaceutical art, and are described, for
example, in Remington's Pharmaceutical Sciences, Mack Publishing
Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical
carrier, excipient or diluent can be selected with regard to the
intended route of administration and standard pharmaceutical
practice. The pharmaceutical compositions may comprise as--or in
addition to--the carrier, excipient or diluent any suitable
binder(s), lubricant(s), suspending agent(s), coating agent(s),
solubilising agent(s).
[0268] Preservatives, stabilizers, dyes and even flavoring agents
may be provided in the pharmaceutical composition. Examples of
preservatives include sodium benzoate, sorbic acid and esters of
p-hydroxybenzoic acid. Antioxidants and suspending agents may be
also used.
[0269] There may be different composition/formulation requirements
dependent on the different delivery systems. By way of example, the
pharmaceutical composition as described here may be formulated to
be delivered using a mini-pump or by a mucosal route, for example,
as a nasal spray or aerosol for inhalation or ingestable solution,
or parenterally in which the composition is formulated by an
injectable form, for delivery, by, for example, an intravenous,
intramuscular or subcutaneous route. Alternatively, the formulation
may be designed to be delivered by both routes.
[0270] Where the agent is to be delivered mucosally through the
gastrointestinal mucosa, it should be able to remain stable during
transit though the gastrointestinal tract; for example, it should
be resistant to proteolytic degradation, stable at acid pH and
resistant to the detergent effects of bile.
[0271] Where appropriate, the pharmaceutical compositions can be
administered by inhalation, in the form of a suppository or
pessary, topically in the form of a lotion, solution, cream,
ointment or dusting powder, by use of a skin patch, orally in the
form of tablets containing excipients such as starch or lactose, or
in capsules or ovules either alone or in admixture with excipients,
or in the form of elixirs, solutions or suspensions containing
flavouring or colouring agents, or they can be injected
parenterally, for example intravenously, intramuscularly or
subcutaneously. For parenteral administration, the compositions may
be best used in the form of a sterile aqueous solution which may
contain other substances, for example enough salts or
monosaccharides to make the solution isotonic with blood. For
buccal or sublingual administration the compositions may be
administered in the form of tablets or lozenges which can be
formulated in a conventional manner.
Vaccines
[0272] Another embodiment relates to a method for inducing an
immunological response in a mammal which comprises inoculating the
mammal with the cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem
polypeptide, or a fragment thereof, adequate to produce antibody
and/or T cell immune response to protect said animal from cgFAIM,
cgFADD, cgPDCD6 and/or cgRequiem associated disease.
[0273] Yet another embodiment relates to a method of inducing
immunological response in a mammal which comprises delivering a
cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem polypeptide via a vector
directing expression of a cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem
polynucleotide in vivo in order to induce such an immunological
response to produce antibody to protect said animal from
diseases.
[0274] A further embodiment relates to an immunological/vaccine
formulation (composition) which, when introduced into a mammalian
host, induces an immunological response in that mammal to a cgFAIM,
cgFADD, cgPDCD6 and/or cgRequiem polypeptide wherein the
composition comprises a cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem
polypeptide or cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem gene. The
vaccine formulation may further comprise a suitable carrier.
[0275] Since the cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem
polypeptide may be broken down in the stomach, it is preferably
administered parenterally (including subcutaneous, intramuscular,
intravenous, intradermal etc. injection). Formulations suitable for
parenteral administration include aqueous and non-aqueous sterile
injection solutions which may contain anti-oxidants, buffers,
bacteriostats and solutes which render the formulation instonic
with the blood of the recipient; and aqueous and non-aqueous
sterile suspensions which may include suspending agents or
thickening agents. The formulations may be presented in unit-dose
or multi-dose containers, for example, sealed ampoules and vials
and may be stored in a freeze-dried condition requiring only the
addition of the sterile liquid carrier immediately prior to use.
The vaccine formulation may also include adjuvant systems for
enhancing the immunogenicity of the formulation, such as oil-in
water systems and other systems known in the art. The dosage will
depend on the specific activity of the vaccine and can be readily
determined by routine experimentation.
[0276] Vaccines may be prepared from one or more polypeptides or
peptides as described here.
[0277] The preparation of vaccines which contain an immunogenic
polypeptide(s) or peptide(s) as active ingredient(s), is known to
one skilled in the art. Typically, such vaccines are prepared as
injectables, either as liquid solutions or suspensions; solid forms
suitable for solution in, or suspension in, liquid prior to
injection may also be prepared. The preparation may also be
emulsified, or the protein encapsulated in liposomes. The active
immunogenic ingredients are often mixed with excipients which are
pharmaceutically acceptable and compatible with the active
ingredient. Suitable excipients are, for example, water, saline,
dextrose, glycerol, ethanol, or the like and combinations
thereof.
[0278] In addition, if desired, the vaccine may contain minor
amounts of auxiliary substances such as wetting or emulsifying
agents, pH buffering agents, and/or adjuvants which enhance the
effectiveness of the vaccine. Examples of adjuvants which may be
effective include but are not limited to: aluminum hydroxide,
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred
to as nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-s-
n-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred
to as MTP-PE), and RIBI, which contains three components extracted
from bacteria, monophosphoryl lipid A, trehalose dimycolate and
cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80
emulsion.
[0279] Further examples of adjuvants and other agents include
aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate
(alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil
emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial
endotoxin, lipid X, Corynebacterium parvum (Propionobacterium
acnes), Bordetella pertussis, polyribonucleotides, sodium alginate,
lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole,
DEAE-dextran, blocked copolymers or other synthetic adjuvants. Such
adjuvants are available commercially from various sources, for
example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.)
or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco
Laboratories, Detroit, Mich.).
[0280] Typically, adjuvants such as Amphigen (oil-in-water),
Alhydrogel (aluminum hydroxide), or a mixture of Amphigen and
Alhydrogel are used. Only aluminum hydroxide is approved for human
use.
[0281] The proportion of immunogen and adjuvant can be varied over
a broad range so long as both are present in effective amounts. For
example, aluminum hydroxide can be present in an amount of about
0.5% of the vaccine mixture (Al.sub.2O.sub.3 basis). Conveniently,
the vaccines are formulated to contain a final concentration of
immunogen in the range of from 0.2 to 200 .mu.g/ml, preferably 5 to
50 .mu.g/ml, most preferably 15 .mu.g/ml.
[0282] After formulation, the vaccine may be incorporated into a
sterile container which is then sealed and stored at a low
temperature, for example 4.degree. C., or it may be freeze-dried.
Lyophilisation permits long-term storage in a stabilised form.
[0283] The vaccines are conventionally administered parenterally,
by injection, for example, either subcutaneously or
intramuscularly. Additional formulations which are suitable for
other modes of administration include suppositories and, in some
cases, oral formulations. For suppositories, traditional binders
and carriers may include, for example, polyalkylene glycols or
triglycerides; such suppositories may be formed from mixtures
containing the active ingredient in the range of 0.5% to 10%,
preferably 1% to 2%. Oral formulations include such normally
employed excipients as, for example, pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, and the like. These compositions
take the form of solutions, suspensions, tablets, pills, capsules,
sustained release formulations or powders and contain 10% to 95% of
active ingredient, preferably 25% to 70%. Where the vaccine
composition is lyophilised, the lyophilised material may be
reconstituted prior to administration, e.g. as a suspension.
Reconstitution is preferably effected in buffer
[0284] Capsules, tablets and pills for oral administration to a
patient may be provided with an enteric coating comprising, for
example, Eudragit "S", Eudragit "L", cellulose acetate, cellulose
acetate phthalate or hydroxypropylmethyl cellulose.
[0285] The polypeptides described here may be formulated into the
vaccine as neutral or salt forms. Pharmaceutically acceptable salts
include the acid addition salts (formed with free amino groups of
the peptide) and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids
such as acetic, oxalic, tartaric and maleic. Salts formed with the
free carboxyl groups may also be derived from inorganic bases such
as, for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, 2-ethylamino ethanol, histidine and procaine.
Administration
[0286] Typically, a physician will determine the actual dosage
which will be most suitable for an individual subject and it will
vary with the age, weight and response of the particular patient.
The dosages below are exemplary of the average case. There can, of
course, be individual instances where higher or lower dosage ranges
are merited.
[0287] The pharmaceutical and vaccine compositions as disclosed
here may be administered by direct injection. The composition may
be formulated for parenteral, mucosal, intramuscular, intravenous,
subcutaneous, intraocular or transdermal administration. Typically,
each protein may be administered at a dose of from 0.01 to 30 mg/kg
body weight, preferably from 0.1 to 10 mg/kg, more preferably from
0.1 to 1 mg/kg body weight.
[0288] The term "administered" includes delivery by viral or
non-viral techniques. Viral delivery mechanisms include but are not
limited to adenoviral vectors, adeno-associated viral (AAV)
vectors, herpes viral vectors, retroviral vectors, lentiviral
vectors, and baculoviral vectors. Non-viral delivery mechanisms
include lipid mediated transfection, liposomes, immunoliposomes,
lipofectin, cationic facial amphiphiles (CFAs) and combinations
thereof. The routes for such delivery mechanisms include but are
not limited to mucosal, nasal, oral, parenteral, gastrointestinal,
topical, or sublingual routes.
[0289] The term "administered" includes but is not limited to
delivery by a mucosal route, for example, as a nasal spray or
aerosol for inhalation or as an ingestable solution; a parenteral
route where delivery is by an injectable form, such as, for
example, an intravenous, intramuscular or subcutaneous route.
[0290] The term "co-administered" means that the site and time of
administration of each of for example, the polypeptide and an
additional entity such as adjuvant are such that the necessary
modulation of the immune system is achieved. Thus, whilst the
polypeptide and the adjuvant may be administered at the same moment
in time and at the same site, there may be advantages in
administering the polypeptide at a different time and to a
different site from the adjuvant. The polypeptide and adjuvant may
even be delivered in the same delivery vehicle--and the polypeptide
and the antigen may be coupled and/or uncoupled and/or genetically
coupled and/or uncoupled.
[0291] The cgFAIM, cgFADD, cgPDCD6 and/or cgRequiem polypeptide,
polynucleotide, peptide, nucleotide, antibody etc and optionally an
adjuvant may be administered separately or co-administered to the
host subject as a single dose or in multiple doses.
[0292] The vaccine composition and pharmaceutical compositions
described here may be administered by a number of different routes
such as injection (which includes parenteral, subcutaneous and
intramuscular injection) intranasal, mucosal, oral, intra-vaginal,
urethral or ocular administration.
[0293] The vaccines and pharmaceutical compositions described here
may be conventionally administered parenterally, by injection, for
example, either subcutaneously or intramuscularly. Additional
formulations which are suitable for other modes of administration
include suppositories and, in some cases, oral formulations. For
suppositories, traditional binders and carriers may include, for
example, polyalkylene glycols or triglycerides; such suppositories
may be formed from mixtures containing the active ingredient in the
range of 0.5% to 10%, may be 1% to 2%. Oral formulations include
such normally employed excipients as, for example, pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate, and the like. These
compositions take the form of solutions, suspensions, tablets,
pills, capsules, sustained release formulations or powders and
contain 10% to 95% of active ingredient, preferably 25% to 70%.
Where the vaccine composition is lyophilised, the lyophilised
material may be reconstituted prior to administration, e.g. as a
suspension. Reconstitution is preferably effected in buffer.
EXAMPLES
Example 1
Cell Lines & Cell Culture
[0294] CHO IFN-.gamma. is a Chinese Hamster Ovary cell line that
had been adapted to grow in suspension. It was originally derived
from dehydroxyfolate reductase negative (DHFR.sup.-), Dukx cells
(Urlaub & Chasin 1980). CHO IFN-.gamma. had been cotransfected
with genes for DHFR and human interferon-.gamma. (Scahill et al.
1983).
[0295] CHO IFN-.gamma. is maintained in glucose/glutamine-free HyQ
CHO MPS media (Hyclone, Logan, Utah) supplemented with 4 mM
glutamine, 20 mM glucose and 0.25 .mu.M methotrexate (Sigma, St.
Louis, Mo.).
Example 2
Total RNA Extraction & First Strand cDNA Synthesis
[0296] Total RNA is extracted from CHO K1 cells using TRIZOL.TM.
(Invitrogen). All reverse transcription reagents are from Promega.
Full length cDNA is synthesized using Moloney Murine Leukaemia
Virus reverse transcriptase for 1 hr at 42.degree. C. in a reaction
mix containing 1.times. reverse transcription buffer, 10 mM of each
dNTPs and 25 units of recombinant RNAsin.RTM. ribonuclease
inhibitor.
Example 3
Gene Specific PCR
[0297] The cDNA prepared from CHO K1 total RNA is used as a
template for gene specific PCR. All PCR reagents are from
Promega.
Example 4
Gene Specific Cloning of Cricetulus griseus FAIM
[0298] The coding region of FAIM is amplified using a 5' PCR
primer, 5'-GCCGCGAGAGCTGCTGACTACGTCGTGG-3' (SEQ ID NO: 17) and a 3'
PCR primer 5'-GTTACTGTG-GTGAGATATGAATGGGTTTGG-3' (SEQ ID NO: 18).
The PCR reaction mix contains 1 .mu.L of cDNA template, 1.times.
Reaction buffer, 200 .mu.M of each dNTP, 2.0 mM MgCl.sub.2, 1 .mu.M
of each primer and Taq DNA polymerase mix (Total 5 U). PCR
conditions are: 94.degree. C. for 5 min, followed by 31 cycles of
94.degree. C. for 1 min, 58.degree. C. for 1 min and 72.degree. C.
for 2 min and a final extension at 72.degree. C. for 10 min. The
PCR product is then subcloned into pCR.RTM.-TOPO.RTM. (Invitrogen,
Grand Island, N.Y.) for sequencing.
[0299] Cricetulus griseus FAIM (cgFAIM)
[0300] The sequence of cgFAIM is set out in SEQ ID NO: 1 and SEQ ID
NO: 5. The Cricetulus griseus sequence encodes for a 179-amino acid
protein.
[0301] Fas which, is also known as CD95 or APO-1, is a receptor
from the tumor necrosis factor (TNF) receptor family that plays a
major role in receptor-mediated apoptosis pathway. The
extracellular region of TNF receptor family have 2-6 repeats of
cysteine-rich subdomain. Fas activation initiates intracellular
signaling cascade through the oligomerization of caspase 8. Caspase
8 has been shown to be one of the initiator caspases responsible
for the cascade-like activation of effector caspases (Srinivasula
et al. 1996). Fas has also been shown to cause mitochondrial
cytochrome c release that results in the activation of caspase 9
and other effector caspases (Li et al. 1997).
[0302] Fas apoptosis inhibitory molecule (FAIM) had been found to
be an inducible protein that can confer resistance to Fas induced
apoptosis (Schneider et al. 1999; Rothstein et al. 2000). It has
been shown that together with sIg signals, FAIM expression in B
cells is able to block Fas killing and does so by blocking a step
in the Fas signaling pathway before the activation of caspase 3
(Schneider et al. 1999). FAIM has been shown to exist in two
alternatively spliced forms with FAIM-S broadly expressed while
FAIM-L is brain tissue-specific (Zhong et al, 2001). FAIM sequence
also seemed to be highly conserved in different species suggesting
an important phylogeny role.
Example 5
Gene Specific Cloning of Cricetulus griseus FADD
[0303] The partial coding region of FADD is amplified using a 5'
PCR primer 5'-CCATGGACCCATTCCTGGTGC-3' (SEQ ID NO: 19) and a 3' PCR
primer 5'-TTCTTCCACCAGGTCAGC-CACC-3' (SEQ ID NO: 20). The PCR
reaction mix contains 1 .mu.L of cDNA template, 1.times. Reaction
buffer, 200 .mu.M of each dNTP, 1.5 mM MgCl.sub.2, 1 .mu.M of each
primer and Taq DNA polymerase mix (Total 5 U).
[0304] PCR conditions are: 94.degree. C. for 5 min, followed by 31
cycles of 94.degree. C. for 1 min, 55.degree. C. for 1 min and
72.degree. C. for 2 min and a final extension at 72.degree. C. for
10 min. The PCR product is then subcloned into pCR.RTM.-TOPO.RTM.
(Invitrogen, Grand Island, N.Y.) for sequencing.
[0305] Cricetulus griseus FADD (cgFADD)
[0306] The sequence of cgFADD is set out in SEQ ID NO: 2 and SEQ ID
NO: 6.
[0307] FADD is common mediator of both CD95 (Fas/APO-1) and tumor
necrosis factor (TNF) receptor-induced apoptosis (Chinnaiyan et al
1996). FADD which contains both death and death effector domains is
an important mediator of caspase 8 activation upon Fas engagement
(Chinnaiyan et al. 1995). We describe the modulation of expression
of cgFADD by use of an artificially engineered FADD molecule, FADD
DN that only contained only the apoptosis receptor binding death
domain and not the death effector domain of FADD. It is the
effector domain which causes the activation of further downstream
apoptosis cascade by recruiting caspase to the death-inducing
signaling complex. The methods described here enable
over-expression of an engineered form of FADD, i.e., a dominant
negative, and use of such an engineered molecule to prevent caspase
recruitment by competing with native FADD and thus breaking the
apoptosis cascade.
Example 6
Gene Specific Cloning of Cricetulus griseus PDCD 6
[0308] The partial coding region of PDCD6 is amplified using a
5'-PCR primer, 5'-GCCCATGGCTGCCTACTCCTA-3' (SEQ ID NO: 21) and a
3'-PCR primer, 5'-AATCCAGCCATCCTGAT-CCGT-3' (SEQ ID NO: 22). The
PCR reaction mix contains 1 .mu.L of cDNA template, 1.times.
Reaction buffer, 200 .mu.M of each dNTP, 1.5 mM MgCl.sub.2, 1 .mu.M
of each primer and Taq DNA polymerase mix (Total 5 U).
[0309] PCR conditions are: 94.degree. C. for 5 min, followed by 31
cycles of 94.degree. C. for 1 min, 52.degree. C. for 1 min and
72.degree. C. for 2 min and a final extension at 72.degree. C. for
10 min. The PCR product is then subcloned into pCR.RTM.-TOPO.RTM.
(Invitrogen, Grand Island, N.Y.) for sequencing. The PCR product
obtained is purified and cloned into pCR 2.1-Topo vector and
sequenced. The 3'-RACE primer is
5'-CAGCGGGTTGATAAAGACAGGAGTGGAGTG-3' (SEQ ID NO: 23).
[0310] The 3'-RACE PCR products are separated by electrophoresis in
1% agarose gel containing ethidium bromide, with the relevant band
excised and gel extracted using the Qiagen kit before being cloned
into pCR.RTM.-TOPO.RTM. (Invitrogen, Grand Island, N.Y.) for
sequencing.
[0311] Cricetulus griseus PDCD6 (cg PDCD6)
[0312] The sequence of cgPDCD6 is set out in SEQ ID NO: 3 and SEQ
ID NO: 7. The Cricetulus griseus sequence encodes a 191-amino acid
protein.
[0313] Also known as apoptosis-linked gene 2 (ALG-2), PDCD6 encodes
for a calcium-binding protein that belonged to the penta-EF-hand
protein family. There are indications that it participates in
receptor-, Fas- and glucocorticoid-induced apoptosis (Vito et al.
1996; Krebs & Klemenz, 2000 and Jung et al., 2001).
Interestingly, Jang et al. 2002 demonstrated that ALG-2 deficiency
resulted in no block of apoptosis induced by TCR, FAS or
dexamethasone signals.
Example 7
Gene Specific Cloning of Cricetulus griseus Requiem
[0314] The partial coding region of Requiem is amplified using a
5'-PCR primer, 5'-ATG-GCGGCTGTGGTGGAGAAT-3' (SEQ ID NO: 24) and a
3'-PCR primer, 5'-GGAGTTCTGGTTCTGGTAG-ATGG-3' (SEQ ID NO: 25). The
PCR reaction mix contained 1 .mu.L of cDNA template, 1.times.
Reaction buffer, 200 .mu.M of each dNTP, 2.0 mM MgCl.sub.2, 1 .mu.M
of each primer and Taq DNA polymerase mix (Total 5 U).
[0315] PCR conditions are: 94.degree. C. for 5 min, followed by 60
cycles of 94.degree. C. for 1 min, 44.degree. C. for 1 min and
72.degree. C. for 2 min and a final extension at 72.degree. C. for
10 min. The PCR product is then subcloned into pCR.RTM.-TOPO.RTM.
(Invitrogen, Grand Island, N.Y.) for sequencing. The PCR product
obtained is purified and cloned into pCR 2.1-Topo vector and
sequenced. The 3'-RACE primer is
5'-GCCTCAGTTACCACTATGCCCATTCCCACC-3' (SEQ ID NO: 26).
[0316] The 3'-RACE PCR products are separated by electrophoresis in
1% agarose gel containing ethidium bromide, with the relevant band
excised and gel extracted using the Qiagen kit before being cloned
into pCR.RTM.-TOPO.RTM. (Invitrogen, Grand Island, N.Y.) for
sequencing.
[0317] Cricetulus griseus Requiem (cgRequiem)
[0318] The sequence of cgRequiem is set out in SEQ ID NO: 4 and SEQ
ID NO: 8. The Cricetulus griseus sequence encodes for a 391-amino
acid protein.
[0319] Requiem, which is also known as ubi-d4, is a zinc finger
gene essential for the activation of caspases in myeloid cells
(Gabig et al. 1994). It was suggested that Requiem is likely to
encode a transcription factor required for apoptosis response
following survival factor withdrawal (Gabig et al. 1994). In
addition, Gabig et al. (1998) later detected the protein both in
cytoplasmic and nuclear subcellular fractions of murine myeloid
cells and human K562 leukemia cells thereby suggesting that the
protein may have a function distinct from that of a transcription
factor.
Example 8
Cricetulus griseus FAIM Expression Vector Construction
[0320] In summary, the verified PCR product from FAIM gene specific
PCR that is cloned into pCR.RTM.-TOPO.RTM. (Invitrogen, Grand
Island, N.Y.), is subcloned into pcDNA3.1(+) (Invitrogen) and
sequenced again. The final plasmid pcDNA3.1(+) FAIM is then
purified using Maxi Plasmid Purification Kit (Qiagen, Hilden,
Germany) and its concentration quantified for transfection into CHO
IFN-.gamma..
[0321] In detail, cgFAIM with artificial kozak sequence and linker
regions is created by using the 5'-PCR primer,
5'-GAATTCGCCACCATGACAGATCTTGTAGC-3'(SEQ ID NO: 43) and the 3'-PCR
primer, 5'-GAATTCGTGAACACATTTAATTACCA-3' (SEQ ID NO: 44). The
underlined sequence consists of a EcoRI restriction site while the
italicized sequence consists of an artificial kozak sequence to
facilitate `in-frame` expression of FAIM. The incorporated regions
of cgFAIM are in bold.
[0322] The PCR reaction mix contained 1 .mu.L of pCR2.1-TOPO cgFAIM
template, 1.times.Reaction buffer, 200 .mu.M of each dNTP, 2.0 mM
MgCl.sub.2, 1 .mu.M of each primer and Taq DNA polymerase mix
(Total 5 U). PCR conditions are: 94.degree. C. for 5 min, followed
by 60 cycles of 94.degree. C. for 1 min, 44.degree. C. for 1 min
and 72.degree. C. for 2 min and a final extension at 72.degree. C.
for 10 min.
[0323] The verified PCR product is then digested with EcoRI
restriction enzyme at 37.degree. C. for approximately 4 hours to
create cgFAIM inserts with sticky ends for further ligation. Blank
pcDNA3.1(+) vector (Invitrogen) is also digested with EcoRI
restriction enzyme at 37.degree. C. for approximately 4 hours.
EcoRI digested cgFAIM insert is then ligated into EcoRI digested
pcDNA3.1(+) by adding 124 of insert to 34 of vector, 24 of
DNase-free water, 24 of 10.times.T4 DNA ligase buffer (Invitrogen)
and 1 .mu.L of T4 DNA ligase (3 U/.mu.L) (Invitrogen). This
ligation mixture is then incubated for approximately 16 hours at
room temperature.
[0324] 10 .mu.L of the ligation mixture is then transformed into
competent DH5.alpha. bacterial cells for plasmid propagation.
Positive transformants are selected for by culturing in LB agar
plates with ampicillin for selection. Plasmid extraction is then
carried out on various DH5.alpha. clones for sequencing to verify
cgFAIM sequence inserted into pcDNA3.1(+) expression vector. The
plasmid pcDNA3.1(+) cgFAIM from a verified clone is then purified
using Maxi Plasmid Purification Kit (Qiagen, Hilden, Germany) and
its concentration quantified for transfection into CHO
IFN-.gamma..
Example 9
Cricetulus griseus FADD Dominant Negative Expression Vector
Construction
[0325] An artificial FADD dominant negative (FADD DN) fragment with
kozak sequence is created by using the 5'-PCR primer,
5'-GATATCGGATCCGCCACC-ATGGCCTTTGACATTGTATGCGACAATGTGGGG-3' (SEQ ID
NO: 11) and the 3'-PCR primer,
5'-CCCGGG-CTCGAGTGCCTCCC-TTCCACCAGGTCAG-3' (SEQ ID NO: 12). The
underlined sequence consists of a BamHI and XhoI restriction site
respectively while the italicized sequence consists of an
artificial kozak and start codon to facilitate `in frame`
expression of cgFADD Dominant Negative. The incorporated coding
regions of cgFADD are in bold.
[0326] The PCR reaction mix contains 1 .mu.L of cDNA template,
1.times. Reaction buffer, 200 .mu.M of each dNTP, 1.5 mM
MgCl.sub.2, 1 .mu.M of each primer and Taq DNA polymerase mix
(Total 5 U). The partial FADD sequence subcloned in
pCR.RTM.-TOPO.RTM. is used as the template. PCR conditions are:
94.degree. C. for 5 min, followed by 31 cycles of 94.degree. C. for
1 min, 50.degree. C. for 1 min and 72.degree. C. for 2 min and a
final extension at 72.degree. C. for 10 min.
[0327] The verified PCR product is then digested with BamHI and
XhoI restriction enzymes at 37.degree. C. for approximately 4 hours
to create cgFADD DN inserts with sticky ends for further ligation.
Blank pcDNA3.1(+) vector (Invitrogen) is also digested with BamHI
and XhoI restriction enzyme at 37.degree. C. for approximately 4
hours. BamHI/XhoI digested cgFADD DN insert is then ligated into
BamHI/XhoI digested pcDNA3.1(+) by adding 12 .mu.L of insert to 3
.mu.L of vector, 2 .mu.L of Dnase-free water, 2 .mu.L of
10.times.T4 DNA ligase buffer (Invitrogen) and 1 .mu.L of T4 DNA
ligase (3 U/.mu.L) (Invitrogen). This ligation mixture is then
incubated for approximately 16 hours at room temperature.
[0328] 10 .mu.L of the ligation mixture is then transformed into
competent DH5.alpha. bacterial cells for plasmid propagation.
Positive transformants are selected for by culturing in LB agar
plates with ampicillin for selection. Plasmid extraction is then
carried out on various DH5.alpha. clones for sequencing to verify
cgFADD DN sequence inserted into pcDNA3.1(+) expression vector. The
plasmid pcDNA3.1(+) FADD Dominant Negative from a verified clone is
then purified using Maxi Plasmid Purification Kit (Qiagen, Hilden,
Germany) and its concentration quantified for transfection into CHO
IFN-.gamma..
Example 10
Cricetulus griseus PDCD6 Suppression Vector Construction
[0329] An oligo insert is designed based on the obtained cgPDCD6
sequence. The oligo insert design is compared to a genomic database
using BLAST to eliminate any significant homology to other genes.
The 5' oligo insert,
5'-GATCCCGTGAGCTTCAGCAAGCATTATTCAAGAGATAATGCTTGCTGAAGC-TCATTTTTTGGAAA-3'
(SEQ ID NO: 13) is annealed to the 3' oligo insert,
5'-AGCTTTTCCAAAAAATGAGCTTCAGCAAGCATTATCTCTTGAATAATGCTTGCTG
AAGCTCACG-3' (SEQ ID NO: 14) is then synthesized and then ligated
into HindIII/BglII digested pSUPER.neo vector (OligoEngine,
Seattle, Wash.).
[0330] 4 .mu.L of annealed oligo insert is added to 3 .mu.L of
HindIII/BglII digested pSUPER.neo, 13 .mu.L of Dnase-free water, 2
.mu.L of 10.times.T4 DNA ligase buffer (Invitrogen) and 1 .mu.L of
T4 DNA ligase (3 U/.mu.L) (Invitrogen). This ligation mixture is
then incubated for approximately 16 hours at room temperature.
[0331] 10 .mu.L of the ligation mixture is then transformed into
competent DH5.alpha. bacterial cells for plasmid propagation.
Positive transformants are selected for by culturing in LB agar
plates with ampicillin for selection. Plasmid extraction is then
carried out on various DH5.alpha. clones for sequencing to verify
cgPDCD6 siRNA sequence inserted into pSUPER.neo expression vector.
The plasmid pSUPER.neo cgPDCD6 siRNA from a verified clone is then
purified using Maxi Plasmid Purification Kit (Qiagen, Hilden,
Germany) and its concentration quantified for transfection into CHO
IFN-.gamma..
Example 11
Cricetulus griseus Requiem Suppression Vector Construction
[0332] Oligo insert is designed based on obtained cgRequiem
sequence. The 5' oligo insert,
5'-GATCCCGCGGATCCTTGAACCTGATTTCAAGAGAATCAGGTTCAAGGATCCGC-TTTTTTGGAAA-3'
(SEQ ID NO: 15) is annealed to the 3' oligo insert,
5'-AGCTTTTCCAAAAAAGCGGATCCTTGAACCTGATTCTCTTGAAATCAGGTTCAAG
GATCCGCGG-3' (SEQ ID NO: 16) and then ligated into HindIII and
BglII digested pSUPER.neo vector (OligoEngine, Seattle, Wash.).
[0333] 4 .mu.L of annealed oligo insert is added to 3 .mu.L of
HindIII/BglII digested pSUPER.neo, 13 .mu.L of Dnase-free water, 2
.mu.L of 10.times.T4 DNA ligase buffer (Invitrogen) and 1 .mu.L of
T4 DNA ligase (3 U/.mu.L) (Invitrogen). This ligation mixture is
then incubated for approximately 16 hours at room temperature.
[0334] 10 .mu.L of the ligation mixture is then transformed into
competent DH5.alpha. bacterial cells for plasmid propagation.
Positive transformants are selected for by culturing in LB agar
plates with ampicillin for selection. Plasmid extraction is then
carried out on various DH5.alpha. clones for sequencing to verify
cgRequiem siRNA sequence inserted into pSUPER.neo expression
vector. The plasmid pSUPER.neo cgRequiem siRNA from a verified
clone is then purified using Maxi Plasmid Purification Kit (Qiagen,
Hilden, Germany) and its concentration quantified for transfection
into CHO IFN-.gamma..
Example 12
Transfection & Selection
[0335] Transfection is carried out using Lipofectamine reagent
(Invitrogen). Cells are grown overnight in 6-well plates with 0.5
million cells per well and transfected with approximately 1 .mu.g
of linearized plasmid per well the next day. The Lipofectamine-DNA
complex is prepared according to manufacturer's instructions in a
3:1 Lipofectamine (.mu.L) to DNA (.mu.g) ratio.
[0336] To generate stable cells, the cells are grown for 24 hr
before the media is changed to selection media containing 1000
.mu.g/mL of Geneticin. The cells are maintained in selection media
for 4 weeks where the untransfected cells in the selection media
died within a week.
Example 12A
Stably Integrated Single Cell Clones
[0337] Stably integrated single cell clones are obtained by serial
dilution of cells into 96-well plates such that there would only be
one cell in each well. Wells are checked under light microscope and
those that only contain a single cell are marked. Single clones are
then expanded into 24-well plates followed by 6 well plates before
going into shake flasks culture.
Example 12B
Batch and Fed-Batch Culture
[0338] An initial working volume of 4.0 L of culture media is
inoculated with a seeding density of 2.5.times.10.sup.5 cells/mL in
a 5.0 L bioreactor (B. Braun, Melsungen, Germany). Batch cultures
are carried out using glucose/glutamine-free HyQ CHO MPS media
(Hyclone, Logan, Utah) supplemented with 20 mM glucose and 4 mM
glutamine while fed-batch cultures are supplemented with 4 mM
glucose and 0.5 mM glutamine. Dissolved oxygen concentration is
maintained at 50% air saturation and culture pH is maintained at
7.15 using intermittent CO.sub.2 addition to the gas mix and/or
7.5% (w/v) NaHCO.sub.3 solution (Sigma).
[0339] Fed-batch operation is performed using a modified online
dynamic feeding strategy (Lee et al., 2003). Online monitoring of
concentrations of the relevant controlled nutrient level are
conducted every 1.5 hr using an automated aseptic online sampling
loop. Basal feed media for fed-batch cultures is prepared from a
custom formulated 10.times. calcium-free, glucose-free and
glutamine-free DMEM/F12 with 1.times. salts (Hyclone) supplemented
with 10 g/L of soybean protein hydrolysate, Hysoy (Quest
International, Hoffman Estate, Ill.), 10 mL/L of chemically defined
lipids (Gibco BRL,Grand Island, N.Y.), 1 mg/L of d-biotin (Sigma),
2 mM L-aspartic acid, 2 mM L-asparagine, 4 mM L-cysteine, 1 mM
L-glutamic acid, 1 mM L-methionine and 5 mM L-serine (Sigma).
[0340] The basal feed media is further supplemented with 100 mM of
glutamine (Sigma) and 500 mM of glucose (Sigma). Every 1.5 hr, an
automated on-line measurement of residual glutamine concentrations
would be taken. If residual glutamine concentration falls below
setpoint control concentrations, feed injections would be effected
with feed media to raise culture glutamine concentrations to 0.3
mM.
Example 13
Gene Expression Quantification Using Real-Time PCR
[0341] Approximately 10 million cells are collected from stable
cells and total RNA is extracted using TRIZOL.TM. reagent
(Invitrogen). RNA Samples are then quantified using GENEQUANT.TM.
Pro RNA/DNA Calculator (Amersham Biosciences, Piscataway, N.J.).
RNA quality is assessed using the absorbance ratio of 260 nm to 280
nm, where a ratio of 1.8 and above is considered as RNA sample of
sufficient purity.
[0342] Quantitative real time PCR is used to ascertain the relative
over-expression or suppression of gene of interest after
transfection experiments. In order to generate standard curves of
transcript copy, quantified pCR.RTM.-TOPO.RTM. (Invitrogen)
plasmids containing either FAIM, FADD, PDCD6 or Requiem are
serially diluted and used for quantitative real time PCR.
[0343] Quantitative real time PCR for FAIM transcripts is carried
out using 5'-primer 5'-TGGAGCTGCGAAAACCAAAG-3' (SEQ ID NO: 27) and
3'-primer 5'-AAACTCGCCTGCTGTCTCCAT-3' (SEQ ID NO: 28). Quantitative
real time PCR for FADD Dominant Negative transcripts is carried
using 5'-primer 5'-GATATCGGATCCGCCACCATGG-3' (SEQ ID NO: 29) and
3'-primer 5'-TGCCTCCCTTCCACCAG-GTCAG'3' (SEQ ID NO: 30).
Quantitative real time PCR for PDCD6 transcripts is carried out
using 5'-primer 5'-CAGCGGGTTGATAAAGACAGG-3' (SEQ ID NO: 31) and
3'-primer 5'-GCCAGCCTTG-TTTTCTCGG-3' (SEQ ID NO: 32). Quantitative
real time PCR for Requiem transcripts is carried out using
5'-primers, 5'-TGGAGTAGCCCAGAGCAATTG-3' (SEQ ID NO: 33) and
3'-primer, 5'-TCGACGCTTTTTACGCCAG-3' (SEQ ID NO: 34).
[0344] Each sample is then normalized against .beta.-actin
transcript expression. Quantitative real time PCR for .beta.-actin
transcripts is carried out using 5'-primer
5'-AGCTGAGAGGGAAATTGTGCG-3' (SEQ ID NO: 35) and 3'-primer
5'-GCAACGG-AACCGCTCATT-3' (SEQ ID NO: 36). Finally, normalized
quantitative gene expression in transfected cells is divided by
normalized quantitative gene expression in null vector transfected
cells to give normalized relative gene expression.
Example 14
Apoptosis Assay
[0345] An Ethidium Bromide/Acridine Orange Assay is used to
classify cells in samples collected into apoptotic or non-apoptotic
populations. Stock solutions of 100 .mu.g/mL of Ethidium Bromide
(Sigma) and Acridine orange (Sigma) are prepared in PBS solution
(Sigma).
[0346] Approximately 1.times.106 cells are sampled from samples and
resuspended in 100 .mu.L of 1:1 Ethidium Bromide: Acridine Orange
stock solution and incubated for 5 minutes at room temperature.
Samples are then loaded onto glass slides and approximately 400
cells are examined under fluorescence microscopy.
[0347] Cells with apoptotic morphology of nuclear condensation are
then classified accordingly. In addition to morphological analysis,
caspases 2, 3, 8 and 9 activity is measured using BD ApoAlert.RTM.
Caspase Assay Plates (BD Biosciences Clontech, CA) according to the
manufacturer's protocol. Activation of caspases is considered as
biochemical hallmarks of apoptosis.
Example 15
IFN-.gamma. Quantification
[0348] IFN-.gamma. concentrations of serially diluted supernatant
samples are analyzed using an enzyme-linked immunosorbent (ELISA)
assay (HyCult Biotechnology, Uden, Netherlands). Samples that had
the highest IFN-.gamma. concentrations during high viability
(>95%) and during low viability (70-80%), are sent for
immunoaffinity purification and further N-glycosylation
characterization.
Example 16
Real Time PCR for Gene Expression Detection
[0349] To determine the expression of targeted genes in cells,
quantitative real-time PCR is carried out. Real-time PCR is
considered as a sensitive method for the detection of transcript
levels.
[0350] Gene expression analysis shows that cells transfected with
pcDNA3.1(+) Faim over-express FAIM by 3 times more than cells
transfected with pcDNA3.1(+) blank (FIG. 1). Cells transfected with
pcDNA3.1(+) FADD Dominant Negative, over-express FADD Dominant
negative by up to 4 times (FIG. 1). The data in FIG. 1 also shows
that siRNA can be used effectively to suppress gene expression.
Transfection with pSUPER PDCD6 siRNA results in suppression of
PDCD6 expression by approximately 60% while pSUPER Requiem siRNA is
able to suppress Requiem expression by up to 70% (FIG. 1).
Example 17
Apoptosis Resistance Conferred by Gene Targeting of Cricetulus
griseus FAIM
[0351] Cells are transfected with a FAIM expression vector (Example
8) and a blank vector as control, and resistance to apoptosis
assayed.
[0352] Compared to cells transfected with just the blank vector,
cells over-expressing FAIM are able to maintain high viable cell
density for a longer period of time (FIG. 2A). Cells with FAIM
over-expression also show a significant extension of viability by
at least 24 hours before viability started to drop below 95% (FIG.
2B). The percentage of apoptotic cells is also significantly lower
than that of control cells without FAIM over-expression (FIG. 2D).
Increase in caspase 2 and 3 activity is also delayed by
approximately 24 hours compared to control cells (FIG. 6B). This
showed that FAIM over-expression could be very effective in
suppressing apoptosis in cell culture processes.
Example 18
Apoptosis Resistance Conferred by Gene Targeting of Cricetulus
griseus FADD
[0353] Cells are transfected with a FADD expression vector (Example
9) and a blank vector as control, and resistance to apoptosis
assayed.
[0354] Compared to cells transfected with just the blank vector,
cells over-expressing FADD dominant negative are able to maintain
high viable cell density for a longer period of time (FIG. 3A).
Cells with FADD dominant negative over-expression also show a
significant extension of viability by around 24 hours before
viability started to drop below 95% (FIG. 3B). The percentage of
apoptotic cells is also significantly lower than that of control
cells without FAIM over-expression (FIG. 3D). Increase in caspase
2, 3 and 8 activity is also delayed significantly (FIG. 6C). The
data showed that caspase 2 and 8 activity only increase after 144
hr in culture while caspase 3 activity increase is suppressed
significantly. This shows that the targeting of FADD signaling
effectively suppresses apoptosis in cell culture processes.
Example 19
Apoptosis Resistance Conferred by Gene Targeting of Cricetulus
griseus PDCD6
[0355] Cells are transfected with a PDCD6 expression vector
(Example 10) and a blank vector as control, and resistance to
apoptosis assayed.
[0356] Compared to cells transfected with just the blank siRNA
vector, cells with PDCD6 suppression are able to maintain high
viable cell density for a longer period of time (FIG. 4A). Cells
with PDCD6 suppression also show a significant decrease in the rate
of viability loss (FIG. 4B). The percentage of apoptotic cells is
also significantly lower than that of control cells without PDCD6
suppression (FIG. 4D). Increase in caspase 2, 3, 8 and 9 activity
is also delayed significantly (FIG. 6D). The data showed that
caspase 2, 3 and 8 activity only increase after 144 hr in culture
while caspase 9 activity is always below the reference point. This
show that the targeting of PDCD6 signalling could effectively
suppress apoptosis in cell culture processes.
Example 20
Apoptosis Resistance Conferred by Gene Targeting of Cricetulus
griseus Requiem
[0357] Cells are transfected with a Requiem expression vector
(Example 11) and a blank vector as control, and resistance to
apoptosis assayed.
[0358] Compared to cells transfected with just the blank siRNA
vector, cells with Requiem suppression is able to maintain higher
viable cell density for a longer period of time (FIG. 5A). Cells
with Requiem suppression also show a significant decrease in the
rate of viability loss (FIG. 5B). The percentage of apoptotic cells
is also significantly lower than that of control cells without
Requiem suppression (FIG. 5D). Increase in caspase 2, 3, and 9
activity is also delayed significantly (FIG. 6E). The data showed
that caspases activity are significantly lower than that of the
control. This show that the targeting of Requiem signalling could
effectively suppress apoptosis in cell culture processes.
Example 21
Improvement in Recombinant Protein Yield
[0359] FIG. 7 shows that gene targeting improves cell culture
processes in terms of final product yield.
[0360] Cells with FAIM over-expression allows for interferon gamma
yields of up to 3.3 mg/L compared to the typical 2.3 mg/L yields
seen in control cells (FIG. 7A).
[0361] Targeting of FADD signalling even allows for interferon
gamma yield concentration of up to 5.0 mg/L, representing more than
200% increase (FIG. 7B).
[0362] Suppression of PDCD6 and Requiem allows interferon gamma
yield to improve to 4.2 and 5.8 mg/L respectively.
[0363] The increased robustness of engineered cells to apoptosis
induction coupled with effective increase in final recombinant
protein yield shows that gene targeting of FADD, FAIM, PDCD6 and
Requiem are effective novel strategies for improving
biotherapeutics production in cell culture systems.
Example 22
Enhanced Viable Culture Density in addition to Viability
Enhancement in Fed-Batch Culture
[0364] Experiments are conducted to determine the ability of an
engineered apoptosis resistant cell line to perform in fed-batch
culture under the different batch and fed-batch stress/nutrient
environment.
[0365] FIG. 8A shows the viable cell densities of stably integrated
CHO IFN-.gamma. cell lines with Requiem or PDCD6 suppression. PDCD6
and Requiem suppression allows for significant increase in viable
cell densities during fed-batch culture.
[0366] Viable cell density of as high as 9.times.10.sup.6 cells/mL
can be achieved compared to 5.times.10.sup.6 cells/mL typically
seen in cells without any apoptosis targeting.
[0367] The experiments are repeated with stably integrated CHO
IFN-.gamma. cell lines showing FADD DN or FAIM over-expression and
similar results are observed.
[0368] This demonstrates that apoptosis resistance engineering by
Requiem or PDCD6 suppression or FAIM and FADD DN overexpression not
only allows for extension of culture viability but confers the
ability to grow to much higher viable cell densities due to their
robustness against factor(s) that inhibit cell growth.
Example 23
Enhanced Recombinant Protein Yield in Fed-Batch Culture
[0369] FIG. 9A shows that gene targeting allows for further
significant improvement in interferon gamma production and
yield.
[0370] Cells with stably suppressed Requiem or PDCD6 give
interferon gamma yields of up to 49 and 41 .mu.g/mL respectively
compared to the typical 20 .mu.g/mL seen in normal fed-batch
culture. This represents a greater than 200% improvement in
recombinant protein yield.
[0371] The experiments are repeated with cells overexpressing FAIM
and similar results are seen.
Example 24
Enhanced Sialylation of Recombinant Human IFN-.gamma. in Modified
CHO Cell Lines--Sialic Acid Content Assay
[0372] Modified CHO cell lines which express IFN-.gamma. are
produced from parental CHO cell lines as described above.
[0373] Recombinant IFN-.gamma. is purified from samples collected
at mid-exponential growth phase and at when the highest IFN-.gamma.
concentrations are detected during high viability (>95%) and
during low viability (70-80%).
[0374] The sialic acid content of the IFN-.gamma. is then
determined using a modified thiobarbituric acid assay as described
in Wong et al. (2005a), Biotechnol Bioeng 89: 164-177).
Example 25
Enhanced Sialylation of Recombinant Human IFN-.gamma. in Modified
CHO Cell Lines--Results
[0375] FIG. 10 shows the sialylation of recombinant human
IFN-.gamma. harvested at three time points during fed-batch
culture, namely at the mid-exponential (>95% viability),
stationary (>95% viability) and death phase (70-80% viability)
for the modified CHO IFN-.gamma. cell lines and parental CHO
IFN-.gamma. cell lines.
[0376] For the latter, the sialic acid content of recombinant human
IFN-.gamma. decreased as the culture progressed from
mid-exponential (2.9 mol of SA/mol of IFN-.gamma.) to stationary
(2.3 mol of SA/mol of IFN-.gamma.) to death phase (2.1 mol of
SA/mol of IFN-.gamma.).
[0377] In contrast, the sialic acid content of IFN-.gamma.
harvested at the three time points for the four modified CHO
IFN-.gamma. cell lines are maintained, and even showed increase in
sialylation, ranging from 2.7 to 3.5 mol of SA/mol of
IFN-.gamma..
[0378] These results show that another potential benefit of
apoptosis-resistant CHO cells is the maintenance/enhancement of
protein glycosylation quality over extended culture time,
regardless of loss in culture viability (70-80%). This is a
distinct advantage for cell lines used for manufacturing
biotherapeutics as a lower degree of sialylation can decrease the
in vivo half-life of protein-based drugs (Varki, 1993, Biotechnol
Bioeng 43:423-428; Gramer et al., 1995, Glycobiology 3:97-130).
TABLE-US-00005 SEQUENCES SEQ ID NO: 1 Cricetulus griseus FAIM amino
acid sequence MTDLVAVWDVALSDGVHKIEFEHGTTSGKRVVYVDGKEEIRKEWMFKLVG
KETFCVGAAKTKATINIDAVSGFAYEYTLEIDGKSLKKYMENRSKTTNTW
VLHLDGQDLRVVLEKDTMDVWCNGQKMETAGEFVDDGTETHFSVGNHDCY
IKAVSSGKRREGIIHTLIVDNREIPELPQ SEQ ID NO: 2 Cricetulus griseus FADD
amino acid sequence
MDPFLVLLHSVSGNLSSSDLLELKFLCRERVSKRKLERVQSGLDLFSVLL
EQNDLERTRTGLLRELLASLRRHDLLQRLDDFEAGTAASAAPGEADLRVA
FDIVCDNVGRDWKRLARQLKVSEAKIDGIEERYPRSLSEQVREALRVWKI
AEREKATVAGLVKALRACRLNLVADLVEGR SEQ ID NO: 3 Cricetulus griseus
PDCD6 amino acid sequence
MAAYSYRPGPGAGPGPSAGAALPDQSFLWNVFQRVDKDRSGVISDNELQQ
ALSNGTWTPFNPVTVRSIISMFDRENKAGVNFSEFTGVWKYITDWQNVFR
TYDRDNSGMIDKNELKQALSGFGYRLSDQFHDILIRKFDRQGRGQIAFDD
FIQGCIVLQRLTDIFRRYDTDQDGWIQVSYEQYLSMVFSIV SEQ ID NO: 4 Cricetulus
griseus Requiem amino acid sequence
MAAVVENVVKLLGEQYYKDAMEQCHNYNARLCAERSVRLPFLDSQTGVAQ
SNCYIWMEKRHRGPGLASGQLYSYPARRWRKKRRAHPPEDPRLSFPSIKP
DTDQTLKKEGLISQDGSSLEALLRTDPLEKRGAPDPRVDDDSLGEFPVTN
SRARKRILEPDDFLDDLDDEDYEEDTPKRRGKGKSKSKGVSSARKKLDAS
ILEDRDKPYACDICGKRYKNRPCLSYHYAYSHLAEEEGEDKEDSQPPTPV
SQRSEEQKSKKGPDGLALPNNYCDFCLGDSKINKKTGQPEELVSCSDCGR
SGHPSCLQFTPVMMAAVKTYRWQCIECKCCNLCGTSENDDQLLFCDDCDR
GYHMYCLTSSMSEPPEGSWSCHLCLDLLKEKASIYQNQSSS SEQ ID NO: 5 Cricetulus
griseus FAIM nucleic acid sequence GCCGCGAGAG CTGCTGACTA CGTCGTGGGA
TCAGGAGCCG GTGGCGGAGC GCCGGGCAGC CCTCTTTATA ACCTGGAAAA AATGACAGAT
CTTGTAGCTG TTTGGGACGT TGCATTAAGT GATGGAGTCC ACAAGATTGA ATTTGAGCAT
GGGACCACAT CAGGCAAACG AGTTGTGTAC GTGGATGGGA AGGAAGAGAT AAGAAAAGAA
TGGATGTTCA AATTGGTGGG CAAAGAAACC TTCTGTGTTG GAGCTGCGAA AACCAAAGCC
ACCATAAATA TAGATGCTGT CAGTGGTTTT GCTTATGAGT ATACCCTGGA AATCGATGGG
AAAAGCCTCA AGAAGTACAT GGAGAACAGA TCAAAGACCA CCAACACCTG GGTACTGCAC
TTGGATGGCC AGGACTTAAG AGTTGTTTTG GAAAAAGATA CTATGGATGT ATGGTGCAAT
GGTCAAAAAA TGGAGACAGC AGGCGAGTTT GTAGATGATG GAACTGAAAC ACACTTCAGT
GTTGGGAACC ATGACTGTTA CATAAAAGCT GTCAGCAGCG GGAAGAGAAG AGAAGGGATT
ATCCACACAC TCATTGTGGA TAACAGGGAG ATCCCAGAGC TCCCTCAGTG ACTGCTGGTT
AGTGGGTTCT GAGCTGAAGA GGAGACATCA GGACTTTCTA ATGGCTGTGG TAATTAAATG
TGTTCACTGT GTACATATTG GTAGATTTAG TCTGCAATGT TTTTATTTTT TGTTACTGGA
AACTGTAATA TTCCAATGGT CAAGAAAAAT GTGGAATCAT AAAAATTTAT TTTTTAACTA
CTGTAAAGTG TTTCTAATTC AAATAGGAAA TAAAATATGG ACCAAACCCA TTCATATCTC
ACCACAGTAA C SEQ ID NO: 6 Cricetulus griseus FADD nucleic acid
sequence CCATGGACCC ATTCCTGGTG CTGCTGCACT CGGTGTCTGG CAACTTGTCG
AGCAGCGATC TGCTGGAGCT AAAGTTCCTG TGCCGTGAGC GCGTGAGCAA ACGAAAGCTG
GAGCGTGTGC AGAGTGGCCT GGACCTGTTC TCAGTGCTGC TGGAGCAGAA CGATCTGGAG
CGCACACGCA CCGGGCTGCT GCGTGAGCTG CTGGCCTCGC TGCGCAGACA CGATCTCCTG
CAACGCCTGG ACGACTTTGA AGCGGGGACG GCGGCCTCGG CCGCACCGGG GGAGGCAGAT
CTGCGGGTGG CCTTTGACAT TGTATGCGAC AATGTGGGGA GAGATTGGAA GAGACTGGCC
CGCCAGCTGA AAGTGTCTGA GGCCAAAATT GATGGGATTG AGGAGAGGTA CCCCCGAAGC
CTGAGTGAGC AGGTAAGGGA GGCTCTGAGA GTCTGGAAGA TTGCCGAGAG GGAGAAAGCC
ACGGTGGCTG GACTGGTAAA GGCACTTCGG GCCTGCCGGC TGAACCTGGT GGCTGACCTG
GTGGAAGGGA GG SEQ ID NO: 7 Cricetulus griseus PDCD6 nucleic acid
sequence GCCCATGGCT GCCTACTCCT ACCGCCCAGG CCCGGGCGCC GGCCCCGGCC
CTTCTGCTGG AGCTGCGCTG CCAGACCAGA GCTTCCTGTG GAACGTCTTC CAGCGGGTTG
ATAAAGACAG GAGTGGAGTG ATTTCAGACA ATGAGCTTCA GCAAGCATTA TCCAATGGTA
CTTGGACTCC GTTTAATCCA GTGACTGTTA GGTCAATCAT TTCTATGTTT GACCGAGAAA
ACAAGGCTGG CGTGAACTTC AGTGAATTTA CAGGCGTGTG GAAGTACATC ACAGACTGGC
AGAATGTCTT CCGAACCTAT GACCGGGACA ACTCTGGGAT GATTGACAAG AACGAGCTCA
AGCAAGCACT CTCAGGTTTT GGCTACCGGC TCTCTGACCA GTTCCATGAC ATCCTCATCC
GCAAATTTGA CAGACAAGGA CGAGGGCAGA TCGCATTTGA TGACTTCATC CAGGGCTGCA
TCGTCTTGCA GAGGTTGACA GACATATTCA GACGCTATGA CACGGATCAG GACGGCTGGA
TTCAGGTGTC TTATGAGCAG TATCTCTCCA TGGTCTTCAG CATCGTATAA CCAGGCCCTG
TGAACAGCAA GCACAGCATG CAAAAAAGAC TGAAAATGCC AAATCCCTTC CCTGTGATGA
AACAGGGCAC AAGTACAGTT AGATGCTGTT CTTCCTGTAG GCTGTATAAT TAATACTTGG
GGACCTGGCT GTACATATGT GAATAAGCTG GTTAGTGATT CTGTAGTGGC ACCCTAGCTA
CACTGTTATA ATACAAACAT TGGGTTTGCT GACTAATTGT GCCACGAGGG GAAACCGAAT
ATTGGTTCAG GATTCTGCTC TCAAACTATC ATGTTCTTTT CTAGCTGTCT CTAATTCTGT
AGTTGAAAAT ACTTTTATTA GCCAATAGGA TTTTAAAATA ATATGGAACT TGCACAGAAG
GCTTTTCATG TGCCTTACTT TTTTAAAAAA GAGTTTATGT ATTCATTGGA ATATGTAACA
TAAGCAATAA AGTAATGATC CAGCCCAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAA SEQ
ID NO: 8 Cricetulus griseus Requiem nucleic acid sequence
GGTATCAACG CAGCCTCCCG GCGGGAGGGA GAGGAGCAGG GAAGATGGCG GCTGTGGTGG
AGAATGTAGT GAAGCTCCTT GGGGAACAGT ACTACAAAGA TGCCATGGAA CAGTGCCACA
ATTACAATGC CCGCCTCTGT GCTGAGCGTA GCGTGCGTCT GCCTTTCTTG GACTCACAGA
CTGGAGTAGC CCAGAGCAAT TGTTATATCT GGATGGAAAA GCGACACCGG GGACCAGGAT
TGGCCTCTGG ACAGTTGTAC TCCTACCCTG CCCGGCGCTG GCGTAAAAAG CGTCGAGCTC
ACCCACCTGA GGATCCCAGG CTTTCCTTTC CATCTATTAA ACCAGACACA GACCAGACCC
TGAAGAAAGA GGGGCTTATA TCTCAGGATG GCAGCAGTTT AGAGGCTCTA CTGCGTACTG
ACCCTCTGGA GAAACGAGGT GCTCCAGATC CCCGTGTTGA TGATGACAGC CTGGGCGAGT
TTCCTGTCAC CAACAGTCGA GCACGGAAGC GGATCCTTGA ACCTGATGAC TTCCTAGATG
ATCTTGATGA TGAAGACTAT GAAGAAGATA CTCCAAAACG TCGGGGAAAG GGGAAATCCA
AGAGTAAGGG TGTGAGCAGT GCCCGGAAGA AACTGGATGC TTCCATCCTG GAGGACCGTG
ATAAGCCCTA TGCCTGTGAC ATTTGTGGAA AACGCTACAA GAATCGACCT TGCCTCAGTT
ACCACTATGC CTATTCCCAC CTGGCTGAGG AGGAGGGAGA GGACAAAGAA GACTCTCAAC
CACCTACTCC TGTTTCCCAG AGGTCTGAGG AGCAGAAATC CAAGAAAGGA CCTGATGGAT
TAGCCCTGCC CAACAACTAC TGTGACTTCT GCCTGGGAGA CTCAAAAATC AACAAGAAGA
CAGGGCAGCC TGAGGAGCTA GTGTCCTGTT CTGACTGTGG CCGCTCAGGG CACCCGTCCT
GCCTGCAGTT CACCCCCGTG ATGATGGCGG CTGTGAAGAC CTACCGCTGG CAGTGCATCG
AGTGCAAGTG CTGCAACCTC TGCGGCACTT CGGAGAATGA CGACCAGCTG CTCTTCTGTG
ATGACTGTGA CCGTGGCTAC CACATGTACT GTCTCACCTC ATCCATGTCG GAGCCTCCTG
AAGGAAGTTG GAGCTGCCAC CTGTGTCTGG ATCTGCTGAA GGAGAAAGCG TCCATCTACC
AGAACCAGAG CTCCTCCTGA TGTGCCACCC GGCTCCCCAC ACACCTAAGG CTGTTGCTCT
CCTCTACCTT GGTTTTCATA CCCCTCTTCT TCTTCTTCTT TCACTCTGGT AGTTCTGCCC
AACTGCCTTT GGCAACAGCA CAGGGAAGGT GGCAACTCTT
GACTGCCTCT GGTCCCAAGC CCTCAGGGAG TAAGGAGCAG CATGCTGCCC CAGGCTGATC
TGTGGGCCCA GCTTCTCTCT GCTCTCCAAG AAGTGCATTC ACTCTGCTTG CCTTGGGCCC
AAGTCCCTGG TAATCACAGG GTTCAAATGG GTTCCTCTAA GAAGTATGAG AGCAGCTCAC
TTGTCTCAAG CCTGGCCTAC CCCTCCTCCC CCTCTGGTGT CCAGAGTTTT ACCCCAGGGG
TGAGCCAGGC CTAACCTTTG CTTGGAGCAC CTGGAGTGAT CAGACTGAGG TGGCACTTGC
TAGGACCCTT TCCTACCCCT TGTTCTGCTT CACTTTGCCT CTGCCAAAGC AGTCCTGTGT
CTTCTGTCAT GCTACATGGG GTCCTGTGCT TGCACTGTGA TGCTCTCAGG CACCTCCTGG
CTCTGTCCTG TTCTGCCCAG TCCCACAAAG AGACAAGCAG CTTCACCTGC CCTTCCCGTG
CTTGGCTGGC GCGCTCACAG GTGGTCTCTG GCAATCCAAA CATTTCCCAT CCTCAGACTT
TTGAGTCTTC TGCCTCCTTC CTTGTTCCCT TTGGTTTTGT GGGGGAGAGG GACAATGTCA
GGGGGCCCTG CCAGAAGCTT GGGGACCACA AGAAGTTGGA TAATGTGCCT GTTTTTTAAC
TCGATAAAAA TGCCTACCTC CAAAATCCCC TTTTCGTTCT TCCTGGACCT GGGCATTCAG
CCTCCTGCCC TTAACTGAAT CAGGAGCCTC TGCCTCCTAC TGTGTATCCT GGCTCCCAGG
AGAGAGGATG GTCCCCTTTC CTTGCACACT AGCTAGCAGC TGGTAAAGTC TTCTTTCCCT
GATTTTTGTT TCCTGCTTAG TGGCACTGAC ATTAAGTAGG AGGGGACAGT CCATGCCAGA
ACACTCTGGA ATGGCCTTCC TCCTTGGCTG TGGGCAGGCC CTGACTTGTT TTCTGCAAAG
TTGAGGCCCC TCCTCCTATC CTTAGTTCCT GTATCCAAAA CATTAGTAAG AATAAACATT
TTTACACAGA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AA SEQ ID NO: 9
Cricetulus griseus FADD dominant negative amino acid sequence
MAFDIVCDNVGRDWKRLARQLKVSEAKIDGIEERYPRSLSEQVREALRVW
KIAEREKATVAGLVKALRACRLNLVADLVEGR SEQ ID NO: 10 Cricetulus griseus
FADD dominant negative nucleic acid sequence
GCCACCATGGCCTTTGACATTGTATGCGACAATGTGGGGAGAGATTGGAA
GAGACTGGCCCGCCAGCTGAAAGTGTCTGAGGCCAAAATTGATGGGATTG
AGGAGAGGTACCCCCGAAGCCTGAGTGAGCAGGTAAGGGAGGCTCTGAGA
GTCTGGAAGATTGCCGAGAGGGAGAAAGCCACGGTGGCTGGACTGGTAAA
GGCACTTCGGGCCTGCCGGCTGAACCTAGTGGCTGACCTGGTGGAAGGGA GGC SEQ ID NO:
11 Cricetulus griseus FADD dominant negative 5'-PCR primer
5'-GATATCGGATCCGCCACCATGGCC-TTTGACATTGTATGCGACAATG TGGGG-3' SEQ ID
NO: 12 Cricetulus griseus FADD dominant negative 3'-PCR primer
5'-CCCGGG-CTCGAGTGCCTCCC-TTCCACCAGGTCAG-3' SEQ ID NO: 13 Cricetulus
griseus PDCD6 suppression vector insert 5'
5'-GATCCCGTGAGCTTCAGCAAGCATTATTCAAGAGATAATGCTTGCTG
AAGC-TCATTTTTTGGAAA-3' SEQ ID NO: 14 Cricetulus griseus PDCD6
suppression vector insert 3'
5'-AGCTTTTCCAAAAAATGAGCTTCAGCAAGCATTATCTCTTGAATAAT
GCTTGCTGAAGCTCACG-3' SEQ ID NO: 15 Cricetulus griseus Requiem
suppression vector insert 5'
5'-GATCCCGCGGATCCTTGAACCTGATTTCAAGAGAATCAGGTTCAAGG
ATCCGC-TTTTTTGGAAA-3' SEQ ID NO: 16 Cricetulus griseus Requiem
suppression vector insert 3'
5'-AGCTTTTCCAAAAAAGCGGATCCTTGAACCTGATTCTCTTGAAATCA
GGTTCAAGGATCCGCGG-3' SEQ ID NO: 17 Cricetulus griseus FAIM 5' PCR
primer 5'-GCCGCGAGAGCTGCTGACTACGTCGTGG-3' SEQ ID NO: 18 Cricetulus
griseus FAIM 3' PCR primer 5'-GTTACTGTG-GTGAGATATGAATGGGTTTGG-3'.
SEQ ID NO: 19 Cricetulus griseus FADD 5' PCR primer
5'-CCATGGACCCATTCCTGGTGC-3' SEQ ID NO: 20 Cricetulus griseus FADD
3' PCR primer 5'-TTCTTCCACCAGGTCAGC-CACC-3' SEQ ID NO: 21
Cricetulus griseus PDCD6 5' PCR primer 5'-GCCCATGGCTGCCTACTCCTA-3'
SEQ ID NO: 22 Cricetulus griseus PDCD6 3' PCR primer
5'-AATCCAGCCATCCTGAT-CCGT-3'. SEQ ID NO: 23 Cricetulus griseus
PDCD6 3'-RACE primer 5'-CAGCGGGTTGATAAAGACAGGAGTGGAGTG-3'. SEQ ID
NO: 24 Cricetulus griseus Requiem 5' PCR primer
5'-ATG-GCGGCTGTGGTGGAGAAT-3' SEQ ID NO: 25 Cricetulus griseus
Requiem 3' PCR primer 5'-GGAGTTCTGGTTCTGGTAG-ATGG-3' SEQ ID NO: 26
Cricetulus griseus Requiem 3'-RACE primer
5'-GCCTCAGTTACCACTATGCCCATTCCCACC-3' SEQ ID NO: 27 Cricetulus
griseus FAIM Quantitative Real Time PCR primer 5'
5'-TGGAGCTGCGAAAACCAAAG-3' SEQ ID NO: 28 Cricetulus griseus FAIM
Quantitative Real Time PCR primer 3' 5'-AAACTCGCCTGCTGTCTCCAT-3'
SEQ ID NO: 29 Cricetulus griseus FADD Quantitative Real Time PCR
primer 5' 5'-GATATCGGATCCGCCACCATGG-3' SEQ ID NO: 30 Cricetulus
griseus FADD Quantitative Real Time PCR primer 3'
5'-TGCCTCCCTTCCACCAG-GTCAG' 3' SEQ ID NO: 31 Cricetulus griseus
PDCD6 Quantitative Real Time PCR primer 5'
5'-CAGCGGGTTGATAAAGACAGG-3' SEQ ID NO: 32 Cricetulus griseus PDCD6
Quantitative Real Time PCR primer 3' 5'-GCCAGCCTTG-TTTTCTCGG-3' SEQ
ID NO: 33 Cricetulus griseus Requiem Quantitative Real Time PCR
primer 5' 5'-TGGAGTAGCCCAGAGCAATTG-3' SEQ ID NO: 34 Cricetulus
griseus Requiem Quantitative Real Time PCR primer 3'
5'-tcgacgctttttacgccag-3' SEQ ID NO: 35 .beta.-actin Quantitative
Real Time PCR primer 5' 5'-AGCTGAGAGGGAAATTGTGCG-3' SEQ ID NO: 36
.beta.-actin Quantitative Real Time PCR primer 3'
5'-GCAACGG-AACCGCTCATT-3' SEQ ID NO: 37 Plasmid pcDNA3.1(+) FAIM
plasmid nucleic acid sequence (underlined sequence denotes cgFAIM
insert) GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATC
TGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTT
GGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAG
GCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCG
CTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGAC
TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATA
TGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCG
CCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGT
AACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGT
AAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCC
CCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTA
CATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCA
TCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGA
TAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA
TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTA
ACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAG
GTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTG
GCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGC
GTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGT
GGAATTCGCCACCATGACAGATCTTGTAGCTGTTTGGGACGTTGCATTAA
GTGATGGAGTCCACAAGATTGAATTTGAGCATGGGACCACATCAGGCAAA
CGAGTTGTGTACGTGGATGGGAAGGAAGAGATAAGAAAAGAATGGATGTT
CAAATTGGTGGGCAAAGAAACCTTCTGTGTTGGAGCTGCGAAAACCAAAG
CCACCATAAATATAGATGCTGTCAGTGGTTTTGCTTATGAGTATACCCTG
GAAATCGATGGGAAAAGCCTCAAGAAGTACATGGAGAACAGATCAAAGAC
CACCAACACCTGGGTACTGCACTTGGATGGCCAGGACTTAAGAGTTGTTT
TGGAAAAAGATACTATGGATGTATGGTGCAATGGTCAAAAAATGGAGACA
GCAGGCGAGTTTGTAGATGATGGAACTGAAACACACTTCAGTGTTGGGAA
CCATGACTGTTACATAAAAGCTGTCAGCAGCGGGAAGAGAAGAGAAGGGA
TTATCCACACACTCATTGTGGATAACAGGGAGATCCCAGAGCTCCCTCAG
TGACTGCTGGTTAGTGGGTTCTGAGCTGAAGAGGAGACATCAGGACTTTC
TAATGGCTGTGGTAATTAAATGTGTTCACGAATTCTGCAGATATCCAGCA
CAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGC
CTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCG
TGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAA
AATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGG
GGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCA
GGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACC
AGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAG
CGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCG
CCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTC
GCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCG
ATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATG
GTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACG
TTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAAC
ACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGA
TTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCG
AATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGC
TCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAAC
CAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCA
TGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATC
CCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACT
AATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTAT
TCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAG
CTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATG
AGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGG
CCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATC
GGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGT
TCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACG
AGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCT
GTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGA
AGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAG
TATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCT
ACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTAC
TCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATC
AGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCC
GACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATAT
CATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGG
GTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCT
GAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTAT
CGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGT
TCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCA
ACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTG
GGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGG
GGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTT
ATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCA
TTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATC
TTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCAT
GGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACAC
AACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGT
GAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGG
GAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGA
GGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCT
GCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGG
TAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGA
GCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGC
GTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCT
CAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTT
CCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTAC
CGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATA
GCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTG
GGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGG
TAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGG
CAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCT
ACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGT
ATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTG
GTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTT
TGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTT
GATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAG
GGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTA
AATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTG
GTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCT
GTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACT
ACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCG
AGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCG
GAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAG
TCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAG
TTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGT
CGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTT
ACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCC
GATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGG
CAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCT
GTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCG
ACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATA
GCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAA
CTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCG
TGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGT
GAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACA
CGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCAT
TTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGA
AAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCT GACGTC SEQ ID
NO: 38 Plasmid pcDNA3.1(+) FADD DN plasmid nucleic acid sequence
(underlined sequence denotes cgFADD dominant negative insert)
GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATC
TGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTT
GGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAG
GCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCG
CTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGAC
TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATA
TGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCG
CCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGT
AACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGT
AAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCC
CCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTA
CATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCA
TCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGA
TAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA
TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTA
ACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAG
GTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTG
GCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGC
GTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCGCCACCATGGCCTTTG
ACATTGTATGCGACAATGTGGGGAGAGATTGGAAGAGACTGGCCCGCCAG
CTGAAAGTGTCTGAGGCCAAAATTGATGGGATTGAGGAGAGGTACCCCCG
AAGCCTGAGTGAGCAGGTAAGGGAGGCTCTGAGAGTCTGGAAGATTGCCG
AGAGGGAGAAAGCCACGGTGGCTGGACTGGTAAAGGCACTTCGGGCCTGC
CGGCTGAACCTAGTGGCTGACCTGGTGGAAGGGAGGCACTCGAGTCTAGA
GGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCA
GCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTG
CCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGT
CTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAA
GGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCT
CTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCC
CACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCG
CAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTT
TCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTA
AATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGA
CCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCT
GATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGT
GGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTC
TTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATG
AGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTC
AGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAA
AGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTC
CCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCA
TAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCC
GCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGC
CGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTT
TTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATT
TTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACA
AGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCG
GCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTC
CGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTC
CGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGG
CCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCG
GGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTC
ATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGC
GGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCG
AAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGA
TCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGT
TCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACC
CATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTC
TGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACA
TAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCT
GACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCAT
CGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTT
CGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATT
CCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGAC
GCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGC
CCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATA
GCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGT
GGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGAC
CTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAA
ATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAG
TGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTT
GCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATT
AATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCT
TCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCG
AGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAG
GGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGG
AACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCC
TGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGA
CAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGC
TCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCC
TTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTT
CGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTT
CAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCC
GGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTA
GCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCT
AACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAA
GCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAA
CCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGA
AAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGC
TCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAA
AAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCA
ATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAAT
CAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTG
CCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCT
GGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGA
TTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTC
CTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCT
AGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGC
TACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCT
CCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAA
AAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGC
CGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTG
TCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAG
TCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTC
AATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCA
TTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTG
AGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATC
TTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATG
CCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTC
TTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAG
CGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGC
GCACATTTCCCCGAAAAGTGCCACCTGACGTC SEQ ID NO: 39 Plasmid
pSUPER.neo.PDCD6 siRNA nucleic acid sequence (underlined sequence
denotes cgPDCD6 siRNA insert)
CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTT
AAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTAT
AAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAA
CAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAA
CCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGT
TTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAG
CCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGG
AAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCG
GTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACA
GGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGAT
CGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCT
GCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTG
TAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAAT
TGGAGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCCCGGG
CTGCATCCCCGATCCTTATCGCTATCGATTCACACAAAAAACCAACACAC
AGATGTAATGAAAATAAAGATATTTTATTGCGGCCATCGTGATGGCAGGT
TGGGCGTCGCTTGGTCGGTCATTTCGAACCCCAGAGTCCCGCTCAGAAGA
ACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCG
ATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTC
AGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACAC
CCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATG
ATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTC
GGGCATGCGCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCT
GATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGA
GTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGT
AGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATA
CTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACT
TCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCAC
AGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCT
CGTCCTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGA
ACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCC
GATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGG
CCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGGTGGCTCTAGCC
TTAAGTTCGAGACTGTTGTGTCAGAAGAATCAAGCTGATCTGAGTCCGGT
AAGCTAGCTTGGGCTGCAGGTCGAAAGGCCCGGAGATGAGGAAGAGGAGA
ACAGCGCGGCAGACGTGCGCTTTTGAAGCGTGCAGAATGCCGGGCCTCCG
GAGGACCTTCGGGCGCCCGCCCCGCCCCTGAGCCCGCCCCTGAGCCCGCC
CCCGGACCCACCCCTTCCCAGCCTCTGAGCCCAGAAAGCGAAGGAGCAAA
GCTGCTATTGGCCGCTGCCCCAAAGGCCTACCCGCTTCCATTGCTCAGCG
GTGCTGTCCATCTGCACGAGACTAGTGAGACGTGCTACTTCCATTTGTCA
CGTCCTGCACGACGCGAGCTGCGGGGCGGGGGGGAACTTCCTGACTAGGG
GAGGAGTAGAAGGTGGCGCGAAGGGGCCACCAAAGAACGGAGCCGGTTGG
CGCCTACCGGCCGGTGGATGTGGAATGTGTGCGAGGCCAGAGGCCACTTG
TGTAGCGCCAAGTGCCCAGCGGGGCTGCTAAAGCGCATGCTCCAGACTGC
CTTGGGAAAAGCGCCTCCCCTACCCGGTAGAATTCGAACGCTGACGTCAT
CAACCCGCTCCAAGGAATCGCGGGCCCAGTGTCACTAGGCGGGAACACCC
AGCGCGCGTGCGCCCTGGCAGGAAGATGGCTGTGAGGGACAGGGGAGTGG
CGCCCTGCAATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAA
CGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGACCAC
AGATCCCGTGAGCTTCAGCAAGCATTATTCAAGAGATAATGCTTGCTGAA
GCTCATTTTTTGGAAAAGCTTATCGATACCGTCGACCTCGAGGGGGGGCC
CGGTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCG
TAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAAT
TCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCT
AATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTC
CAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGC
GGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTG
ACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCA
AAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAA
CATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGT
TGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAAT
CGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCA
GGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGC
CGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTT
TCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTC
CAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCT
TATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCG
CCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGG
CGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAA
GGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAA
AGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGG
TTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAG
AAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAAC
TCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTA
GATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATG
AGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATC
TCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGT
GTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAA
TGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAAC
CAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGC
CTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGC
CAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTG
TCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATC
AAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCT
TCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTC
ATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAG
ATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGT
GTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACC
GCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTC
GGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGT
AACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGC
GTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAAT
AAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATT
ATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAA
TGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAA AGTGCCAC SEQ ID
NO: 40 Plasmid pSUPER.neo.Requeim siRNA nucleic acid sequence
(underlined sequence denotes cgREQUIEM siRNA insert)
CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTT
AAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTAT
AAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAA
CAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAA
CCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGT
TTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAG
CCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGG
AAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCG
GTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACA
GGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGAT
CGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCT
GCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTG
TAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAAT
TGGAGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCCCGGG
CTGCATCCCCGATCCTTATCGCTATCGATTCACACAAAAAACCAACACAC
AGATGTAATGAAAATAAAGATATTTTATTGCGGCCATCGTGATGGCAGGT
TGGGCGTCGCTTGGTCGGTCATTTCGAACCCCAGAGTCCCGCTCAGAAGA
ACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCG
ATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTC
AGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACAC
CCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATG
ATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTC
GGGCATGCGCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCT
GATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGA
GTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGT
AGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATA
CTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACT
TCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCAC
AGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCT
CGTCCTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGA
ACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCC
GATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGG
CCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGGTGGCTCTAGCC
TTAAGTTCGAGACTGTTGTGTCAGAAGAATCAAGCTGATCTGAGTCCGGT
AAGCTAGCTTGGGCTGCAGGTCGAAAGGCCCGGAGATGAGGAAGAGGAGA
ACAGCGCGGCAGACGTGCGCTTTTGAAGCGTGCAGAATGCCGGGCCTCCG
GAGGACCTTCGGGCGCCCGCCCCGCCCCTGAGCCCGCCCCTGAGCCCGCC
CCCGGACCCACCCCTTCCCAGCCTCTGAGCCCAGAAAGCGAAGGAGCAAA
GCTGCTATTGGCCGCTGCCCCAAAGGCCTACCCGCTTCCATTGCTCAGCG
GTGCTGTCCATCTGCACGAGACTAGTGAGACGTGCTACTTCCATTTGTCA
CGTCCTGCACGACGCGAGCTGCGGGGCGGGGGGGAACTTCCTGACTAGGG
GAGGAGTAGAAGGTGGCGCGAAGGGGCCACCAAAGAACGGAGCCGGTTGG
CGCCTACCGGCCGGTGGATGTGGAATGTGTGCGAGGCCAGAGGCCACTTG
TGTAGCGCCAAGTGCCCAGCGGGGCTGCTAAAGCGCATGCTCCAGACTGC
CTTGGGAAAAGCGCCTCCCCTACCCGGTAGAATTCGAACGCTGACGTCAT
CAACCCGCTCCAAGGAATCGCGGGCCCAGTGTCACTAGGCGGGAACACCC
AGCGCGCGTGCGCCCTGGCAGGAAGATGGCTGTGAGGGACAGGGGAGTGG
CGCCCTGCAATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAA
CGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGACCAC
AGATCCCGCGGATCCTTGAACCTGATTTCAAGAGAATCAGGTTCAGGATC
CGCTTTTTTGGAAAAGCTTATCGATACCGTCGACCTCGAGGGGGGGCCCG
GTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTA
ATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTC
CACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAA
TGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCA
GTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGG
GGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGAC
TCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAA
GGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACA
TGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTG
CTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCG
ACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGG
CGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCG
CTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTC
TCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCA
AGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTA
TCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCC
ACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCG
GTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGG
ACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAG
AGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTT
TTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAA
GATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTC
ACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGA
TCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAG
TAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTC
AGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGT
AGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATG
ATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCA
GCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCT
CCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCA
GTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTC
ACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAA
GGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTC
GGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCAT
GGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGAT
GCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGT
ATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGC
GCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGG
GGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAA
CCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGT
TTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAA
GGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTAT
TGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATG
TATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAG TGCCAC
REFERENCES
[0379] Altschul S F, Thomas L M, Alejandro A S, Jinghui Z, Zheng Z,
Webb M, and David J L (1997) Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs. Nucleic Acids Res.
25:3389-3402. [0380] Chestkov, A. V., Baka, I. D., Kost, M. V.,
Georgiev, G. P., Buchman, V. L. (1996) The d4 gene family in the
human genome. Genomics 36: 174-177 [0381] Chinnaiyan A M, O'Rourke
K, Tewari M and Dixit V M. (1995) FADD, a novel death
domain-containing protein, interacts with the death domain of Fas
and initiates apoptosis. Cell, May 81(4):505-12 [0382] Chinnaiyan A
M, Tepper C G, Seldin M F, O'Rourke K, Kischkel F C, Hellbardt S,
Krammer P H, Peter M E, and Dixit V M. (1996) FADD/MORT1 is a
common mediator of CD95(Fas/APO-1 and TNF receptor-induced
apoptosis. The Journal of Biological Chemistry. 271(9):4961-4965
[0383] Cohen G M (1997) Caspases: the executioners of apoptosis.
Biochem J. 326: 1-16 [0384] Fussenegger M., Fassnacht D., Schwartz
R., Zanghi J. A., Graf M., Bailey J. E. and Portner R. (2000)
Regulated over-expression of the survival factor bcl-2 in CHO cells
increases viable cell density in batch culture and decrease DNA
release in extended fixed-bed cultivation. Cytotechnology 32:45-61
[0385] Gabig T G, Mantel P L, Rosli R and Crean C D. (1994)
Requiem: a novel zinc finger gene essential for apoptosis in
myeloid cells. J. Biol. Chem. 269(47):29515-9 [0386] Gabig, T. G.,
Crean, C. D., Klenk, A., Long, H., Copeland, N. G., Gilbert, D. J.,
Jenkins, N. A., Quincey, D., Parente, F., Lespinasse, F., Carle, G.
F., Gaudray, P., et al. (1998) Expression and chromosomal
localization of the Requiem gene. Mammalian Genome 9: 660-665
[0387] Goswami J., Sinskey A.J., Steller H., Stephanopoulos and
Wang DIC. (1999) Apoptosis in batch cultures of Chinese hamster
ovary cells. Biotechnol Bioeng. 62: 632-640 [0388] Gramer M J,
Goochee C F. 1994. Glycosidase activities of the 293 and NSO cell
lines and of an antibody-producing hybridoma cell line. Biotechnol
Bioeng 43:423-428 [0389] Hu Y, Benedict M A, Ding L and Nunez G
(1999) Role of cytochrome c and dATP/ATP hydrolysis in APAF-1
mediated caspase-9 activation and apoptosis. EMBO J. 18: 3586-3595
[0390] Jong, I. K., Hu, R., Lacana, E., D'Adamio, L., Gu, H. (2002)
Apoptosis-linked gene 2-deficient mice exhibit normal T-cell
development and function. Molec. Cell. Biol. 22: 4094-4100 [0391]
Jung, Y. S., Kim, K. S., Kim, K. D., Lim, J. S., Kim, J. W. and
Kim, E. (2001) Apoptosis-linked gene 2 binds to the death domain of
Fas and dissociates from Fas during Fas-mediated apoptosis in
Jurkat cells. Biochem. Biophys. Res. Commun. 288(2):420-426 [0392]
Kerr J F R, Wyllie A H and Currie A R (1972) Apoptosis: a basic
biological phenomenon with wide-ranging implications in tissue
kinetics. Br. J. Cancer 26:239-257 [0393] Krebs, J. and Klemenz, R.
(2000) The ALG-2/AIP-complex, a modulator at the interface between
cell proliferation and cell death? A hypothesis. Biochim. Biophys.
Acta 1498(2-3): 153-161 [0394] Laken H A and Leonard M W. (2001)
Understanding and modulation apoptosis in industrial cell culture.
Cur. Opinion. in Biotechnol. 12:1/5-1/9 [0395] Lee Y Y, Yap M G S,
Hu W S and Wong K T Y. 2003. Low-glutamine fed-batch cultures of
293-HEK serum-free suspension cells for adenovirus production.
Biotechnol. Prog. 19:501-509 [0396] Li P, Nijhawan D., Budihardjo
I., Srinivasula S. M., Ahmad M., Alnemri E.S, and Wang X. (1997)
Cytochorme c and dATP-dependent formation of Apaf-1/caspase-9
complex initiates an apoptotic protease cascade. Cell. 91:479-489
[0397] Mastrangelo A. J., Hardwich J. M., Zou S, and Betenbaugh M.
J. (2000) Over-expression of bcl-2 family members enhances survival
of mammalian cells in response to various culture insults.
Biotechnol Bioeng. 67:555-564 [0398] Nicholson D W and Thornberry N
A (1997) Caspases: killer proteases. Trends Biochem Sci. 272:
2952-2956 [0399] Rothstein T L, Zhong X, Schram B R, Negm R S,
Donohoe T J, Cabral D S, Foote L C, Schneider T J. (2000)
Receptor-specific regulation of B-cell susceptibility to
Fas-mediated apoptosis and a novel Fas apoptosis inhibitory
molecule. Immunol Rev. 176:116-33. [0400] Scahill, S J, Devos R,
Der Heyden V J and Fiers W. 1983. Expression and characterization
of the product of a human immune interferon cDNA gene in Chinese
hamster ovary cells. Proc. Natl. Acad. Sci. USA. 80: 4654-4658
[0401] Schneider T J, Fischer G M, Donohoe T J, Colarusso T P and
Thomas L. Rothstein (1999) A Novel Gene Coding for a Fas Apoptosis
Inhibitory Molecule (FAIM) Isolated from Inducibly Fas-resistant B
Lymphocytes J. Exp. Med., Volume 189, Number 6, Mar. 15, 1999
949-956 [0402] Srinivasula S. M., Ahmand M., Hernandes-Alnemri T.,
Litwack G. and Alnemri E. S. (1996) Molecular ordering of the
Fas-apoptotic pathway: The Fas/APO-1 protease MchS is a
CrmA-inhibitable protease that activates multiple Ced-3/ICE-like
cysteine proteases. Proc. Natl. Acad. Sci. USA. 93:14486-14491
[0403] Tey B. T., Singh R. P., Piredda L., Piacentini M. and
Al-Rubeai M. (2000) Influcence of Bc1-2 on cell death during the
cultivation of a Chinese hamser ovary cell line expressing a
chimeric antibody. Biotechnol Bioeng. 68:31-43 [0404] Thornberry N
A and Littlewood Y (1998) Caspases: Enemies within. Science
281:1312-1316 [0405] Urlaub & Chasin. 1980. Isolation of
Chinese hamster cell mutants deficient in dihydrofolate reductase
activity Proc. Natl. Acad. Sci. USA. 77: 4216-4220 [0406] Varki A.
1993. Biological roles of oligosaccharides: All of the theories are
correct. Glycobiology 3:97-130 [0407] Vito P, Lacana E and D'Adamio
L. (1996) Interfering with apoptosis: Ca.sup.2+ binding protein
ALG-2 and Alzheimer's disease gene ALG-3. Science. 271(5248):521-5
[0408] Wong C F D, Wong T K K, Goh L T, Heng C K, Yap G S M. 2005a.
Impact of dynamic online fed-batch strategies on metabolism,
productivity and N-glycosylation quality in CHO cell cultures.
Biotechnol Bioeng 89: 164-177 [0409] Zhong X, Schneider T J, Cabral
D S, Donohoe T J, Rothstein T L. (2001) An alternatively spliced
long form of Fas apoptosis inhibitory molecule (FAIM) with
tissue-specific expression in the brain. Mol. Immunol. 2001
January; 38(1):65-72
[0410] Zou H, Li Y, Liu X and Wang X (1999) An APAF-1 cytochrome c
multimeric complex is a functional apoptosome that activates
procaspase-9. J Biol Chem 274: 11549-11556
[0411] Each of the applications and patents mentioned in this
document, and each document cited or referenced in each of the
above applications and patents, including during the prosecution of
each of the applications and patents ("application cited
documents") and any manufacturer's instructions or catalogues for
any products cited or mentioned in each of the applications and
patents and in any of the application cited documents, are hereby
incorporated herein by reference. Furthermore, all documents cited
in this text, and all documents cited or referenced in documents
cited in this text, and any manufacturer's instructions or
catalogues for any products cited or mentioned in this text, are
hereby incorporated herein by reference.
[0412] Various modifications and variations of the described
methods and system of the invention will be apparent to those
skilled in the art without departing from the scope and spirit of
the invention. Although the invention has been described in
connection with specific preferred embodiments, it should be
understood that the invention as claimed should not be unduly
limited to such specific embodiments. Indeed, various modifications
of the described modes for carrying out the invention which are
obvious to those skilled in molecular biology or related fields are
intended to be within the scope of the claims.
Sequence CWU 1
1
481179PRTCricetulus griseus 1Met Thr Asp Leu Val Ala Val Trp Asp
Val Ala Leu Ser Asp Gly Val1 5 10 15His Lys Ile Glu Phe Glu His Gly
Thr Thr Ser Gly Lys Arg Val Val 20 25 30Tyr Val Asp Gly Lys Glu Glu
Ile Arg Lys Glu Trp Met Phe Lys Leu 35 40 45Val Gly Lys Glu Thr Phe
Cys Val Gly Ala Ala Lys Thr Lys Ala Thr 50 55 60Ile Asn Ile Asp Ala
Val Ser Gly Phe Ala Tyr Glu Tyr Thr Leu Glu65 70 75 80Ile Asp Gly
Lys Ser Leu Lys Lys Tyr Met Glu Asn Arg Ser Lys Thr 85 90 95Thr Asn
Thr Trp Val Leu His Leu Asp Gly Gln Asp Leu Arg Val Val 100 105
110Leu Glu Lys Asp Thr Met Asp Val Trp Cys Asn Gly Gln Lys Met Glu
115 120 125Thr Ala Gly Glu Phe Val Asp Asp Gly Thr Glu Thr His Phe
Ser Val 130 135 140Gly Asn His Asp Cys Tyr Ile Lys Ala Val Ser Ser
Gly Lys Arg Arg145 150 155 160Glu Gly Ile Ile His Thr Leu Ile Val
Asp Asn Arg Glu Ile Pro Glu 165 170 175Leu Pro Gln2180PRTCricetulus
griseus 2Met Asp Pro Phe Leu Val Leu Leu His Ser Val Ser Gly Asn
Leu Ser1 5 10 15Ser Ser Asp Leu Leu Glu Leu Lys Phe Leu Cys Arg Glu
Arg Val Ser 20 25 30Lys Arg Lys Leu Glu Arg Val Gln Ser Gly Leu Asp
Leu Phe Ser Val 35 40 45Leu Leu Glu Gln Asn Asp Leu Glu Arg Thr Arg
Thr Gly Leu Leu Arg 50 55 60Glu Leu Leu Ala Ser Leu Arg Arg His Asp
Leu Leu Gln Arg Leu Asp65 70 75 80Asp Phe Glu Ala Gly Thr Ala Ala
Ser Ala Ala Pro Gly Glu Ala Asp 85 90 95Leu Arg Val Ala Phe Asp Ile
Val Cys Asp Asn Val Gly Arg Asp Trp 100 105 110Lys Arg Leu Ala Arg
Gln Leu Lys Val Ser Glu Ala Lys Ile Asp Gly 115 120 125Ile Glu Glu
Arg Tyr Pro Arg Ser Leu Ser Glu Gln Val Arg Glu Ala 130 135 140Leu
Arg Val Trp Lys Ile Ala Glu Arg Glu Lys Ala Thr Val Ala Gly145 150
155 160Leu Val Lys Ala Leu Arg Ala Cys Arg Leu Asn Leu Val Ala Asp
Leu 165 170 175Val Glu Gly Arg 1803191PRTCricetulus griseus 3Met
Ala Ala Tyr Ser Tyr Arg Pro Gly Pro Gly Ala Gly Pro Gly Pro1 5 10
15Ser Ala Gly Ala Ala Leu Pro Asp Gln Ser Phe Leu Trp Asn Val Phe
20 25 30Gln Arg Val Asp Lys Asp Arg Ser Gly Val Ile Ser Asp Asn Glu
Leu 35 40 45Gln Gln Ala Leu Ser Asn Gly Thr Trp Thr Pro Phe Asn Pro
Val Thr 50 55 60Val Arg Ser Ile Ile Ser Met Phe Asp Arg Glu Asn Lys
Ala Gly Val65 70 75 80Asn Phe Ser Glu Phe Thr Gly Val Trp Lys Tyr
Ile Thr Asp Trp Gln 85 90 95Asn Val Phe Arg Thr Tyr Asp Arg Asp Asn
Ser Gly Met Ile Asp Lys 100 105 110Asn Glu Leu Lys Gln Ala Leu Ser
Gly Phe Gly Tyr Arg Leu Ser Asp 115 120 125Gln Phe His Asp Ile Leu
Ile Arg Lys Phe Asp Arg Gln Gly Arg Gly 130 135 140Gln Ile Ala Phe
Asp Asp Phe Ile Gln Gly Cys Ile Val Leu Gln Arg145 150 155 160Leu
Thr Asp Ile Phe Arg Arg Tyr Asp Thr Asp Gln Asp Gly Trp Ile 165 170
175Gln Val Ser Tyr Glu Gln Tyr Leu Ser Met Val Phe Ser Ile Val 180
185 1904391PRTCricetulus griseus 4Met Ala Ala Val Val Glu Asn Val
Val Lys Leu Leu Gly Glu Gln Tyr1 5 10 15Tyr Lys Asp Ala Met Glu Gln
Cys His Asn Tyr Asn Ala Arg Leu Cys 20 25 30Ala Glu Arg Ser Val Arg
Leu Pro Phe Leu Asp Ser Gln Thr Gly Val 35 40 45Ala Gln Ser Asn Cys
Tyr Ile Trp Met Glu Lys Arg His Arg Gly Pro 50 55 60Gly Leu Ala Ser
Gly Gln Leu Tyr Ser Tyr Pro Ala Arg Arg Trp Arg65 70 75 80Lys Lys
Arg Arg Ala His Pro Pro Glu Asp Pro Arg Leu Ser Phe Pro 85 90 95Ser
Ile Lys Pro Asp Thr Asp Gln Thr Leu Lys Lys Glu Gly Leu Ile 100 105
110Ser Gln Asp Gly Ser Ser Leu Glu Ala Leu Leu Arg Thr Asp Pro Leu
115 120 125Glu Lys Arg Gly Ala Pro Asp Pro Arg Val Asp Asp Asp Ser
Leu Gly 130 135 140Glu Phe Pro Val Thr Asn Ser Arg Ala Arg Lys Arg
Ile Leu Glu Pro145 150 155 160Asp Asp Phe Leu Asp Asp Leu Asp Asp
Glu Asp Tyr Glu Glu Asp Thr 165 170 175Pro Lys Arg Arg Gly Lys Gly
Lys Ser Lys Ser Lys Gly Val Ser Ser 180 185 190Ala Arg Lys Lys Leu
Asp Ala Ser Ile Leu Glu Asp Arg Asp Lys Pro 195 200 205Tyr Ala Cys
Asp Ile Cys Gly Lys Arg Tyr Lys Asn Arg Pro Cys Leu 210 215 220Ser
Tyr His Tyr Ala Tyr Ser His Leu Ala Glu Glu Glu Gly Glu Asp225 230
235 240Lys Glu Asp Ser Gln Pro Pro Thr Pro Val Ser Gln Arg Ser Glu
Glu 245 250 255Gln Lys Ser Lys Lys Gly Pro Asp Gly Leu Ala Leu Pro
Asn Asn Tyr 260 265 270Cys Asp Phe Cys Leu Gly Asp Ser Lys Ile Asn
Lys Lys Thr Gly Gln 275 280 285Pro Glu Glu Leu Val Ser Cys Ser Asp
Cys Gly Arg Ser Gly His Pro 290 295 300Ser Cys Leu Gln Phe Thr Pro
Val Met Met Ala Ala Val Lys Thr Tyr305 310 315 320Arg Trp Gln Cys
Ile Glu Cys Lys Cys Cys Asn Leu Cys Gly Thr Ser 325 330 335Glu Asn
Asp Asp Gln Leu Leu Phe Cys Asp Asp Cys Asp Arg Gly Tyr 340 345
350His Met Tyr Cys Leu Thr Ser Ser Met Ser Glu Pro Pro Glu Gly Ser
355 360 365Trp Ser Cys His Leu Cys Leu Asp Leu Leu Lys Glu Lys Ala
Ser Ile 370 375 380Tyr Gln Asn Gln Ser Ser Ser385
3905881DNACricetulus griseus 5gccgcgagag ctgctgacta cgtcgtggga
tcaggagccg gtggcggagc gccgggcagc 60cctctttata acctggaaaa aatgacagat
cttgtagctg tttgggacgt tgcattaagt 120gatggagtcc acaagattga
atttgagcat gggaccacat caggcaaacg agttgtgtac 180gtggatggga
aggaagagat aagaaaagaa tggatgttca aattggtggg caaagaaacc
240ttctgtgttg gagctgcgaa aaccaaagcc accataaata tagatgctgt
cagtggtttt 300gcttatgagt ataccctgga aatcgatggg aaaagcctca
agaagtacat ggagaacaga 360tcaaagacca ccaacacctg ggtactgcac
ttggatggcc aggacttaag agttgttttg 420gaaaaagata ctatggatgt
atggtgcaat ggtcaaaaaa tggagacagc aggcgagttt 480gtagatgatg
gaactgaaac acacttcagt gttgggaacc atgactgtta cataaaagct
540gtcagcagcg ggaagagaag agaagggatt atccacacac tcattgtgga
taacagggag 600atcccagagc tccctcagtg actgctggtt agtgggttct
gagctgaaga ggagacatca 660ggactttcta atggctgtgg taattaaatg
tgttcactgt gtacatattg gtagatttag 720tctgcaatgt ttttattttt
tgttactgga aactgtaata ttccaatggt caagaaaaat 780gtggaatcat
aaaaatttat tttttaacta ctgtaaagtg tttctaattc aaataggaaa
840taaaatatgg accaaaccca ttcatatctc accacagtaa c
8816542DNACricetulus griseus 6ccatggaccc attcctggtg ctgctgcact
cggtgtctgg caacttgtcg agcagcgatc 60tgctggagct aaagttcctg tgccgtgagc
gcgtgagcaa acgaaagctg gagcgtgtgc 120agagtggcct ggacctgttc
tcagtgctgc tggagcagaa cgatctggag cgcacacgca 180ccgggctgct
gcgtgagctg ctggcctcgc tgcgcagaca cgatctcctg caacgcctgg
240acgactttga agcggggacg gcggcctcgg ccgcaccggg ggaggcagat
ctgcgggtgg 300cctttgacat tgtatgcgac aatgtgggga gagattggaa
gagactggcc cgccagctga 360aagtgtctga ggccaaaatt gatgggattg
aggagaggta cccccgaagc ctgagtgagc 420aggtaaggga ggctctgaga
gtctggaaga ttgccgagag ggagaaagcc acggtggctg 480gactggtaaa
ggcacttcgg gcctgccggc tgaacctggt ggctgacctg gtggaaggga 540gg
54271068DNACricetulus griseus 7gcccatggct gcctactcct accgcccagg
cccgggcgcc ggccccggcc cttctgctgg 60agctgcgctg ccagaccaga gcttcctgtg
gaacgtcttc cagcgggttg ataaagacag 120gagtggagtg atttcagaca
atgagcttca gcaagcatta tccaatggta cttggactcc 180gtttaatcca
gtgactgtta ggtcaatcat ttctatgttt gaccgagaaa acaaggctgg
240cgtgaacttc agtgaattta caggcgtgtg gaagtacatc acagactggc
agaatgtctt 300ccgaacctat gaccgggaca actctgggat gattgacaag
aacgagctca agcaagcact 360ctcaggtttt ggctaccggc tctctgacca
gttccatgac atcctcatcc gcaaatttga 420cagacaagga cgagggcaga
tcgcatttga tgacttcatc cagggctgca tcgtcttgca 480gaggttgaca
gacatattca gacgctatga cacggatcag gacggctgga ttcaggtgtc
540ttatgagcag tatctctcca tggtcttcag catcgtataa ccaggccctg
tgaacagcaa 600gcacagcatg caaaaaagac tgaaaatgcc aaatcccttc
cctgtgatga aacagggcac 660aagtacagtt agatgctgtt cttcctgtag
gctgtataat taatacttgg ggacctggct 720gtacatatgt gaataagctg
gttagtgatt ctgtagtggc accctagcta cactgttata 780atacaaacat
tgggtttgct gactaattgt gccacgaggg gaaaccgaat attggttcag
840gattctgctc tcaaactatc atgttctttt ctagctgtct ctaattctgt
agttgaaaat 900acttttatta gccaatagga ttttaaaata atatggaact
tgcacagaag gcttttcatg 960tgccttactt ttttaaaaaa gagtttatgt
attcattgga atatgtaaca taagcaataa 1020agtaatgatc cagcccaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaa 106882482DNACricetulus griseus
8ggtatcaacg cagcctcccg gcgggaggga gaggagcagg gaagatggcg gctgtggtgg
60agaatgtagt gaagctcctt ggggaacagt actacaaaga tgccatggaa cagtgccaca
120attacaatgc ccgcctctgt gctgagcgta gcgtgcgtct gcctttcttg
gactcacaga 180ctggagtagc ccagagcaat tgttatatct ggatggaaaa
gcgacaccgg ggaccaggat 240tggcctctgg acagttgtac tcctaccctg
cccggcgctg gcgtaaaaag cgtcgagctc 300acccacctga ggatcccagg
ctttcctttc catctattaa accagacaca gaccagaccc 360tgaagaaaga
ggggcttata tctcaggatg gcagcagttt agaggctcta ctgcgtactg
420accctctgga gaaacgaggt gctccagatc cccgtgttga tgatgacagc
ctgggcgagt 480ttcctgtcac caacagtcga gcacggaagc ggatccttga
acctgatgac ttcctagatg 540atcttgatga tgaagactat gaagaagata
ctccaaaacg tcggggaaag gggaaatcca 600agagtaaggg tgtgagcagt
gcccggaaga aactggatgc ttccatcctg gaggaccgtg 660ataagcccta
tgcctgtgac atttgtggaa aacgctacaa gaatcgacct tgcctcagtt
720accactatgc ctattcccac ctggctgagg aggagggaga ggacaaagaa
gactctcaac 780cacctactcc tgtttcccag aggtctgagg agcagaaatc
caagaaagga cctgatggat 840tagccctgcc caacaactac tgtgacttct
gcctgggaga ctcaaaaatc aacaagaaga 900cagggcagcc tgaggagcta
gtgtcctgtt ctgactgtgg ccgctcaggg cacccgtcct 960gcctgcagtt
cacccccgtg atgatggcgg ctgtgaagac ctaccgctgg cagtgcatcg
1020agtgcaagtg ctgcaacctc tgcggcactt cggagaatga cgaccagctg
ctcttctgtg 1080atgactgtga ccgtggctac cacatgtact gtctcacctc
atccatgtcg gagcctcctg 1140aaggaagttg gagctgccac ctgtgtctgg
atctgctgaa ggagaaagcg tccatctacc 1200agaaccagag ctcctcctga
tgtgccaccc ggctccccac acacctaagg ctgttgctct 1260cctctacctt
ggttttcata cccctcttct tcttcttctt tcactctggt agttctgccc
1320aactgccttt ggcaacagca cagggaaggt ggcaactctt gactgcctct
ggtcccaagc 1380cctcagggag taaggagcag catgctgccc caggctgatc
tgtgggccca gcttctctct 1440gctctccaag aagtgcattc actctgcttg
ccttgggccc aagtccctgg taatcacagg 1500gttcaaatgg gttcctctaa
gaagtatgag agcagctcac ttgtctcaag cctggcctac 1560ccctcctccc
cctctggtgt ccagagtttt accccagggg tgagccaggc ctaacctttg
1620cttggagcac ctggagtgat cagactgagg tggcacttgc taggaccctt
tcctacccct 1680tgttctgctt cactttgcct ctgccaaagc agtcctgtgt
cttctgtcat gctacatggg 1740gtcctgtgct tgcactgtga tgctctcagg
cacctcctgg ctctgtcctg ttctgcccag 1800tcccacaaag agacaagcag
cttcacctgc ccttcccgtg cttggctggc gcgctcacag 1860gtggtctctg
gcaatccaaa catttcccat cctcagactt ttgagtcttc tgcctccttc
1920cttgttccct ttggttttgt gggggagagg gacaatgtca gggggccctg
ccagaagctt 1980ggggaccaca agaagttgga taatgtgcct gttttttaac
tcgataaaaa tgcctacctc 2040caaaatcccc ttttcgttct tcctggacct
gggcattcag cctcctgccc ttaactgaat 2100caggagcctc tgcctcctac
tgtgtatcct ggctcccagg agagaggatg gtcccctttc 2160cttgcacact
agctagcagc tggtaaagtc ttctttccct gatttttgtt tcctgcttag
2220tggcactgac attaagtagg aggggacagt ccatgccaga acactctgga
atggccttcc 2280tccttggctg tgggcaggcc ctgacttgtt ttctgcaaag
ttgaggcccc tcctcctatc 2340cttagttcct gtatccaaaa cattagtaag
aataaacatt tttacacaga aaaaaaaaaa 2400aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2460aaaaaaaaaa
aaaaaaaaaa aa 2482982PRTCricetulus griseus 9Met Ala Phe Asp Ile Val
Cys Asp Asn Val Gly Arg Asp Trp Lys Arg1 5 10 15Leu Ala Arg Gln Leu
Lys Val Ser Glu Ala Lys Ile Asp Gly Ile Glu 20 25 30Glu Arg Tyr Pro
Arg Ser Leu Ser Glu Gln Val Arg Glu Ala Leu Arg 35 40 45Val Trp Lys
Ile Ala Glu Arg Glu Lys Ala Thr Val Ala Gly Leu Val 50 55 60Lys Ala
Leu Arg Ala Cys Arg Leu Asn Leu Val Ala Asp Leu Val Glu65 70 75
80Gly Arg10253DNACricetulus griseus 10gccaccatgg cctttgacat
tgtatgcgac aatgtgggga gagattggaa gagactggcc 60cgccagctga aagtgtctga
ggccaaaatt gatgggattg aggagaggta cccccgaagc 120ctgagtgagc
aggtaaggga ggctctgaga gtctggaaga ttgccgagag ggagaaagcc
180acggtggctg gactggtaaa ggcacttcgg gcctgccggc tgaacctagt
ggctgacctg 240gtggaaggga ggc 2531151DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
11gatatcggat ccgccaccat ggcctttgac attgtatgcg acaatgtggg g
511234DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 12cccgggctcg agtgcctccc ttccaccagg tcag
341365DNAArtificial SequenceDescription of Artificial Sequence
Synthetic suppression vector insert 13gatcccgtga gcttcagcaa
gcattattca agagataatg cttgctgaag ctcatttttt 60ggaaa
651464DNAArtificial SequenceDescription of Artificial Sequence
Synthetic suppression vector insert 14agcttttcca aaaaatgagc
ttcagcaagc attatctctt gaataatgct tgctgaagct 60cacg
641564DNAArtificial SequenceDescription of Artificial Sequence
Synthetic suppression vector insert 15gatcccgcgg atccttgaac
ctgatttcaa gagaatcagg ttcaaggatc cgcttttttg 60gaaa
641664DNAArtificial SequenceDescription of Artificial Sequence
Synthetic suppression vector insert 16agcttttcca aaaaagcgga
tccttgaacc tgattctctt gaaatcaggt tcaaggatcc 60gcgg
641728DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 17gccgcgagag ctgctgacta cgtcgtgg
281830DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 18gttactgtgg tgagatatga atgggtttgg
301921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 19ccatggaccc attcctggtg c 212022DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
20ttcttccacc aggtcagcca cc 222121DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 21gcccatggct gcctactcct a
212221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 22aatccagcca tcctgatccg t 212330DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
23cagcgggttg ataaagacag gagtggagtg 302421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
24atggcggctg tggtggagaa t 212523DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 25ggagttctgg ttctggtaga tgg
232630DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 26gcctcagtta ccactatgcc cattcccacc
302720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 27tggagctgcg aaaaccaaag 202821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
28aaactcgcct gctgtctcca t 212922DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 29gatatcggat ccgccaccat gg
223022DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 30tgcctccctt ccaccaggtc ag 223121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
31cagcgggttg ataaagacag g 213219DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 32gccagccttg ttttctcgg
193321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 33tggagtagcc cagagcaatt g 213419DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
34tcgacgcttt ttacgccag 193521DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 35agctgagagg gaaattgtgc g
213618DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 36gcaacggaac cgctcatt 18376056DNAArtificial
SequenceDescription of Artificial Sequence Synthetic plasmid
37gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa
ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa
cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
420attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg
taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc
ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg
cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct
ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga
ctcactatag ggagacccaa gctggctagc 900gtttaaactt aagcttggta
ccgagctcgg atccactagt ccagtgtggt ggaattcgcc 960accatgacag
atcttgtagc tgtttgggac gttgcattaa gtgatggagt ccacaagatt
1020gaatttgagc atgggaccac atcaggcaaa cgagttgtgt acgtggatgg
gaaggaagag 1080ataagaaaag aatggatgtt caaattggtg ggcaaagaaa
ccttctgtgt tggagctgcg 1140aaaaccaaag ccaccataaa tatagatgct
gtcagtggtt ttgcttatga gtataccctg 1200gaaatcgatg ggaaaagcct
caagaagtac atggagaaca gatcaaagac caccaacacc 1260tgggtactgc
acttggatgg ccaggactta agagttgttt tggaaaaaga tactatggat
1320gtatggtgca atggtcaaaa aatggagaca gcaggcgagt ttgtagatga
tggaactgaa 1380acacacttca gtgttgggaa ccatgactgt tacataaaag
ctgtcagcag cgggaagaga 1440agagaaggga ttatccacac actcattgtg
gataacaggg agatcccaga gctccctcag 1500tgactgctgg ttagtgggtt
ctgagctgaa gaggagacat caggactttc taatggctgt 1560ggtaattaaa
tgtgttcacg aattctgcag atatccagca cagtggcggc cgctcgagtc
1620tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
gccagccatc 1680tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
ggtgccactc ccactgtcct 1740ttcctaataa aatgaggaaa ttgcatcgca
ttgtctgagt aggtgtcatt ctattctggg 1800gggtggggtg gggcaggaca
gcaaggggga ggattgggaa gacaatagca ggcatgctgg 1860ggatgcggtg
ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
1920tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
cgcgcagcgt 1980gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
gctttcttcc cttcctttct 2040cgccacgttc gccggctttc cccgtcaagc
tctaaatcgg gggctccctt tagggttccg 2100atttagtgct ttacggcacc
tcgaccccaa aaaacttgat tagggtgatg gttcacgtag 2160tgggccatcg
ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
2220tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
attcttttga 2280tttataaggg attttgccga tttcggccta ttggttaaaa
aatgagctga tttaacaaaa 2340atttaacgcg aattaattct gtggaatgtg
tgtcagttag ggtgtggaaa gtccccaggc 2400tccccagcag gcagaagtat
gcaaagcatg catctcaatt agtcagcaac caggtgtgga 2460aagtccccag
gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
2520accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
ttccgcccat 2580tctccgcccc atggctgact aatttttttt atttatgcag
aggccgaggc cgcctctgcc 2640tctgagctat tccagaagta gtgaggaggc
ttttttggag gcctaggctt ttgcaaaaag 2700ctcccgggag cttgtatatc
cattttcgga tctgatcaag agacaggatg aggatcgttt 2760cgcatgattg
aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
2820ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
gttccggctg 2880tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
tgtccggtgc cctgaatgaa 2940ctgcaggacg aggcagcgcg gctatcgtgg
ctggccacga cgggcgttcc ttgcgcagct 3000gtgctcgacg ttgtcactga
agcgggaagg gactggctgc tattgggcga agtgccgggg 3060caggatctcc
tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
3120atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
agcgaaacat 3180cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
tcgatcagga tgatctggac 3240gaagagcatc aggggctcgc gccagccgaa
ctgttcgcca ggctcaaggc gcgcatgccc 3300gacggcgagg atctcgtcgt
gacccatggc gatgcctgct tgccgaatat catggtggaa 3360aatggccgct
tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
3420gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
ggctgaccgc 3480ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
gcatcgcctt ctatcgcctt 3540cttgacgagt tcttctgagc gggactctgg
ggttcgaaat gaccgaccaa gcgacgccca 3600acctgccatc acgagatttc
gattccaccg ccgccttcta tgaaaggttg ggcttcggaa 3660tcgttttccg
ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
3720tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
aatagcatca 3780caaatttcac aaataaagca tttttttcac tgcattctag
ttgtggtttg tccaaactca 3840tcaatgtatc ttatcatgtc tgtataccgt
cgacctctag ctagagcttg gcgtaatcat 3900ggtcatagct gtttcctgtg
tgaaattgtt atccgctcac aattccacac aacatacgag 3960ccggaagcat
aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
4020cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
cattaatgaa 4080tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
ctcttccgct tcctcgctca 4140ctgactcgct gcgctcggtc gttcggctgc
ggcgagcggt atcagctcac tcaaaggcgg 4200taatacggtt atccacagaa
tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 4260agcaaaaggc
caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
4320cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
ccgacaggac 4380tataaagata ccaggcgttt ccccctggaa gctccctcgt
gcgctctcct gttccgaccc 4440tgccgcttac cggatacctg tccgcctttc
tcccttcggg aagcgtggcg ctttctcata 4500gctcacgctg taggtatctc
agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 4560acgaaccccc
cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
4620acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
attagcagag 4680cgaggtatgt aggcggtgct acagagttct tgaagtggtg
gcctaactac ggctacacta 4740gaagaacagt atttggtatc tgcgctctgc
tgaagccagt taccttcgga aaaagagttg 4800gtagctcttg atccggcaaa
caaaccaccg ctggtagcgg tttttttgtt tgcaagcagc 4860agattacgcg
cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
4920acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
tcaaaaagga 4980tcttcaccta gatcctttta aattaaaaat gaagttttaa
atcaatctaa agtatatatg 5040agtaaacttg gtctgacagt taccaatgct
taatcagtga ggcacctatc tcagcgatct 5100gtctatttcg ttcatccata
gttgcctgac tccccgtcgt gtagataact acgatacggg 5160agggcttacc
atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
5220cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
ggtcctgcaa 5280ctttatccgc ctccatccag tctattaatt gttgccggga
agctagagta agtagttcgc 5340cagttaatag tttgcgcaac gttgttgcca
ttgctacagg catcgtggtg tcacgctcgt 5400cgtttggtat ggcttcattc
agctccggtt cccaacgatc aaggcgagtt acatgatccc 5460ccatgttgtg
caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
5520tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
actgtcatgc 5580catccgtaag atgcttttct gtgactggtg agtactcaac
caagtcattc tgagaatagt 5640gtatgcggcg accgagttgc tcttgcccgg
cgtcaatacg ggataatacc gcgccacata 5700gcagaacttt aaaagtgctc
atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 5760tcttaccgct
gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
5820catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
aatgccgcaa 5880aaaagggaat aagggcgaca cggaaatgtt gaatactcat
actcttcctt tttcaatatt 5940attgaagcat ttatcagggt tattgtctca
tgagcggata catatttgaa tgtatttaga 6000aaaataaaca aataggggtt
ccgcgcacat ttccccgaaa agtgccacct gacgtc 6056385632DNAArtificial
SequenceDescription of Artificial Sequence Synthetic plasmid
38gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa
ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa
cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
420attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg
taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc
ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg
cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct
ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga
ctcactatag ggagacccaa gctggctagc 900gtttaaactt aagcttggta
ccgagctcgg atccgccacc atggcctttg acattgtatg 960cgacaatgtg
gggagagatt ggaagagact ggcccgccag ctgaaagtgt ctgaggccaa
1020aattgatggg attgaggaga ggtacccccg aagcctgagt gagcaggtaa
gggaggctct 1080gagagtctgg aagattgccg agagggagaa agccacggtg
gctggactgg taaaggcact 1140tcgggcctgc cggctgaacc tagtggctga
cctggtggaa gggaggcact cgagtctaga 1200gggcccgttt aaacccgctg
atcagcctcg actgtgcctt ctagttgcca gccatctgtt 1260gtttgcccct
cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
1320taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
tctggggggt 1380ggggtggggc aggacagcaa gggggaggat tgggaagaca
atagcaggca tgctggggat 1440gcggtgggct ctatggcttc tgaggcggaa
agaaccagct ggggctctag ggggtatccc 1500cacgcgccct gtagcggcgc
attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 1560gctacacttg
ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
1620acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
gttccgattt 1680agtgctttac ggcacctcga ccccaaaaaa cttgattagg
gtgatggttc acgtagtggg 1740ccatcgccct gatagacggt ttttcgccct
ttgacgttgg agtccacgtt ctttaatagt 1800ggactcttgt tccaaactgg
aacaacactc aaccctatct cggtctattc ttttgattta 1860taagggattt
tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
1920aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
ccaggctccc 1980cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
agcaaccagg tgtggaaagt 2040ccccaggctc cccagcaggc agaagtatgc
aaagcatgca tctcaattag tcagcaacca 2100tagtcccgcc cctaactccg
cccatcccgc ccctaactcc gcccagttcc gcccattctc 2160cgccccatgg
ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
2220agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
aaaaagctcc 2280cgggagcttg tatatccatt ttcggatctg atcaagagac
aggatgagga tcgtttcgca 2340tgattgaaca agatggattg cacgcaggtt
ctccggccgc ttgggtggag aggctattcg 2400gctatgactg ggcacaacag
acaatcggct gctctgatgc cgccgtgttc cggctgtcag 2460cgcaggggcg
cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
2520aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
gcagctgtgc 2580tcgacgttgt cactgaagcg ggaagggact ggctgctatt
gggcgaagtg ccggggcagg 2640atctcctgtc atctcacctt gctcctgccg
agaaagtatc catcatggct gatgcaatgc 2700ggcggctgca tacgcttgat
ccggctacct gcccattcga ccaccaagcg aaacatcgca 2760tcgagcgagc
acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
2820agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc
atgcccgacg 2880gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
gaatatcatg gtggaaaatg 2940gccgcttttc tggattcatc gactgtggcc
ggctgggtgt ggcggaccgc tatcaggaca 3000tagcgttggc tacccgtgat
attgctgaag agcttggcgg cgaatgggct gaccgcttcc 3060tcgtgcttta
cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
3120acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
cgcccaacct 3180gccatcacga gatttcgatt ccaccgccgc cttctatgaa
aggttgggct tcggaatcgt 3240tttccgggac gccggctgga tgatcctcca
gcgcggggat ctcatgctgg agttcttcgc 3300ccaccccaac ttgtttattg
cagcttataa tggttacaaa taaagcaata gcatcacaaa 3360tttcacaaat
aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
3420tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
aatcatggtc 3480atagctgttt cctgtgtgaa attgttatcc gctcacaatt
ccacacaaca tacgagccgg 3540aagcataaag tgtaaagcct ggggtgccta
atgagtgagc taactcacat taattgcgtt 3600gcgctcactg cccgctttcc
agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg 3660ccaacgcgcg
gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
3720ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
aggcggtaat 3780acggttatcc acagaatcag gggataacgc aggaaagaac
atgtgagcaa aaggccagca 3840aaaggccagg aaccgtaaaa aggccgcgtt
gctggcgttt ttccataggc tccgcccccc 3900tgacgagcat cacaaaaatc
gacgctcaag tcagaggtgg cgaaacccga caggactata 3960aagataccag
gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
4020gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
ctcatagctc 4080acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
aagctgggct gtgtgcacga 4140accccccgtt cagcccgacc gctgcgcctt
atccggtaac tatcgtcttg agtccaaccc 4200ggtaagacac gacttatcgc
cactggcagc agccactggt aacaggatta gcagagcgag 4260gtatgtaggc
ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
4320aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
gagttggtag 4380ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtttt
tttgtttgca agcagcagat 4440tacgcgcaga aaaaaaggat ctcaagaaga
tcctttgatc ttttctacgg ggtctgacgc 4500tcagtggaac gaaaactcac
gttaagggat tttggtcatg agattatcaa aaaggatctt 4560cacctagatc
cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
4620aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
cgatctgtct 4680atttcgttca tccatagttg cctgactccc cgtcgtgtag
ataactacga tacgggaggg 4740cttaccatct ggccccagtg ctgcaatgat
accgcgagac ccacgctcac cggctccaga 4800tttatcagca ataaaccagc
cagccggaag ggccgagcgc agaagtggtc ctgcaacttt 4860atccgcctcc
atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
4920taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
gctcgtcgtt 4980tggtatggct tcattcagct ccggttccca acgatcaagg
cgagttacat gatcccccat 5040gttgtgcaaa aaagcggtta gctccttcgg
tcctccgatc gttgtcagaa gtaagttggc 5100cgcagtgtta tcactcatgg
ttatggcagc actgcataat tctcttactg tcatgccatc 5160cgtaagatgc
ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
5220gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
cacatagcag 5280aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
cgaaaactct caaggatctt 5340accgctgttg agatccagtt cgatgtaacc
cactcgtgca cccaactgat cttcagcatc 5400ttttactttc accagcgttt
ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa 5460gggaataagg
gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
5520aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
tttagaaaaa 5580taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
ccacctgacg tc 5632394758DNAArtificial SequenceDescription of
Artificial Sequence Synthetic plasmid 39ctaaattgta agcgttaata
ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60attttttaac caataggccg
aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120gatagggttg
agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
180caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
aaccatcacc 240ctaatcaagt tttttggggt cgaggtgccg taaagcacta
aatcggaacc ctaaagggag 300cccccgattt agagcttgac ggggaaagcc
ggcgaacgtg gcgagaaagg aagggaagaa 360agcgaaagga gcgggcgcta
gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 420cacacccgcc
gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
480caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
tggcgaaagg 540gggatgtgct gcaaggcgat taagttgggt aacgccaggg
ttttcccagt cacgacgttg 600taaaacgacg gccagtgagc gcgcgtaata
cgactcacta tagggcgaat tggagctcca 660ccgcggtggc ggccgctcta
gaactagtgg atcccccggg ctgcatcccc gatccttatc 720gctatcgatt
cacacaaaaa accaacacac agatgtaatg aaaataaaga tattttattg
780cggccatcgt gatggcaggt tgggcgtcgc ttggtcggtc atttcgaacc
ccagagtccc 840gctcagaaga actcgtcaag aaggcgatag aaggcgatgc
gctgcgaatc gggagcggcg 900ataccgtaaa gcacgaggaa gcggtcagcc
cattcgccgc caagctcttc agcaatatca 960cgggtagcca acgctatgtc
ctgatagcgg tccgccacac ccagccggcc acagtcgatg 1020aatccagaaa
agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc
1080acgacgagat cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag
ttcggctggc 1140gcgagcccct gatgctcttc gtccagatca tcctgatcga
caagaccggc ttccatccga 1200gtacgtgctc gctcgatgcg atgtttcgct
tggtggtcga atgggcaggt agccggatca 1260agcgtatgca gccgccgcat
tgcatcagcc atgatggata ctttctcggc aggagcaagg 1320tgagatgaca
ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct
1380tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag
ccacgatagc 1440cgcgctgcct cgtcctgcag ttcattcagg gcaccggaca
ggtcggtctt gacaaaaaga 1500accgggcgcc cctgcgctga cagccggaac
acggcggcat cagagcagcc gattgtctgt 1560tgtgcccagt catagccgaa
tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat 1620ccatcttgtt
caatcatggt ggctctagcc ttaagttcga gactgttgtg tcagaagaat
1680caagctgatc tgagtccggt aagctagctt gggctgcagg tcgaaaggcc
cggagatgag 1740gaagaggaga acagcgcggc agacgtgcgc ttttgaagcg
tgcagaatgc cgggcctccg 1800gaggaccttc gggcgcccgc cccgcccctg
agcccgcccc tgagcccgcc cccggaccca 1860ccccttccca gcctctgagc
ccagaaagcg aaggagcaaa gctgctattg gccgctgccc 1920caaaggccta
cccgcttcca ttgctcagcg gtgctgtcca tctgcacgag actagtgaga
1980cgtgctactt ccatttgtca cgtcctgcac gacgcgagct gcggggcggg
ggggaacttc 2040ctgactaggg gaggagtaga aggtggcgcg aaggggccac
caaagaacgg agccggttgg 2100cgcctaccgg ccggtggatg tggaatgtgt
gcgaggccag aggccacttg tgtagcgcca 2160agtgcccagc ggggctgcta
aagcgcatgc tccagactgc cttgggaaaa gcgcctcccc 2220tacccggtag
aattcgaacg ctgacgtcat caacccgctc caaggaatcg cgggcccagt
2280gtcactaggc gggaacaccc agcgcgcgtg cgccctggca ggaagatggc
tgtgagggac 2340aggggagtgg cgccctgcaa tatttgcatg tcgctatgtg
ttctgggaaa tcaccataaa 2400cgtgaaatgt ctttggattt gggaatctta
taagttctgt atgagaccac agatcccgtg 2460agcttcagca agcattattc
aagagataat gcttgctgaa gctcattttt tggaaaagct 2520tatcgatacc
gtcgacctcg agggggggcc cggtacccag
cttttgttcc ctttagtgag 2580ggttaattgc gcgcttggcg taatcatggt
catagctgtt tcctgtgtga aattgttatc 2640cgctcacaat tccacacaac
atacgagccg gaagcataaa gtgtaaagcc tggggtgcct 2700aatgagtgag
ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
2760acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
ggtttgcgta 2820ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
ctcggtcgtt cggctgcggc 2880gagcggtatc agctcactca aaggcggtaa
tacggttatc cacagaatca ggggataacg 2940caggaaagaa catgtgagca
aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 3000tgctggcgtt
tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
3060gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
cctggaagct 3120ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
atacctgtcc gcctttctcc 3180cttcgggaag cgtggcgctt tctcatagct
cacgctgtag gtatctcagt tcggtgtagg 3240tcgttcgctc caagctgggc
tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 3300tatccggtaa
ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
3360cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
gagttcttga 3420agtggtggcc taactacggc tacactagaa ggacagtatt
tggtatctgc gctctgctga 3480agccagttac cttcggaaaa agagttggta
gctcttgatc cggcaaacaa accaccgctg 3540gtagcggtgg tttttttgtt
tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 3600aagatccttt
gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
3660ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
aattaaaaat 3720gaagttttaa atcaatctaa agtatatatg agtaaacttg
gtctgacagt taccaatgct 3780taatcagtga ggcacctatc tcagcgatct
gtctatttcg ttcatccata gttgcctgac 3840tccccgtcgt gtagataact
acgatacggg agggcttacc atctggcccc agtgctgcaa 3900tgataccgcg
agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
3960gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
tctattaatt 4020gttgccggga agctagagta agtagttcgc cagttaatag
tttgcgcaac gttgttgcca 4080ttgctacagg catcgtggtg tcacgctcgt
cgtttggtat ggcttcattc agctccggtt 4140cccaacgatc aaggcgagtt
acatgatccc ccatgttgtg caaaaaagcg gttagctcct 4200tcggtcctcc
gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
4260cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
gtgactggtg 4320agtactcaac caagtcattc tgagaatagt gtatgcggcg
accgagttgc tcttgcccgg 4380cgtcaatacg ggataatacc gcgccacata
gcagaacttt aaaagtgctc atcattggaa 4440aacgttcttc ggggcgaaaa
ctctcaagga tcttaccgct gttgagatcc agttcgatgt 4500aacccactcg
tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
4560gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
cggaaatgtt 4620gaatactcat actcttcctt tttcaatatt attgaagcat
ttatcagggt tattgtctca 4680tgagcggata catatttgaa tgtatttaga
aaaataaaca aataggggtt ccgcgcacat 4740ttccccgaaa agtgccac
4758404756DNAArtificial SequenceDescription of Artificial Sequence
Synthetic plasmid 40ctaaattgta agcgttaata ttttgttaaa attcgcgtta
aatttttgtt aaatcagctc 60attttttaac caataggccg aaatcggcaa aatcccttat
aaatcaaaag aatagaccga 120gatagggttg agtgttgttc cagtttggaa
caagagtcca ctattaaaga acgtggactc 180caacgtcaaa gggcgaaaaa
ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240ctaatcaagt
tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
300cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
aagggaagaa 360agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
gtcacgctgc gcgtaaccac 420cacacccgcc gcgcttaatg cgccgctaca
gggcgcgtcc cattcgccat tcaggctgcg 480caactgttgg gaagggcgat
cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540gggatgtgct
gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
600taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat
tggagctcca 660ccgcggtggc ggccgctcta gaactagtgg atcccccggg
ctgcatcccc gatccttatc 720gctatcgatt cacacaaaaa accaacacac
agatgtaatg aaaataaaga tattttattg 780cggccatcgt gatggcaggt
tgggcgtcgc ttggtcggtc atttcgaacc ccagagtccc 840gctcagaaga
actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg
900ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc caagctcttc
agcaatatca 960cgggtagcca acgctatgtc ctgatagcgg tccgccacac
ccagccggcc acagtcgatg 1020aatccagaaa agcggccatt ttccaccatg
atattcggca agcaggcatc gccatgggtc 1080acgacgagat cctcgccgtc
gggcatgcgc gccttgagcc tggcgaacag ttcggctggc 1140gcgagcccct
gatgctcttc gtccagatca tcctgatcga caagaccggc ttccatccga
1200gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga atgggcaggt
agccggatca 1260agcgtatgca gccgccgcat tgcatcagcc atgatggata
ctttctcggc aggagcaagg 1320tgagatgaca ggagatcctg ccccggcact
tcgcccaata gcagccagtc ccttcccgct 1380tcagtgacaa cgtcgagcac
agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc 1440cgcgctgcct
cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga
1500accgggcgcc cctgcgctga cagccggaac acggcggcat cagagcagcc
gattgtctgt 1560tgtgcccagt catagccgaa tagcctctcc acccaagcgg
ccggagaacc tgcgtgcaat 1620ccatcttgtt caatcatggt ggctctagcc
ttaagttcga gactgttgtg tcagaagaat 1680caagctgatc tgagtccggt
aagctagctt gggctgcagg tcgaaaggcc cggagatgag 1740gaagaggaga
acagcgcggc agacgtgcgc ttttgaagcg tgcagaatgc cgggcctccg
1800gaggaccttc gggcgcccgc cccgcccctg agcccgcccc tgagcccgcc
cccggaccca 1860ccccttccca gcctctgagc ccagaaagcg aaggagcaaa
gctgctattg gccgctgccc 1920caaaggccta cccgcttcca ttgctcagcg
gtgctgtcca tctgcacgag actagtgaga 1980cgtgctactt ccatttgtca
cgtcctgcac gacgcgagct gcggggcggg ggggaacttc 2040ctgactaggg
gaggagtaga aggtggcgcg aaggggccac caaagaacgg agccggttgg
2100cgcctaccgg ccggtggatg tggaatgtgt gcgaggccag aggccacttg
tgtagcgcca 2160agtgcccagc ggggctgcta aagcgcatgc tccagactgc
cttgggaaaa gcgcctcccc 2220tacccggtag aattcgaacg ctgacgtcat
caacccgctc caaggaatcg cgggcccagt 2280gtcactaggc gggaacaccc
agcgcgcgtg cgccctggca ggaagatggc tgtgagggac 2340aggggagtgg
cgccctgcaa tatttgcatg tcgctatgtg ttctgggaaa tcaccataaa
2400cgtgaaatgt ctttggattt gggaatctta taagttctgt atgagaccac
agatcccgcg 2460gatccttgaa cctgatttca agagaatcag gttcaggatc
cgcttttttg gaaaagctta 2520tcgataccgt cgacctcgag ggggggcccg
gtacccagct tttgttccct ttagtgaggg 2580ttaattgcgc gcttggcgta
atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 2640ctcacaattc
cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
2700tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
gtcgggaaac 2760ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
ggagaggcgg tttgcgtatt 2820gggcgctctt ccgcttcctc gctcactgac
tcgctgcgct cggtcgttcg gctgcggcga 2880gcggtatcag ctcactcaaa
ggcggtaata cggttatcca cagaatcagg ggataacgca 2940ggaaagaaca
tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
3000ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
acgctcaagt 3060cagaggtggc gaaacccgac aggactataa agataccagg
cgtttccccc tggaagctcc 3120ctcgtgcgct ctcctgttcc gaccctgccg
cttaccggat acctgtccgc ctttctccct 3180tcgggaagcg tggcgctttc
tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 3240gttcgctcca
agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
3300tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
actggcagca 3360gccactggta acaggattag cagagcgagg tatgtaggcg
gtgctacaga gttcttgaag 3420tggtggccta actacggcta cactagaagg
acagtatttg gtatctgcgc tctgctgaag 3480ccagttacct tcggaaaaag
agttggtagc tcttgatccg gcaaacaaac caccgctggt 3540agcggtggtt
tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
3600gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
acgttaaggg 3660attttggtca tgagattatc aaaaaggatc ttcacctaga
tccttttaaa ttaaaaatga 3720agttttaaat caatctaaag tatatatgag
taaacttggt ctgacagtta ccaatgctta 3780atcagtgagg cacctatctc
agcgatctgt ctatttcgtt catccatagt tgcctgactc 3840cccgtcgtgt
agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
3900ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
gccagccgga 3960agggccgagc gcagaagtgg tcctgcaact ttatccgcct
ccatccagtc tattaattgt 4020tgccgggaag ctagagtaag tagttcgcca
gttaatagtt tgcgcaacgt tgttgccatt 4080gctacaggca tcgtggtgtc
acgctcgtcg tttggtatgg cttcattcag ctccggttcc 4140caacgatcaa
ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
4200ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
ggttatggca 4260gcactgcata attctcttac tgtcatgcca tccgtaagat
gcttttctgt gactggtgag 4320tactcaacca agtcattctg agaatagtgt
atgcggcgac cgagttgctc ttgcccggcg 4380tcaatacggg ataataccgc
gccacatagc agaactttaa aagtgctcat cattggaaaa 4440cgttcttcgg
ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
4500cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
ttctgggtga 4560gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
gggcgacacg gaaatgttga 4620atactcatac tcttcctttt tcaatattat
tgaagcattt atcagggtta ttgtctcatg 4680agcggataca tatttgaatg
tatttagaaa aataaacaaa taggggttcc gcgcacattt 4740ccccgaaaag tgccac
47564178PRTCricetulus griseus 41Phe Asp Ile Val Cys Asp Asn Val Gly
Arg Asp Trp Lys Arg Leu Ala1 5 10 15Arg Gln Leu Lys Val Ser Glu Ala
Lys Ile Asp Gly Ile Glu Glu Arg 20 25 30Tyr Pro Arg Ser Leu Ser Glu
Gln Val Arg Glu Ala Leu Arg Val Trp 35 40 45Lys Ile Ala Glu Arg Glu
Lys Ala Thr Val Ala Gly Leu Val Lys Ala 50 55 60Leu Arg Ala Cys Arg
Leu Asn Leu Val Ala Asp Leu Val Glu65 70 7542234DNACricetulus
griseus 42tttgacattg tatgcgacaa tgtggggaga gattggaaga gactggcccg
ccagctgaaa 60gtgtctgagg ccaaaattga tgggattgag gagaggtacc cccgaagcct
gagtgagcag 120gtaagggagg ctctgagagt ctggaagatt gccgagaggg
agaaagccac ggtggctgga 180ctggtaaagg cacttcgggc ctgccggctg
aacctggtgg ctgacctggt ggaa 2344329DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 43gaattcgcca ccatgacaga
tcttgtagc 294426DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 44gaattcgtga acacatttaa ttacca
2645634DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 45gaattcgcca ccatgacaga tcttgtagct
gtttgggacg ttgcattaag tgatggagtc 60cacaagattg aatttgagca tgggaccaca
tcaggcaaac gagttgtgta cgtggatggg 120aaggaagaga taagaaaaga
atggatgttc aaattggtgg gcaaagaaac cttctgtgtt 180ggagctgcga
aaaccaaagc caccataaat atagatgctg tcagtggttt tgcttatgag
240tataccctgg aaatcgatgg gaaaagcctc aagaagtaca tggagaacag
atcaaagacc 300accaacacct gggtactgca cttggatggc caggacttaa
gagttgtttt ggaaaaagat 360actatggatg tatggtgcaa tggtcaaaaa
atggagacag caggcgagtt tgtagatgat 420ggaactgaaa cacacttcag
tgttgggaac catgactgtt acataaaagc tgtcagcagc 480gggaagagaa
gagaagggat tatccacaca ctcattgtgg ataacaggga gatcccagag
540ctccctcagt gactgctggt tagtgggttc tgagctgaag aggagacatc
aggactttct 600aatggctgtg gtaattaaat gtgttcacga attc
63446266DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 46ggatccgcca ccatggcctt tgacattgta
tgcgacaatg tggggagaga ttggaagaga 60ctggcccgcc agctgaaagt gtctgaggcc
aaaattgatg ggattgagga gaggtacccc 120cgaagcctga gtgagcaggt
aagggaggct ctgagagtct ggaagattgc cgagagggag 180aaagccacgg
tggctggact ggtaaaggca cttcgggcct gccggctgaa cctagtggct
240gacctggtgg aagggaggca ctcgag 2664770DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 47gatcccgtga gcttcagcaa gcattattca agagataatg
cttgctgaag ctcatttttt 60ggaaaagctt 704868DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 48gatcccgcgg atccttgaac ctgatttcaa gagaatcagg
ttcaggatcc gcttttttgg 60aaaagctt 68
* * * * *