U.S. patent application number 09/997905 was filed with the patent office on 2003-04-17 for control of gene expression.
Invention is credited to Graham, Michael Wayne, Rice, Robert Norman.
Application Number | 20030074684 09/997905 |
Document ID | / |
Family ID | 25645735 |
Filed Date | 2003-04-17 |
United States Patent
Application |
20030074684 |
Kind Code |
A1 |
Graham, Michael Wayne ; et
al. |
April 17, 2003 |
Control of gene expression
Abstract
The present invention relates generally to synthetic genes for
modifying endogenous gene expression in a cell, tissue or organ of
a transgenic organism, in particular a transgenic animal or plant.
More particularly, the present invention provides novel synthetic
genes and genetic constructs which are capable of repressing
delaying or otherwise reducing the expression of an endogenous gene
or a target gene in an organism when introduced thereto.
Inventors: |
Graham, Michael Wayne; (St.
Lucia, AU) ; Rice, Robert Norman; (Sinnamon Park,
AU) |
Correspondence
Address: |
Leopold Presser
Scully, Scott, Murphy & Presser
400 Garden City Plaza
Garden City
NY
11530
US
|
Family ID: |
25645735 |
Appl. No.: |
09/997905 |
Filed: |
November 30, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09997905 |
Nov 30, 2001 |
|
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09100812 |
Jun 19, 1998 |
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Current U.S.
Class: |
800/278 ;
800/288 |
Current CPC
Class: |
C12N 15/85 20130101;
C12N 2830/60 20130101; C12N 2830/42 20130101; C12N 15/63 20130101;
C12N 9/1051 20130101; C12N 2830/00 20130101; C12N 15/8216 20130101;
C12N 15/111 20130101; C12N 15/8283 20130101; A61P 31/12 20180101;
C12N 2330/51 20130101; C12N 2830/15 20130101; C12N 2310/111
20130101; A01K 2217/05 20130101; C12N 9/503 20130101; C12N 15/1131
20130101; C12N 2330/50 20130101; C12N 2830/002 20130101; C12N
2830/55 20130101; C12N 9/127 20130101; C12N 2310/14 20130101; C12N
2320/10 20130101; A61P 43/00 20180101; A61K 48/00 20130101; C12N
15/113 20130101; C12N 15/69 20130101; C12N 2800/108 20130101; C12N
2840/20 20130101; A61P 31/04 20180101; C12N 15/8218 20130101; C12N
2320/30 20130101; C12N 2330/30 20130101; C12N 2310/531 20130101;
C12N 2830/38 20130101 |
Class at
Publication: |
800/278 ;
800/288 |
International
Class: |
A01H 001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 20, 1998 |
AU |
PP2492 |
Mar 20, 1998 |
AU |
PP2499 |
Claims
1. A method of modulating (as defined) the expression of a target
gene (as defined) in a plant cell, tissue or organ comprising (a)
providing one or more dispersed or foreign nucleic acid molecules
(as defined) which include multiple copies (as defined) of a
nucleotide sequence, each of which is substantially identical (as
defined) to or complementary to the nucleotide sequence of the
target gene or a region thereof, and (b) transfecting the plant
cell, tissue or organ with the dispersed or foreign nucleic acid
molecules for a time and under conditions sufficient for expression
of at least two of the multiple copies.
2. The method of claim 1 wherein either (a) at least two of the
copies are in tandem and the same orientation, or (b) at least one
of the copies is in the sense orientation and one is in the
antisense orientation and these two copies are located relative to
each other such that the two copies may form a hairpin RNA
structure when transcribed.
3. A method according to claim 2 wherein the modulating is at least
partly post-transcriptional.
4. The method according to claim 2 wherein at least one of the
copies is a reverse complement of another of the copies.
5. The method according to claim 2 wherein the copies include both
direct and inverted repeats of the target gene sequence or a region
thereof or complementary thereto.
6. The method of claim 1 wherein at least two of the copies are
separated by a stuffer fragment which comprises a sequence of
nucleotides, or a homologue, analogue or derivative thereof.
7. A method according to claim 6 wherein the modulating is at least
partly post-transcriptional.
8. The method according to claim 2, wherein the number of copies is
two.
9. The method according to claim 2, wherein the number of copies is
four.
10. The method according to claim 2, wherein the number of copies
is ten.
11. The method according to claim 1 wherein at least one of the
copies is capable of encoding an amino acid sequence encoded by the
target gene.
12. The method according to claim 1 wherein the plant is a tobacco
plant.
13. The method according to claim 1 wherein the target gene is a
gene which is contained within the genome of the cell, tissue or
organ.
14. The method according to claim I wherein the target gene is
derived from the genome of a pathogen of the cell, tissue or organ
or an organism comprising said cell, tissue or organ.
15. The method according to claim 14 wherein the pathogen is a
virus.
16. The method according to claim 15 wherein the virus is a plant
pathogen.
17. The method according to claim 15 wherein the virus is PVY.
18. A method of modulating (as defined) the expression of a target
gene (as defined) in a plant cell, tissue or organ, said method
comprising: (v) selecting one or more dispersed nucleic acid
molecules or foreign nucleic acid molecules (as defined) which
comprise multiple copies (as defined) of a nucleotide sequence
which is substantially identical (as defined) to the nucleotide
sequence of said target gene or a region thereof or which is
complementary thereto; (vi) producing a synthetic gone comprising
said dispersed nucleic acid molecules or foreign nucleic acid
molecules operably connected to a promoter sequence operable in
said cell, tissue or organ; (vii) introducing said synthetic gene
to said call, tissue or organ; and (viii) expressing said synthetic
gene in said cell, tissue or organ for a time and under conditions
sufficient for expression of at least two of the copies.
19. The method of claim 18 wherein either (a) at least two of the
copies are in tandem and the same orientation, or (b) at least one
of the copies is in the sense orientation and one is in the
antisense orientation and these two copies are located relative to
each other such that the two copies may form a hairpin RNA
structure when transcribed.
20. A method according to claim 19 wherein the modulating is at
least partly post-transcriptional.
21. The method of claim 20 wherein at least two of the copies are
separated by a stuffer fragment which comprises a sequence of
nucleotides, or a homologues, analogues or derivatives thereof.
22. A method of conferring resistance or immunity to a viral
pathogen upon a plant cell, tissue, organ or whole organism,
comprising: (v) selecting one or more dispersed nucleic acid
molecules or foreign nucleic acid molecules (as defined) which
comprise multiple copies (as defined) substantially identical to a
nucleotide sequence derived from the viral pathogen or a
complementary sequence thereto or a region thereof; (vi) producing
a synthetic gene comprising said dispersed nucleic acid molecules
or foreign nucleic acid molecules operably connected to a promoter
sequence operable in said cell, tissue, organ or whole organism;
(vii) introducing said synthetic gene to said cell, tissue, organ
or whole organism; and (viii) expressing said synthetic gene in
said cell, tissue or organ for a time and under conditions
sufficient for expression of at least two of the copies.
23. The method according to claim 22, wherein the dispersed nucleic
acid molecules or foreign nucleic acid molecules comprise multiple
copies of nucleotide sequence encoding a viral replicase,
polymerase, coat protein or uncoating gene.
24. The method according to claim 23 wherein the dispersed nucleic
acid molecules or foreign nucleic acid molecules comprise multiple
copies of nucleotide sequence encoding a viral polymerase or viral
coat protein.
25. A genetic construct comprising multiple structural gene
sequences (as defined), wherein each of said structural gene
sequences is substantially identical (as defined) to a target gene
(as defined) in a plant cell, and wherein said multiple structural
gene sequences are placed operably under the control of a single
promoter sequence which is operable in said cell, wherein at least
one of said structural gene sequences is placed operably in the
sense orientation under the control of said promoter sequence and
at least one other of said structural gene sequences is placed
operably in the antisense orientation under the control of said
promoter sequence, and wherein at least one structural gene
sequence that is placed in the sense orientation relative to said
promoter and at least one structural gene sequence that is placed
in the antisense orientation relative to said promoter are spaced
from each other by a nucleic acid stuffer fragment.
26. The genetic construct of claim 25, wherein the transcription
product of at least one structural gene sequence that is in the
sense orientation relative to the promoter, said stuffer fragment
and at least one structural gene sequence that is in the antisense
orientation relative to the promoter may form a hairpin RNA
structure when transcribed.
27. The genetic construct of claim 26 further comprising at least
one of an origin of replication or a selectable marker gene.
28. The genetic construct according to claim 25 selected from the
list comprising plasmid pSP72.PVYx2; plasmid pBC.PVYx2; plasmid
pBC.PVYx3; plasmid pBC.PVYx4; plasmid pART27.PVYx2; plasmid
pART27.PVYx3; plasmid pART27.PVYx4; plasmid pBC.PVY.LNYV.YVPA;
plasmid pBC.PVY.LNYV.YVP; plasmid pBC.PVY.LNYV.PVY; plasmid
pART27.PVY.LNYV.PVY; plasmid pART27.PVY.LNYV.YVPA; plasmid
pART27.PVY.LNYV.YVP; plasmid pART27.35S.PVY.SCBV.YVP; plasmid
pART27.35S.PVYx3.SCBV.YVPx3; plasmid pART27.PVYx3.LNYV.YVPx3; and
plasmid pART27.PVYx10.
29. Use of the genetic construct according to claim 28 to confer
immunity or resistance against PVY upon a plant cell, tissue, organ
or whole plant.
30. Use according to claim 29, wherein the plant is tobacco.
31. A genetic construct which is capable of modulating (as defined)
the expression of a target gene in a plant cell, which is
transfected with said construct, wherein said construct comprises
multiple copies (as defined) of a structural gene sequence (as
defined), wherein each copy comprises a nucleotide sequence which
is substantially identical to said target gene or a derivative of
said target gene and wherein said multiple copies are placed
operably under the control of a single promoter sequence which is
operable in said cell, wherein at least two of said copies are
placed operably in the sense orientation under the control of said
promoter sequence.
32. A genetic construct which is capable of modulating (as defined)
the expression of a target gene (as defined) in a plant cell, which
is transfected with said construct, wherein said construct
comprises multiple structural gene sequences (as defined) wherein
each of said structural gene sequences is separately placed under
the control of a promoter which is operable in said cell, and
wherein each of said structural gene sequences comprises a
nucleotide sequence which is substantially identical to said target
gene or a derivative of said target gene, wherein at least one of
said structural gene sequences is placed operably in the sense
orientation under the control of an individual promoter sequence.
Description
[0001] This application is a continuation-in-part of U.S.
Application Ser. No. 09/100,812.
FIELD OF THE INVENTION
[0002] The present invention relates generally to a method of
modifying gene expression and to synthetic genes for modifying
endogenous gene expression in a call, tissue or organ of a
transgenic organism, in particular a transgenic animal or plant.
More particularly, the present invention utilises recombinant DNA
technology to post-transcriptionally modify or modulate the
expression of a target gene in a call, tissue, organ or whole
organism, thereby producing novel phenotypes. Novel synthetic genes
and genetic constructs which are capable of repressing delaying or
otherwise reducing the expression of an endogenous gene or a target
gene in an organism when introduced thereto are also provided.
GENERAL
[0003] Bibliographic details of the publications referred to in
this specification are collected at the end of the description.
[0004] As used herein the term "derived from" shall be taken to
indicate that a specified integer may be obtained from a particular
specified source or species, albeit not necessarily directly from
that specified source or species.
[0005] Throughout this specification, unless the context requires
otherwise, the word "comprise", or variations such as "comprises"
or "comprising", Will be understood to imply the inclusion of a
stated step or element or integer or group of steps or elements or
integers but not the exclusion of any other step or element or
integer or group of elements or integers.
[0006] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications.
The invention also includes all of the steps, features,
compositions and compounds referred to or indicated in this
specification, individually or collectively, and any and all
combinations or any two or more of said steps or features.
[0007] The present invention is not to be limited in scope by the
specific embodiments described herein, which are intended for the
purposes of exemplification only. Functionally-equivalent products,
compositions and methods are clearly within the scope of the
invention, as described herein.
[0008] Sequence identity numbers (SEQ ID NOS.) containing
nucleotide and amino acid sequence information included in this
specification are collected after the Abstract and have been
prepared using the programme Patentln Version 2.0. . Each
nucleotide or amino acid sequence is identified in the sequence
listing by the numeric indicator <210>followed by the
sequence identifier (e.g. <210>1, <210 >2, etc). The
length, type of sequence (DNA, protein (PRT), etc) and source
organism for each nucleotide or amino acid sequence are indicated
by information provided in the numeric indicator fields
<0211>, <212>and <213>, respectively. Nucleotide
and amino acid sequences referred to in the specification are
defined by the information provided in numeric indicator field
<400>followed by the sequence identifier (eg. <400>1,
<400>2, etc).
[0009] The designation of nucleotide residues referred to herein
are those recommended by the IUPAC-IUB Biochemical Nomenclature
Commission, wherein A represents Adenine, C represents Cytosine, G
represents Guanine, T represents thymine, Y represents a pyrimidine
residue, R represents a purine residue, M represents Adenine or
Cytosine, K represents Guanine or Thymine, S represents Guanine or
Cytosine, W represents Adenine or Thymine, H represents a
nucleotide other than Guanine, B represents a nucleotide other than
Adenine, V represents a nucleotide other than Thymine, D represents
a nucleotide other than Cytosine and N represents any nucleotide
residue.
[0010] The designation of amino acid residues referred to herein,
as recommended by the IUPAC-IUB Biochemical Nomenclature
Commission, are listed in Table 1.
1 TABLE 1 Amino Acid Three-letter code One-letter code Alanine Ala
A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys
C Glutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His H
Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M
Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T
Tryptophan Trp W Tyrosine Tyr Y Valine Val V Aspartate/Asparagine
Baa B Glutamate/Glutamine Zaa Z Any amino acid Xaa X
BACKGROUND TO THE INVENTION
[0011] Controlling metabolic pathways in eukaryotic organisms is
desirable for the purposes of producing novel traits therein or
introducing novel traits into a particular cell, tissue or organ of
said organism. Whilst recombinant DNA technology has provided
significant progress in an understanding of the mechanisms
regulating eukaryotic gene expression, much less progress has been
made in the actual manipulation of gene expression to produce novel
traits. Moreover, there are only limited means by which human
intervention may lead to a modulation of the level of eukaryotic
gene expression.
[0012] One approach to repressing, delaying or otherwise reducing
gene expression utilises an RNA molecule which is transcribed from
the complementary strand of a nuclear gene to that which is
normally transcribed and capable of being translated into a
polypeptide. Although the precise mechanism involved in this
approach is not established, it has been postulated that a
double-stranded RNA may form by base pairing between the
complementary nucleotide sequences, to produce a complex which is
translated at low efficiency and/or degraded by intracellular
ribonuclease enzymes prior to being translated.
[0013] Alternatively, the expression of an endogenous gene in a
plant cell, tissue or organ may be suppressed when one or more
copies of said gene, or one or more copies of a substantially
similar gene are introduced into the cell. Whilst the mechanism
involved in this phenomenon has not been established it appears to
involve mechanistically heterogeneous processes. For example, this
approach has been postulated to involve transcriptional repression,
in which case somatically-heritable repressed states of chromatin
are formed or alternatively, a post-transcriptional silencing
wherein transcription occurs normally but the RNA products of the
co-suppressed genes are subsequently eliminated.
[0014] The efficiency of both of these approaches in targeting the
expression of specific genes is very low and highly variable
results are usually obtained. Inconsistent results are obtained
using different regions of genes, for example 5'-untranslated
regions, 3'-untranslated regions, coding regions or intron
sequences to target gene expression. Accordingly, there currently
exists no consensus as to the nature of genetic sequences which
provide the most efficient means for repressing, delaying or
otherwise reducing gene expression using existing technologies.
Moreover, such a high degree of variation exists between
generations that it is not possible to predict the level of
repression of a specific gene in the progeny of an organism in
which gene expression was markedly modified.
[0015] Recently, Dorer and Henikoff (1994) demonstrated the
silencing of tandemly repeated gene copies in the Drosophila genome
and the transcriptional repression of dispersed Drosophila Adh
genes by Polycomb genes (i.e. the Pc-G system; Pal-Bhadra et al,
1997). However, such silencing of tandemly repeated gene copies is
of little utility in an attempt to manipulate gene expression in an
animal cell by recombinant means, wherein the sequences capable of
targeting the expression of a particular gene are introduced at
dispersed locations in the genome, absent the combination of this
approach with gene-targeting technology. Whilst theoretically
possible, such combinations would be expected to work at only
low-efficiency, based upon the low efficiency of gene-targeting
approaches used in isolation and further, would require complicated
vector systems. Additionally, the utilisation of transcriptional
repression, such as the Drosophila Pc-G system, would appear to
require some knowledge of the regulatory mechanisms capable of
modulating the expression of any specific target gene and, as a
consequence, would be difficult to implement in practice as a
general technology for repressing, delaying or reducing gene
expression in animal cells.
[0016] The poor understanding of the mechanisms involved in these
phenomena has meant that there have been few improvements in
technologies for modulating the level of gene expression , in
particular technologies for delaying, repressing or otherwise
reducing the expression of specific genes using recombinant DNA
technology. Furthermore, as a consequence of the unpredictability
of these approaches, there is currently no commercially-viable
means for modulating the level of expression of a specific gene in
a eukaryotic or prokaryotic organism.
[0017] Thus, there exists a need for improved methods of modulating
gene expression, in particular repressing, delaying or otherwise
reducing gene expression in animal cells for the purpose of
introducing novel phenotypic traits thereto. In particular, these
methods should provide general means for phenotypic modification,
without the necessity for performing concomitant gene-targeting
approaches.
SUMMARY OF THE INVENTION
[0018] The invention is based in part on the surprising discovery
by the inventors that cells which exhibit one or more desired
traits can be produced and selected from transformed cells
comprising a nucleic acid molecule operably linked to a promoter,
wherein the transcription product of the nucleic acid molecule
comprises a nucleotide sequence which is substantially identical to
the nucleotide sequence of a transcript of an endogenous or
non-endogenous target gene, the expression of which is intended to
be modulated. The transformed cells are regenerated into whole
tissues, organs or organisms capable of exhibiting novel traits, in
particular virus resistance and modified expression of endogenous
genes.
[0019] Accordingly, one aspect of the present invention provides a
method of modulating the expression of a target gene in a plant
cell, tissue or organ comprising (a) providing one or more
dispersed or foreign nucleic acid molecules which include multiple
copies of a nucleotide sequence, each of which is substantially
identical to or complementary to the nucleotide sequence of the
target gene or a region thereof, and (b) transfecting the plant
cell, tissue or organ with the dispersed or foreign nucleic acid
molecules for a time and under conditions sufficient for expression
of at least two of the multiple copies.
[0020] In a particularly preferred embodiment, either (a) at least
two of the copies are in tandem and the same orientation, or (b) at
least one of the copies is in the sense orientation and one is in
the antisense orientation and these two copies are located relative
to each other such that the two copies may form a hairpin RNA
structure when transcribed.
[0021] The target gene may be a gene which is endogenous to the
cell or alternatively, a foreign gene such as a viral or foreign
genetic sequence, amongst others. Preferably, the target gene is a
viral genetic sequence.
[0022] The invention is particularly useful in the modulation of
eukaryotic gene expression, in particular the modulation of plant
or animal gene expression and even more particularly in the
modulation of expression of genes derived from crops, vertebrate
and invertebrate animals, such as insects, aquatic animals (eg.
fish, shellfish, molluscs, crustaceans such as crabs, lobsters and
prawns, avian animals and mammals, amongst others).
[0023] A variety of traits are selectable with appropriate
procedures and sufficient numbers of transformed cells. Such traits
include, but are not limited to, visible traits, disease-resistance
traits, and pathogen-resistance traits. The modulatory effect is
applicable to a variety of genes expressed in animals including,
for example, endogenous genes responsible for cellular metabolism
or cellular transformation, including oncogenes, transcription
factors and other genes which encode polypeptides involved in
cellular metabolism.
[0024] For example, an alteration in the pigment production in mice
can be engineered by targeting the expression of the tyrosinase
gene therein. This provides a novel phenotype of albinism in black
mice. By targeting genes required for virus replication in a plant
cell or an animal cell, a genetic construct which comprises
multiple copies of nucleotide sequence encoding a viral replicase,
polymerase, coat protein or uncoating gene, or protease protein,
may be introduced into a cell where it is expressed, to confer
immunity against the virus upon the cell.
[0025] In performance of the present invention, the dispersed
nucleic acid molecule or foreign nucleic acid molecule will
generally comprise a nucleotide sequence having greater than about
85% identity to the target gene sequence, however, a higher
homology might produce a more effective modulation of expression of
the target gene sequence. Substantially greater homology, or more
than about 90% is preferred, and even more preferably about 95% to
absolute identity is desirable.
[0026] The introduced dispersed nucleic acid molecule or foreign
nucleic acid molecule sequence, needing less than absolute
homology, also need not be full length, relative to either the
primary transcription product or fully processed mRNA of the target
gene. A higher homology in a shorter than full length sequence
compensates for a longer less homologous sequence. Furthermore, the
introduced sequence need not have the same intron or exon pattern,
and homology of non-coding segments will be equally effective.
[0027] A second aspect of the present invention provides a method
of modulating the expression of a target gene in a plant cell,
tissue or organ, said method comprising:
[0028] (i) selecting one or more dispersed nucleic acid molecules
or foreign nucleic acid molecules which comprise multiple copies of
a nucleotide sequence which is substantially identical to the
nucleotide sequence of said target gene or a region thereof or
which is complementary thereto;
[0029] (ii) producing a synthetic gene comprising said dispersed
nucleic acid molecules or foreign nucleic acid molecules operably
connected to a promoter sequence operable in said cell, tissue or
organ;
[0030] (iii) introducing said synthetic gene to said cell, tissue
or organ; and
[0031] (iv) expressing said synthetic gene in said cell, tissue or
organ for a time and under conditions sufficient for expression of
at least two of the copies.
[0032] A third aspect of the invention provides a method of
conferring resistance or immunity to a viral pathogen upon a plant
cell, tissue, organ or whole organism, comprising:
[0033] (i) selecting one or more dispersed nucleic acid molecules
or foreign nucleic acid molecules which comprise multiple copies
substantially identical to a nucleotide sequence derived from the
viral pathogen or a complementary sequence thereto or a region
thereof;
[0034] (ii) producing a synthetic gene comprising said dispersed
nucleic acid molecules or foreign nucleic acid molecules operably
connected to a promoter sequence operable in said cell, tissue,
organ or whole organism;
[0035] (iii) introducing said synthetic gene to said cell, tissue,
organ or whole organism; and
[0036] (iv) expressing said synthetic gene in said cell, tissue or
organ for a time and under conditions sufficient for expression of
at least two of the copies.
[0037] A fourth aspect of the present invention provides a genetic
construct comprising multiple structural gene sequences, wherein
each of said structural gene sequences is substantially identical
to a target gene in a plant cell, and wherein said multiple
structural gene sequences are placed operably under the control of
a single promoter sequence which is operable in said cell, wherein
at least one of said structural gene sequences is placed operably
in the sense orientation under the control of said promoter
sequence and at least one other of said structural gene sequences
is placed operably in the antisense orientation under the control
of said promoter sequence, and wherein at least one structural gene
sequence that is placed in the sense orientation relative to said
promoter and at least one structural gene sequence that is placed
in the antisense orientation relative to said promoter are spaced
from each other by a nucleic acid stuffer fragment.
[0038] A fifth aspect of the present invention provides a genetic
construct which is capable of modulating the expression of a target
gene in a plant cell, which is transfected with said construct,
wherein said construct comprises multiple copies of a structural
gene sequence, wherein each copy comprises a nucleotide sequence
which is substantially identical to said target gene or a
derivative of said target gene and wherein said multiple copies are
placed operably under the control of a single promoter sequence
which is operable in said cell, wherein at least two of said copies
are placed operably in the sense orientation under the control of
said promoter sequence.
[0039] A sixth aspect of the present invention provides a genetic
construct which is capable of modulating the expression of a target
gene in a plant cell, which is transfected with said construct,
wherein said construct comprises multiple structural gene sequences
wherein each of said structural gene sequences is separately placed
under the control of a promoter which is operable in said cell, and
wherein each of said structural gene sequences comprises a
nucleotide sequence which is substantially identical to said target
gene or a derivative of said target gene, wherein at least one of
said structural gene sequences is placed operably in the sense
orientation under the control of an individual promoter
sequence.
[0040] In order to observe many novel traits in multicellular
organisms in particular those which are tissue-specific or
organ-specific or developmentally-regulated, regeneration of a
transformed cell carrying the synthetic genes and genetic
constructs described herein into a whole organism will be required.
Those skilled in the art will be aware that this means growing a
whole organism from a transformed animal cell, a group of such
cells, a tissue or organ. Standard methods for the regeneration of
certain animals from isolated cells and tissues are known to those
skilled in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1 is a diagrammatic representation of the plasmid
pEGFP-N1 MCS.
[0042] FIG. 2 is a diagrammatic representation of the plasmid
pCMV.cass.
[0043] FIG. 3 is a diagrammatic representation of the plasmid
pCMV.SV40L.cass.
[0044] FIG. 4 is a diagrammatic representation of the plasmid
pCMV.SV40LR.cass.
[0045] FIG. 5 is a diagrammatic representation of the plasmid
pCR.Bgl-GFP-Bam.
[0046] FIG. 6 is a diagrammatic representation of the plasmid
pBSII(SK+).EGFP.
[0047] FIG. 7 is a diagrammatic representation of the plasmid
pCMV.EGFP.
[0048] FIG. 8 is a diagrammatic representation of the plasmid
pCR.SV40L.
[0049] FIG. 9 is a diagrammatic representation of the plasmid
pCR.BEV.1.
[0050] FIG. 10 is a diagrammatic representation of the piasmid
pCR.BEV.2.
[0051] FIG. 11 is a diagrammatic representation of the plasmid
pCR.BEV.3.
[0052] FIG. 12 is a diagrammatic representation of the plasmid
pCMV,EGFP.BEV2.
[0053] FIG. 13 is a diagrammatic representation of the plasmid
pCMV.BEV.2.
[0054] FIG. 14 is a diagrammatic representation of the plasmid
pCMV.BEV.3.
[0055] FIG. 15 is a diagrammatic representation of the plasmid
pCMV.VEB.
[0056] FIG. 16 is a diagrammatic representation of the piasmid
pCMV.BEV.GFP.
[0057] FIG. 17 is a diagrammatic representation of the plasmid
pCMV.BEV.SV40L-0.
[0058] FIG. 18 is a diagrammatic representation of the plasmid
pCMV.O.SV40L.BEV.
[0059] FIG. 19 is a diagrammatic representation of the plas mid
pCMV.O.SV40L.VEB,
[0060] FIG. 20 is a diagrammatic representation of the plasmid
pCMV.BEVX2.
[0061] FIG. 21 is a diagrammatic representation of the plasmid
pCMV.BEVx3.
[0062] FIG. 22 is a diagrammatic representation of the plasmid
pCMV.BEVx4.
[0063] FIG. 23 is a diagrammatic representation of the plasmid
pCMV.BEV.SV40L.BEV.
[0064] FIG. 24 is a diagrammatic representation of the plasmid
pCMV.BEV.SV40L.VEB.
[0065] FIG. 25 is a diagrammatic representation of the plasmid
pCMV.BEV.GFP.VEB.
[0066] FIG. 26 is a diagrammatic representation of the plasmid
pCMV.EGFP.BEV2.PFG.
[0067] FIG. 27 is a diagrammatic representation of the plasmid
pCMV.BEV.SV40LR.
[0068] FIG. 28 is a diagrammatic representation of the plasmid
pCDNA3.Galt.
[0069] FIG. 29 is a diagrammatic representation of the plasmid
pCMV.Galt.
[0070] FIG. 30 is a diagrammatic representation of the plasmid
pCMV.EGFP.Gait.
[0071] FIG. 31 is a diagrammatic representation of the plasmid
pCMV.GaIt.GFP.
[0072] FIG. 32 is a diagrammatic representation of the plasmid
pCMV.Galt.SV40L.0,
[0073] FIG. 33 is a diagrammatic representation of the plasmid
pCMV.Gait.SV40L.tiaG.
[0074] FIG. 34 is a diagrammatic representation of the plasmid
pCMV.O.SV40L.Galt.
[0075] FIG. 35 is a diagrammatic representation of the plasmid
pCMV.Gaitx2.
[0076] FIG. 36 is a diagrammatic representation of the plasmid
pCMV.Galtx4.
[0077] FIG. 37 is a diagrammatic representation of the plasmid
pCMV.Galt.SV40L.Gait.
[0078] FIG. 38 is a diagrammatic representation of the plasmid
pCMV.Galt.SV40L.tIaG.
[0079] FIG. 39 is a diagrammatic representation of the plasmid
pCMV.Galt.GFP.tlaG.
[0080] FIG. 40 is a diagrammatic representation of the plasmid
pCMV.EGFP.GaIt.PFG.
[0081] FIG. 41 is a diagrammatic representation of the plasmid
pCMV.Galt.SV40LR.
[0082] FIG. 42 is a diagrammatic representation of the plasmid
pART7.
[0083] FIG. 43 is a diagrammatic representation of the plasmid
pART7.35S.SCBV.cass.
[0084] FIG. 44 Is a diagrammatic representation of the p.asmid
pBCPVY.
[0085] FIG. 45 is a diagrammatic representation of the pfasmid
pSP72.PVY.
[0086] FIG. 46 is a diagrammatic representation of the plasmid
pCIapBC.PVY.
[0087] FIG. 47 is a diagrammatic representation of the plasmid
pBC.PVYx2.
[0088] FIG. 48 is a diagrammatic representation of the piasmid
pSP72.PVYx2.
[0089] FIG. 49 is a diagrammatic representation of the plasmid
pBC.PVYx3.
[0090] FIG. 50 is a diagrammatic representation of the plasmid
pBC.PVYx4.
[0091] FIG. 51 is a diagrammatic representation of the plasmid
pBC.PVY.LNYV.
[0092] FIG. 52 is a diagrammatic representation of the plasmid
pBC.PVY.LNYV.PVY.
[0093] FIG. 53 is a diagrammatic representation of the plasmid
pBC.PVY.LNYV.YVP.
[0094] FIG. 54 is a diagrammatic representation of the plasmid
pBC.PVY.LNYV.YVP.
[0095] FIG. 55 is a diagrammatic representation of the plasmid
pART27.PVY
[0096] FIG. 56 is a diagrammatic representation of the plasmid
pART27.35S.PVY.SCBV.O.
[0097] FIG. 57 is a diagrammatic representation of the plasmid
pART27.35S.O.SCBV.PVY.
[0098] FIG. 58 is a diagrammatic representation of the plasmid
pART27.35S.O.SCBV.YVP.
[0099] FIG. 59 is a diagrammatic representation of the plasmid
pART7.PVYx2.
[0100] FIG. 60 is a diagrammatic representation of the plasmid
pART7.PVYx3.
[0101] FIG. 61 is a diagrammatic representation of the plasmid
pART7.PVYx4.
[0102] FIG. 62 is a diagrammatic representation of the plasmid
pART7.PVY.LNYV.PVY.
[0103] FIG. 63 is a diagrammatic representation of the plasmid
pART7.PVY.LNYV.YVP.
[0104] FIG. 64 is a diagrammatic representation of the piasmid
pART7. PVY.LNYV.YVP.
[0105] FIG. 65 is a diagrammatic representation of
pART7.35S.PVY.SCBV.YVP.
[0106] FIG. 66 is a diagrammatic representation of
pART7.35S.PVYx3.SCBV.YV- Px3.
[0107] FIG. 67 is a diagrammatic representation of
pART7.PVYx3.LNYV.YVPx3.
[0108] FIG. 68 is a diagrammatic representation of the plasmid
pART7,PVYMULTI.
[0109] FIG. 69 shows micrographs of PK-1 cell lines transformed
with pCMV.EGFP, viewed under normal light and under fluorescence
conditions (excitation .lambda.=488 nm, emission .lambda.=507 nm)
designed to detect GFP. A: PK EGFP 2.11 cells under normal light;
B: PK EGFP 2.11 cells under fluorescence conditions; C: PK EGFP
2.18 cells under normal light; D: PK EGFP 2.18 cells under
fluorescence conditions.
[0110] FIG. 70 shows an example of Southern blot analysis of
transgenic porcine kidney cells (PK) which had been transformed
with the construct pCMV.EGFP. Genomic DNA was isolated from PK-1
cells and transformed lines, digested with the restriction
endonuclease BamHl and probed with a .sup.32P-dCTP labeled EGFP DNA
fragment. Lane A is a molecular weight marker where sizes of each
fragment are indicated in kilobases (kb); Lane B is the parental
cell line PK-1. Lane C is A4, a transgenic EGFP-expressing PK-1
cell line; Lane D is C9, a transgenic non-expressing PK-1 cell
line.
[0111] FIG. 71 shows micrographs of CRIB-1 cells and a CRIB-1
transformed line [CRIB-1 BGI2 # 19(tol)] prior to and 48 hrs after
infection with identical titres of BEV. A: CRIB-1 cells prior to
BEV infection; B: CRIB-1 cells 48 hrs after BEV infection; C:
CRIB-1 BGI2 # 19(tol) cells prior to infection with BEV; D: CRIB-1
BGI2 # 19(tol) 48 hrs after BEV infection. For further details
refer to Example 8.
[0112] FIG. 72 shows levels of pigmentation in B16 cells and B16
cells transformed with pCMV.TYR.BGI2.RYT. Cell lines are, from left
to right: B16, B16 2.1.6, B16 2.1.11, B16 3.1.4, B16 3.1.15, B16
4.12.2 and B16 4.12.3. For further details refer to Example 9.
[0113] FIG. 73 shows immunofluorescent micrographs of MDA-MB-468
cells and MDA-MB-468 cells transformed with pCMV.HER2.BGI2.2REH
stained for HER-2. A: MDA-MB-468 cells; B: MDA-MB-468 cells stained
with only the secondary antibody; C: MDA-MB-468 1.4 cells stained
for HER-2; D: MDA-MB-468 1.10 cells stained for HER-2. For further
details refer to Example 10.
[0114] FIG. 74 shows FACS analyses of HER-2 expression in (A)
MDA-MB-468 cells; (B) MDA-MB-468 1.4 cells; (C) MDA-MB-468 1.10
cells. For further details refer to Example 10.
[0115] FIG. 75 is a histograph showing viable cell counts after
transfection with YB-1-related gene constructs and
oligonucleotides. Viable cells were counted in quadruplicate
samples with a haemocytometer following staining with trypan blue.
Column heights show the average cell count of two independent
transfection experiments and vertical bars indicate the standard
deviation. (A) Viable 810.2 cell counts 72 hr after transfection
with gene constructs: (i) control: pCMV.EGFP; (ii)
pCMV.YB1.BGI2.1BY; (iii) pCMV.YB1.p53.BGI2.35p.1BY. All materials
and procedures used are described in the text for Example 1 1. (B)
Viable Pam 212 cell counts 72 hr after transfection with gene
constructs: (i) control: PCMV.EGFP; (ii) pCMV.YB1.BGI2.1BY; (iii)
pCMV.YB1.p53.BGI2.35p.1- BY. All materials and procedures used are
described in the text for Example 11. (C) Viable B10.2 cell counts
18 hr after transfection with oligonucleotides: (i) control.
Lipofectin (trademark) only; (ii) control: non-specific
oligonucleotide; (iii) decoy Y-box oligonucleotide. All materials
and procedures used are described in the text for Example 11. (D)
Viable Pam 212 cell counts 18 hr after transfection with
oligonucleotides: (i) control: Lipofectin (trademark) only; (ii)
control: non-specific oligonucleotide; (iii) decoy Y-box
oligonucleotide. All materials and procedures used are described in
the text for Example 11.
[0116] FIG. 76: A Northern blot showing levels of GFP expression in
MM96L cells and MM96L lines transformed with pCMV.EGFP. 10 .mu.g of
total RNA from the indicated cell lines were electrophoresed on
agarose gels and transferred to a nylon membrane. The filter was
probed with a radio-labelled fragment derived from the EGFP gene. B
Photograph showing ethidium bromide-stained ribosomal RNAs from the
RNA samples probed in A; the equal intensities indicated similar
amounts of RNA from each cell line were probed.
[0117] FIG. 77: Real-Time RT-PCR analysis of transformed cell lines
for EGFP mRNA levels and EGFP RNA transcribed from the EGFP
transgene in nuclear run-on assays. A EGFP mRNA levels in MM96L
lines 3, 9 and 18 (as in FIG. 76). B EGFP gene run-on transcripts
in nuclei of MM96L lines 3, 9 and 18 (as in FIG. 76). C
Glyceraldehyde phosphate dehydrogenase (GAPD) mRNA levels in MM96L
lines 3, 9 and 18(as in FIG. 76). D GAPD gene run-on transcripts in
nuclei of MM96L lines 3, 9 and 18 (as in FIG. 76).
[0118] FIG. 78: Relative mRNA levels and RNA transcribed from the
EGFP transgene in nuclear run-on assays, from the data shown in
FIG. 77. The EGFP gene in line 9 is transcribed but EGFP mRNA
levels are extremely low, signifying post-transcriptional gene
silencing (co-suppression).
DETAILED DESCRIPTION OF THE INVENTION
[0119] The present invention provides a method of modulating the
expression of a target gene in a cell, tissue or organ, said method
at least comprising the step of introducing to said cell, tissue or
organ one or more dispersed nucleic acid molecules or foreign
nucleic acid molecules comprising multiple copies of a nucleotide
sequence which is substantially identical to the nucleotide
sequence of said target gene or a region thereof or complementary
thereto for a time and under conditions sufficient for translation
of the mRNA product of said target gene to be modified, subject to
the proviso that the transcription of said mRNA product is not
exclusively repressed or reduced.
[0120] By "multiple copies" is meant that two or more substantially
identical (as defined below) copies of a nucleotide sequence are
present in the same or different orientation, on the same nucleic
acid molecule. As would be appreciated by one skilled in the art,
the term "direct repeat" is used in contradistinction to the term
"inverted repeat" such that a direct repeat is a 5'-3'.5'-3'repeat
(with or without other nucleotides between the repeated sequences).
An inverted repeat is a 5'-3',3'-5'sequence (the 3'-5'sequence may
also be called a "reverse complement" or antisense of the
5'-3'sequence) such that the transcription product of the inverted
repeat (e.g. mRNA) may form a hairpin RNA structure. Further, the
term "tandem" is used by those skilled in the art to indicate
repeats separated by no or relatively few (relative to the length
of the repeated sequence) intervening nucleotides.
[0121] Repeats may optionally be separated by a stuffer fragment or
intergenic region to facilitate secondary structure formation
between each repeat where this is required. The stuffer fragment
may comprise any combination of nucleotide or amino acid residues,
carbohydrate molecules or oligosaccharide molecules or carbon atoms
or a homologue, analogue or derivative thereof which is capable of
being linked covalently to a nucleic acid molecule.
[0122] Preferably, the stuffer fragment comprises a sequence of
nucleotides or a homologue, analogue or derivative thereof.
[0123] Where the dispersed or foreign nucleic acid molecule
includes intron/exon splice junction sequences, the stuffer
fragment may serve as an intron sequence placed between the
3'-splice site of the structural gene nearer the 5'-end of the gene
and the 5'-splice site of the next downstream unit thereof.
Alternatively, where it is desirable for more than two adjacent
nucleotide sequence units of the dispersed foreign nucleic acid
molecule to be translated, the stuffer fragment placed there
between should not include an in-frame translation stop codon,
absent intron/exon splice junction sequences at both ends of the
stuffer fragment or the addition of a translation start codon at
the 5'end of each unit, as will be obvious to those skilled in the
art.
[0124] Stuffer fragments can include those which encode detectable
marker proteins or biologically-active analogues and derivatives
thereof, for example luciferase, .beta.-galacturonase,
.beta.-galactosidase, chloramphenicol acetyltransferase or green
fluorescent protein, amongst others. Additional stuffer fragments
are not excluded.
[0125] According to this embodiment, the detectable marker or an
analogue or derivative thereof serves to indicate the expression of
the synthetic gene of the invention in a cell, tissue or organ by
virtue of its ability to confer a specific detectable phenotype
thereon, preferably a visually-detectable phenotype.
[0126] As used herein, the term "modulating" shall be taken to mean
that expression of the target gene is reduced in amplitude and/or
the timing of gene expression is delayed and/or the developmental
or tissue-specific or cell-specific pattern of target gene
expression is altered, compared to the expression of said gene in
the absence of the inventive method described herein.
[0127] Whilst not limiting the scope of the invention described
herein, the present invention is directed to a modulation of gene
expression which comprises the repression, delay or reduction in
amplitude of target gene expression in a specified cell, tissue or
organ of a eukaryotic organism, in particular a plant such as a
monocotyledonous or dicotyledonous plant, or a human or other
animal and even more particularly a vertebrate and invertebrate
animal, such as an insect, aquatic animal (eg. fish, shellfish,
mollusc, crustacean such as a crab, lobster or prawn, an avian
animal or a mammal, amongst others).
[0128] More preferably, target gene expression is completely
inactivated by the dispersed nucleic acid molecules or foreign
nucleic acid molecules which has been introduced to the cell,
tissue or organ.
[0129] Whilst not being bound by any theory or mode of action, the
reduced or eliminated expression of the target gene which results
from the performance of the invention may be attributed to reduced
or delayed translation of the RNA transcription product of the
target gene or alternatively, the prevention of translation of said
RNA, as a consequence of sequence-specific degradation of the RNA
transcript of the target gene by an endogenous host cell
system.
[0130] It is particularly preferred that, for optimum results,
sequence-specific degradation of the RNA transcript of the target
gene occurs either prior to the time or stage when the RNA
transcript of the target gene would normally be translated or
alternatively, at the same time as the RNA transcript of the target
gene would normally be translated. Accordingly, the selection of an
appropriate promoter sequence to regulate expression of the
introduced dispersed nucleic acid molecule or foreign nucleic acid
molecule is an important consideration to optimum performance of
the invention. For this reason, strong constitutive promoters or
inducible promoter systems are especially preferred for use in
regulating expression of the introduced dispersed nucleic acid
molecules or foreign nucleic acid molecules.
[0131] The present invention clearly encompasses reduced expression
wherein reduced expression of the target gene is effected by
lowered transcription, subject to the proviso that a reduction in
transcription is not the sole mechanism by which this occurs and
said reduction in transcription is at least accompanied by reduced
translation of the steady-state mRNA pool of the target gene.
[0132] The target gene may be a genetic sequence which is
endogenous to the cell or alternatively, a non-endogenous genetic
sequence, such as a genetic sequence which is derived from a virus
or other foreign pathogenic organism and is capable of entering a
cell and using the cell's machinery following infection.
[0133] Where the target gene is a non-endogenous genetic sequence
to the cell, it is desirable that the target gene encodes a
function which is essential for replication or reproduction of the
viral or other pathogen. In such embodiments, the present invention
is particularly useful in the prophylactic and therapeutic
treatment of viral infection of an animal cell or for conferring or
stimulating resistance against said pathogen.
[0134] Preferably, the target gene comprises one or more nucleotide
sequences of a viral pathogen of a plant or an animal cell, tissue
or organ.
[0135] For example, in the case of animals and humans, the viral
pathogen may be a retrovirus, for example a lentivirus such as the
immunodeficiency viruses, a single-stranded (+) RNA virus such as
bovine enterovirus (BEV) or Sinbis alphavirus.
[0136] Alternatively, the target gene can comprise one or more
nucleotide sequences of a viral pathogen of an animal cell, tissue
or organ, such as but not limited to a double-stranded DNA virus
such as bovine herpes virus or herpes simplex virus I (HSV II),
amongst others.
[0137] In the case of plants, the virus pathogen is preferably a
potyvirus, caulimovirus, badnavirus, geminivirus, reovirus,
rhabdovirus, bunyavirus, tospovirus, tenuivirus, tombusvirus,
luteovirus, sobemovirus, bromovirus, cucomovirus, ilavirus,
alfamovirus, tobamovirus, tobravirus, potexvirus and clostrovirus,
such as but not limited to CaMV, SCSV, PVX, PVY, PLRV, and TMV,
amongst others.
[0138] With particular regard to viral pathogens, those skilled in
the art are aware that virus-encoded functions may be complemented
in trans by polypeptides encoded by the host cell. For example, the
replication of the bovine herpes virus genome in the host cell may
be facilitated by host cell DNA polymerases which are capable of
complementing an inactivated viral DNA polymerase gene.
[0139] Accordingly, where the target gene is a non-endogenous
genetic sequence to the animal cell, a further alternative
embodiment of the invention provides for the target gene to encode
a viral or foreign polypeptide which is not capable of being
complemented by a host cell function, such as a virus-specific
genetic sequence. Exemplary target genes according to this
embodiment of the invention include, but are not limited to genes
which encode virus coat proteins, uncoating proteins and
RNA-dependent DNA polymerases and RNA-dependent RNA polymerases,
amongst others.
[0140] In a particularly preferred embodiment of the present
invention, the target gene is the BEV RNA-dependent RNA polymerase
gene or a homologue, analogue or derivative thereof or PVY Nia
protease-encoding sequences.
[0141] The cell in which expression of the target gene is modified
may be any cell which is derived from a multicellular animal,
including cell and tissue cultures thereof. Preferably, the animal
cell is derived from an anthropod, nematode, reptile, amphibian,
bird, human or other mammal. Exemplary animal cells include
embryonic stem cells, cultured skin fibroblasts, neuronal cells,
somatic cells, haematopoietic stem cells, T-cells and immortalised
cell lines such as COS, VERO, HeLa, mouse C127, Chinese hamster
ovary (CHO), WI-38, baby hamster kidney (BHK) or MDBK cell lines,
amongst others. Such cells and cell lines are readily available to
those skilled in the art. Accordingly, the tissue or organ in which
expression of the target gene is modified may be any tissue or
organ comprising such animal cells.
[0142] Preferably the plant cell is derived from a monocotyledonous
or dicotyledonous plant species or a cell line derived
therefrom.
[0143] As used herein, the term "dispersed nucleic acid molecule"
shall be taken to refer to a nucleic acid molecule which comprises
one or more multiple copies of a nucleotide sequence which is
substantially identical or complementary to the nucleotide sequence
of a gene which originates from the cell, tissue or organ into
which said nucleic acid molecule is introduced, wherein said
nucleic acid molecule is non-endogenous in the sense that it is
introduced to the cell, tissue or organ of an animal via
recombinant means and will generally be present as extrachromosomal
nucleic acid or alternatively, as integrated chromosomal nucleic
acid which is genetically-unlinked to said gene. More particularly,
the "dispersed nucleic acid molecule" will comprise chromosomal or
extrachromosomal nucleic acid which is unlinked to the target gene
against which it is directed in a physical map, by virtue of their
not being tandemly-linked or alternatively, occupying a different
chromosomal position on the same chromosome or being localised on a
different chromosome or present in the cell as an episome, plasmid,
cosmid or virus particle.
[0144] By "foreign nucleic acid molecule" is meant an isolated
nucleic acid molecule which has one or more multiple copies of a
rucleotide sequence which originates from the genetic sequence of
an organism which is different from the organism to which the
foreign nucleic acid molecule is introduced. This definition
encompasses a nucleic acid molecule which originates from a
different individual of the same lowest taxonomic grouping (i.e.
the same population) as the taxonomic grouping to which said
nucleic acid molecule is introduced, as well as a nucleic acid
molecule which originates from a different individual of a
different taxonomic grouping as the taxonomic grouping to which
said nucleic acid molecule is introduced, such as a gene derived
from a viral pathogen.
[0145] Accordingly, a target gene against which a foreign nucleic
acid molecule acts in the performance of the invention may be a
nucleic acid molecule which has been introduced from one organism
to another organism using transformation or introgression
technologies. Exemplary target genes according to this embodiment
of the invention include the green fluorescent protein-encoding
gene derived from the jellyfish Aequoria Victoria (Prasher et
aL.,1992; International Patent Publication No. WO 95/07463),
tyrosinase genes and in particular the murine tyrosinase gene (Kwon
et aL,1988), the Escherichia coli tacd gene which is capable of
encoding a polypeptide repressor of the lacZ gene, the porcine
.beta.1,3-galactosyltransferase gene (NCBI Accession No. L36535)
exemplified herein, and the PVY and BEV structural genes
exemplified herein or a homologue, analogue or derivative of said
genes or a complementary nucleotide sequence thereto.
[0146] The present invention is further useful for simultaneously
targeting the expression of several target genes which are
co-expressed in a particular cell, for example by using a dispersed
nucleic acid molecule or foreign nucleic acid molecule which
comprises nucleotide sequences which are substantially identical to
each of said co-expressed target genes.
[0147] By "substantially identical" is meant that the introduced
dispersed or foreign nucleic acid molecule of the invention and the
target gene sequence are sufficiently identical at the nucleotide
sequence level to permit base-pairing there between under standard
intracellular conditions.
[0148] Preferably, the nucleotide sequence of each repeat in the
dispersed or foreign nucleic acid molecule of the invention and the
nucleotide sequence of a part of the target gene sequence are at
least about 80-85% identical at the nucleotide sequence level, more
preferably at least about 85-90% identical, even more preferably at
least about 90-95% identical and still even more preferably at
least about 95-99% or 100% identical at the nucleotide sequence
level.
[0149] Notwithstanding that the present invention is not limited by
the precise number of repeated sequences in the dispersed nucleic
acid molecule or the foreign nucleic acid molecule of the
invention, it is to be understood that the present invention
requires at least two copies of the dispersed foreign nucleic acid
molecule of the target gene sequence to be expressed in the
cell.
[0150] Preferably, the multiple copies of the target gene sequence
are presented in the dispersed nucleic acid molecule or the foreign
nucleic acid molecule as tandem inverted repeat sequences and/or
tandem direct repeat sequences. Such configurations are exemplified
by the "test plasmids" described herein that comprise Galt, BEV or
PVY gene regions.
[0151] Preferably, the dispersed or foreign nucleic acid molecule
which is introduced to the cell, tissue or organ comprises RNA or
DNA.
[0152] Preferably, the dispersed or foreign nucleic acid molecule
further comprises a nucleotide sequence or is complementary to a
nucleotide sequence which is capable of encoding an amino acid
sequence encoded by the target gene.
[0153] Standard methods may be used to introduce the dispersed
nucleic acid molecule or foreign nucleic acid molecule into the
cell, tissue or organ for the purposes of modulating the expression
of the target gene. For example, the nucleic acid molecule may be
introduced as naked DNA or RNA, optionally encapsulated in a
liposome, in a virus particle as attenuated virus or associated
with a virus coat or a transport protein or inert carrier such as
gold or as a recombinant viral vector or bacterial vector or as a
genetic construct, amongst others.
[0154] Administration means include injection and oral ingestion
(e.g. in medicated food material), amongst others.
[0155] The subject nucleic acid molecules may also be delivered by
a live delivery system such as using a bacterial expression system
optimised for their expression in bacteria which can be
incorporated into gut flora. Alternatively, a viral expression
system can be employed. In this regard, one form of viral
expression is the administration of a live vector generally by
spray, feed or water where an infecting effective amount of the
live vector (e.g. virus or bacterium) is provided to an animal.
Another form of viral expression system is a non-replicating virus
vector which is capable of infecting a cell but not replicating
therein. The non-replicating viral vector provides a means of
introducing to the human or animal subject genetic material for
transient expression therein. The mode of administering such a
vector is the same as a live viral vector.
[0156] The carriers, excipients and/or diluents utilised in
delivering the subject nucleic acid molecules to a host cell should
be acceptable for human or veterinary applications. Such carriers,
excipients and/or diluents are well-known to those skilled in the
art. Carriers and/or diluents suitable for veterinary use include
any and all solvents, dispersion media, aqueous solutions,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, use thereof in the composition is contemplated.
Supplementary active ingredients can also be incorporated into the
compositions.
[0157] In an alternative embodiment, the invention provides a
method of modulating the Expression of a target gene in a cell,
tissue or organ, said method at least comprising the steps of:
[0158] (i) selecting one or more dispersed nucleic acid molecules
or foreign nucleic acid molecules which comprise multiple copies of
a nucleotide sequence which is substantially identical to the
nucleotide sequence of said target gene or a region thereof or
which is complementary thereto; and
[0159] (ii) introducing said dispersed nucleic acid molecules or
foreign nucleic acid molecules to said cell, tissue or organ for a
time and under conditions sufficient for translation of the RNA
product of said target gene to be modified, subject to the proviso
that the transcription of said RNA product is not exclusively
repressed or reduced.
[0160] To select appropriate nucleotide sequences for targeting
expression of the target gene, several approaches may be employed.
In one embodiment, multiple copies of specific regions of
characterised genes may be cloned in operable connection with a
suitable promoter and assayed for efficacy in reducing target gene
expression. Alternatively, shotgun libraries comprising multiple
copies of genetic sequences may be produced and assayed for their
efficacy in reducing target gene expression. The advantage
associated with the latter approach is that it is not necessary to
have any prior knowledge of the significance of any particular
target gene in specifying. an undesirable phenotype in the cell.
For example, shotgun libraries comprising virus sub-genomic
fragments may be employed and tested directly for their ability to
confer virus immunity on the animal host cell, without prior
knowledge of the role which any virus genes play in pathogenesis of
the host cell.
[0161] As used herein, the term "shotgun library" is a set of
diverse nucleotide sequences wherein each member of said set is
preferably contained within a suitable plasmid, cosmid,
bacteriophage or virus vector molecule which is suitable for
maintenance and/or replication in a cellular host. The term
"shotgun library" includes a representative library, in which the
extent of diversity between the nucleotide sequences is numerous
such that all sequences in the genome of the organism from which
said nucleotide sequences is derived are present in the "set" or
alternatively, a limited library in which there is a lesser degree
of diversity between said sequences. The term "shotgun library"
further encompasses random nucleotide sequences, wherein the
nucleotide sequence comprises viral or cellular genome fragments,
amongst others obtained for example by shearing or partial
digestion of genomic DNA using restriction endonucleases, amongst
other approaches. A "shotgun library" further includes cells, virus
particles and bacteriophage particles comprising the individual
nucleotide sequences of the diverse set.
[0162] Preferred shotgun libraries according to this embodiment of
the invention are "representative libraries", comprising a set of
tandem repeated nucleotide sequences derived from a viral pathogen
of a plant or an animal.
[0163] In a particularly preferred embodiment of the invention, the
shotgun library comprises cells, virus particles or bacteriophage
particles comprising a diverse set of tandem-repeated nucleotide
sequences which encode a diverse set of amino acid sequences,
wherein the members of said diverse set of nucleotide sequences are
placed operably under the control of a promoter sequence which is
capable of directing the expression of said tandem-repeated
nucleotide sequence in the cell.
[0164] Accordingly, the nucleotide sequence of each unit in the
tandem-repeated sequence may comprise at least about 20 to 200
nucleotides in length. The use of larger fragments, particularly
employing randomly sheared nucleic acid derived from viral, plant
or animal genomes, is not excluded.
[0165] The introduced nucleic acid molecule is preferably in an
expressible form.
[0166] By "expressible form" is meant that the subject nucleic acid
molecule is presented in an arrangement such that it may be
expressed in the cell, tissue, organ or whole organism, at least at
the transcriptional level (i.e. it is expressed in the animal cell
to yield at least an RNA product which is optionally translatable
or translated to produce a recombinant peptide, oligopeptide or
polypeptide molecule).
[0167] By way of exemplification, in order to obtain expression of
the dispersed nucleic acid molecule or foreign nucleic acid
molecule in the cell, tissue or organ of interest, a synthetic gene
or a genetic construct comprising said synthetic gene is produced,
wherein said synthetic gene comprises a nucleotide sequence as
described supra in operable connection with a promoter sequence
which is capable of regulating expression therein. Thus, the
subject nucleic acid molecule will be operably connected to one or
more regulatory elements sufficient for eukaryotic transcription to
occur.
[0168] Reference herein to a "gene" or "genes" is to be taken in
its broadest context and includes:
[0169] (i) a classical genomic gene consisting of transcriptional
and/or translational regulatory sequences and/or a coding region
and/or non-translated sequences (i.e. introns, 5'- and
3'-untranslated sequences, or DNA and RNA viruses); and/or
[0170] (ii) mRNA or cDNA corresponding to the coding regions (i.e.
exons) or 5'-and 3'-untranslated sequences of the gene; and/or
[0171] (iii) a structural region corresponding to the coding
regions (i.e. exons) optionally further comprising untranslated
sequences and/or a heterologous promoter sequence which consists of
transcriptional and/or translational regulatory regions capable of
conferring expression characteristics on said structural
region.
[0172] Thus, "gene" includes a nucleotide sequence coding for RNA
other than mRNA.
[0173] The term "gene" is also used to describe synthetic or fusion
molecules encoding all or part of a functional product, in
particular a sense or antisense RNA product or a peptide,
oligopeptide or polypeptide or a biologically-active protein.
[0174] The term "synthetic gene" refers to a non-naturally
occurring gene as hereinbefore defined which preferably comprises
at least one or more transcriptional and/or translational
regulatory sequences operably linked to a structural gene
sequence.
[0175] The term "structural gene" shall be taken to refer to a
nucleotide sequence which is capable of being transcribed to
produce RNA and optionally, encodes a peptide, oligopeptide,
polypeptide or biologically active protein molecule. Those skilled
in the art will be aware that not all RNA is capable of being
translated into a peptide, oligopeptide, polypeptide or protein,
for example if the RNA lacks a functional translation start signal
or alternatively, if the RNA is antisense RNA, The present
invention clearly encompasses synthetic genes comprising nucleotide
sequences which are not capable of encoding peptides,
oligopeptides, polypeptides or biologically-active proteins. In
particular, the present inventors have found that such synthetic
genes may be advantageous in modifying target gene expression in
cells, tissues or organs of a prokaryotic or eukaryotic
organism.
[0176] The term "structural gene region" refers to that part of a
synthetic gene which comprises a dispersed nucleic acid molecule or
foreign nucleic acid molecule as described herein which is
expressed in a cell, tissue or organ under the control of a
promoter sequence to which it is operably connected. A structural
gene region may comprise one or more dispersed nucleic acid
molecules and/or foreign nucleic acid molecules operably under the
control of a single promoter sequence or multiple promoter
sequences. Accordingly, the structural gene region of a synthetic
gene may comprise a nucleotide sequence which is capable of
encoding an amino acid sequence or is complementary thereto. In
this regard, a structural gene region which is used in the
performance of the instant invention may also comprise a nucleotide
sequence which encodes an animo acid sequence yet lacks a
functional translation initiation codon and/or a functional
translation stop codon and, as a consequence, does not comprise a
complete open reading frame. In the present context, the term
"structural gene region" also extends to a non-coding nucleotide
sequences, such as 5'-upstream or 3'-downstream sequences of a gene
which would not normally be translated in a eukaryotic cell which
expresses said gene.
[0177] Accordingly, in the context of the present invention, a
structural gene region may also comprise a fusion between two or
more open reading frames of the same or different genes. In such
embodiments, the invention may be used to modulate the expression
of one gene, by targeting different non-contiguous regions thereof
or alternatively, to simultaneously modulate the expression of
several different genes, including different genes of a multigene
family. In the case of a fusion nucleic acid molecule which is
non-endogenous to the animal cell and in particular comprises two
or more nucleotide sequences derived from a viral pathogen, the
fusion may provide the added advantage of conferring simultaneous
immunity or protection against several pathogens, by targeting the
expression of genes in said several pathogens. Alternatively or in
addition, the fusion may provide more effective immunity against
any pathogen by targeting the expression of more than one gene of
that pathogen.
[0178] The optimum number of structural gene sequences which may be
involved in the synthetic gene of the present invention will vary
considerably, depending upon the length of each of said structural
gene sequences, their orientation and degree of identity to each
other. For example, those skilled in the art will be aware of the
inherent instability of palindromic nucleotide sequences in vivo
and the difficulties associated with constructing long synthetic
genes comprising inverted repeated nucleotide sequences, because of
the tendency for such sequences to recombine in vivo.
Notwithstanding such difficulties, the optimum number of structural
gene sequences to be included in the synthetic genes of the present
invention may be determined empirically by those skilled in the
art, without any undue experimentation and by following standard
procedures such as the construction of the synthetic gene of the
invention using recombinase-deficient cell lines, reducing the
number of repeated sequences to a level which eliminates or
minimises recombination events and by keeping the total length of
the multiple structural gene sequence to an acceptable limit,
preferably no more than 5-10kb, more preferably no more than 2-5kb
and even more preferably no more than 0.5-2.0kb in length.
Alternatively, as explained above, a stuffer fragment can be
inserted between copies forming the palindrome.
[0179] Wherein the structural gene region comprises more than one
dispersed nucleic acid molecule or foreign nucleic acid molecule it
shall be referred to herein as a "multiple structural gene region"
or similar term. The present invention clearly extends to the use
of multiple structural gene regions which preferably comprise a
direct repeat sequence, inverted repeat sequence or interrupted
palindrome sequence of a particular structural gene, dispersed
nucleic acid molecule or foreign nucleic acid molecule, or a
fragment thereof.
[0180] Each dispersed or foreign nucleic acid molecule contained
within the multiple structural gene unit of the subject synthetic
gene may comprise a nucleotide sequence which is substantially
identical to a different target gene in the same organism. Such an
arrangement may be of particular utility when the synthetic gene is
intended to provide protection against a pathogen in a cell, tissue
or organ, in particular a viral pathogen, by modifying the
expression of viral target genes. For example, the multiple
structural gene may comprise nucleotide sequences (i.e. two or more
dispersed or foreign nucleic acid molecules) which are
substantially identical to two or more target genes selected from
the list comprising DNA polymerase, RNA polymerase, Nia protease,
and coat protein or other target gene which is essential for viral
infectivity, replication or reproduction. However, it is preferred
with this arrangement that the structural gene units are selected
such that the target genes to which they are substantially
identical are normally expressed at approximately the same time (or
later) in an infected call, tissue or organ as (than) the multiple
structural gene of the subject synthetic gene is expressed under
control of the promoter sequence. This means that the promoter
controlling expression of the multiple structural gene will usually
be selected to confer expression in the cell, tissue or organ over
the entire life cycle of the virus when the viral target genes are
expressed at different stages of infection.
[0181] As with the individual sequence units of a dispersed or
foreign nucleic acid molecule, the individual units of the multiple
structural gene may be spatially connected in any orientation
relative to each other, for example head-to-head, head-to-tail or
tail-to-tail and all such configurations are within the scope of
the invention.
[0182] For expression in eukaryotic cells, the synthetic gene
generally comprises, in addition to the nucleic acid molecule of
the invention, a promoter and optionally other regulatory sequences
designed to facilitate expression of the dispersed nucleic acid
molecule or foreign nucleic acid molecule.
[0183] Reference herein to a "promoter" is to be taken in its
broadest context and includes the transcriptional regulatory
sequences of a classical genomic gene, including the TATA box which
is required for accurate transcription initiation, with or without
a CCAAT box sequence and additional regulatory elements (i.e.
upstream activating sequences, enhancers and silencers) which alter
gene expression in response to developmental and/or external
stimuli, or in a tissue-specific manner. A promoter is usually, but
not necessarily, positioned upstream or 5', of a structural gene
region, the expression of which it regulates. Furthermore, the
regulatory elements comprising a promoter are usually positioned
within 2 kb of the start site of transcription of the gene.
[0184] In the present context, the term "promoter" is also used to
describe a synthetic or fusion molecule, or derivative which
confers, activates or enhances expression of a nucleic acid
molecule in a cell.
[0185] Preferred promoters may contain additional copies of one or
more specific regulatory elements, to further enhance expression of
the sense molecule and/or to alter the spatial expression and/or
temporal expression of said sense molecule. For example, regulatory
elements which confer copper inducibility may be placed adjacent to
a heterologous promoter sequence driving expression of a sense
molecule, thereby conferring copper ion inducibility on the
expression of said molecule.
[0186] Placing a dispersed or foreign nucleic acid molecule under
the regulatory control of a promoter sequence means positioning the
said molecule such that expression is controlled by the promoter
sequence. Promoters are generally positioned 5'(upstream) to the
genes that they control. In the construction of heterologous
promoter/structural gene combinations it is generally preferred to
position the promoter at a distance from the gene transcription
start site that is approximately the same as the distance between
that promoter and the gene it controls in its natural setting,
i.e., the gene from which the promoter is derived, As is known in
the art, some variation in this distance can be accommodated
without loss of promoter function. Similarly, the preferred
positioning of a regulatory sequence element with respect to a
heterologous gene to be placed under its control is defined by the
positioning of the element in its natural setting, i.e., the genes
from which it is derived. Again, as is known in the art, some
variation in this distance can also occur.
[0187] Examples of promoters suitable for use in the synthetic
genes of the present invention include viral, fungal, bacterial,
animal and plant derived promoters capable of functioning in plant,
animal, insect, fungal, yeast, or bacterial cells. The promoter may
regulate the expression of the structural gene component
constitutively, or differentially with respect to the cell, the
tissue or organ in which expression occurs or, with respect to the
developmental stage at which expression occurs, or in response to
external stimuli such as physiological stresses, or pathogens, or
metal ions, or antibiotics, amongst others.
[0188] Preferably, the promoter is capable of regulating expression
of a nucleic acid molecule in a eukaryotic cell, tissue or organ,
at least during the period of time over which the target gene is
expressed therein and more preferably also immediately preceding
the commencement of detectable expression of the target gene in
said cell, tissue or organ.
[0189] Accordingly, strong constitutive promoters are particularly
preferred for the purposes of the present invention or promoters
which may be induced by virus infection or the commencement of
target gene expression.
[0190] Plant-operable and animal-operable promoters are
particularly preferred for use in the synthetic genes of the
present invention. Examples of preferred promoters include the
bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter,
lac operator-promoter, tac promoter, SV40 late promoter, SV40 early
promoter, RSV-LTR promoter, CMV IE promoter, CaMV 35S promoter,
SCSV promoter, SCBV promoter and the like.
[0191] In consideration of the preferred requirement for high-level
expression which coincides with expression of the target gene or
precedes expression of the target gene, it is highly desirable that
the promoter sequence is a constitutive strong promoter such as the
CMV-IE promoter or the SV40 early promoter sequence, the SV40 late
promoter sequence, the CaMV 35S promoter, or the SCBV promoter,
amongst others. Those skilled in the art will readily be aware of
additional promoter sequences other than those specifically
described.
[0192] In the present context, the terms "in operable connection
with" or "operably under the control" or similar shall be taken to
indicate that expression of the structural gene region or multiple
structural gene region is under the control of the promoter
sequence with which it is spatially connected; in a cell, tissue,
organ or whole organism.
[0193] In a preferred embodiment of the invention, a structural
gene region (i.e. dispersed nucleic acid molecule or foreign
nucleic acid molecule) or multiple structural gene region is placed
operably in connection with a promoter orientation relative to the
promoter sequence, such that when it is transcribed an RNA product
is synthesized which is capable of encoding a polypeptide product
of the target gene or a fragment thereof.
[0194] However, the present invention is not to be limited to the
use of such an arrangement and the invention clearly extends to the
use of synthetic genes and genetic constructs wherein the a
structural gene region or multiple structural gene region is placed
in the "antisense" orientation relative to the promoter sequence,
such that at least a part of the RNA transcription product thereof
is complementary to the RNA encoded by the target gene or a
fragment thereof.
[0195] Clearly, as the dispersed nucleic acid molecule, foreign
nucleic acid molecule or multiple structural gene region comprises
tandem direct and/or inverted repeat sequences of the target gene,
all combinations of the above-mentioned configurations are
encompassed by the invention.
[0196] In an alternative embodiment of the invention, the
structural gene region or multiple structural gene region is
operably connected to both a first promoter sequence and a second
promoter sequence, wherein said promoters are located at the distal
and proximal ends thereof such that at least one unit of said
structural gene region or multiple structural gene region is placed
in the "sense" orientation relative to the first promoter sequence
and in the "antisense" orientation relative to the second promoter
sequence. According to this embodiment, it is also preferred that
the first and second promoters be different, to prevent competition
there between for cellular transcription factors which bind
thereto. The advantage of this arrangement is that the effects of
transcription from the first and second promoters in reducing
target gene expression in the cell may be compared to determine the
optimum orientation for each nucleotide sequence tested.
[0197] The synthetic gene preferably contains additional regulatory
elements for efficient transcription, for example a transcription
termination sequence.
[0198] The term "terminator" refers to a DNA sequence at the end of
a transcriptional unit which signals termination of transcription.
Terminators are 3'-non-translated DNA sequences which may contain a
polyadenylation signal, which facilitates the addition of
polyadenylate sequences to the 3'-end of a primary transcript.
Terminators active in plant cells are known and described in the
literature. They may be isolated from bacteria, fungi, viruses,
animals and/or plants or synthesized de novo.
[0199] As with promoter sequences, the terminator may be any
terminator sequence which is operable in the cells, tissues or
organs in which it is intended to be used.
[0200] Examples of terminators particularly suitable for use in the
synthetic genes of the present invention include the SV40
polyadenylation signal, the HSV TK polyadenylation signal, the CYCl
terminator, ADH terminator, SPA terminator, nopaline synthase (NOS)
gene terminator of Agrobacterium tumefaciens, the terminator of the
Cauliflower mosaic virus (CaMV) 35S gene, the zein gene terminator
from Zea mays, the Rubisco small subunit gene (SSU) gene terminator
sequences, subclover stunt virus (SCSV) gene sequence terminators,
any rho-independent E coli terminator, or the lacZ alpha
terminator, amongst others.
[0201] In a particularly preferred embodiment, the terminator is
the SV40 polyadenylation signal or the HSV TK polyadenylation
signal which are operable in animal cells, tissues and organs,
octopine synthase (OCS) or nopaline synthase (NOS) terminator
active in plant cells, tissues or organs, or the lacZ alpha
terminator which is active in prokaryotic cells.
[0202] Those skilled in the art will be aware of additional
terminator sequences which may be suitable for use in performing
the invention. Such sequences may readily be used without any undue
experimentation.
[0203] Means for introducing (i.e. transfecting or transforming)
cells with the synthetic genes described herein or a genetic
construct comprising same are well-known to those skilled in the
art.
[0204] In a further alternative embodiment, a genetic construct is
used which comprises two or more structural gene regions or
multiple structural gene regions wherein each of said structural
gene regions is placed operably under the control of its own
promoter sequence. As with other embodiments described herein, the
orientation of each structural gene region may be varied to
maximise its modulatory effect on target gene expression.
[0205] According to this embodiment, the promoters controlling
expression of the structural gene unit are preferably different
promoter sequences, to reduce competition there between for
cellular transcription factors and RNA polymerases. Preferred
promoters are selected from those referred to supra.
[0206] Those skilled in the art will know how to modify the
arrangement or configuration of the individual structural genes as
described supra to regulate their expression from separate promoter
sequences.
[0207] The synthetic genes described supra are capable of being
modified further, for example by the inclusion of marker nucleotide
sequences encoding a detectable marker enzyme or a functional
analogue or derivative thereof, to facilitate detection of the
synthetic gene in a cell, tissue or organ in which it is expressed.
According to this embodiment, the marker nucleotide sequences will
be present in a translatable format and expressed, for example as a
fusion polypeptide with the translation product(s) of any one or
more of the structural genes or alternatively as a non-fusion
polypeptide.
[0208] Those skilled in the art will be aware of how to produce the
synthetic genes described herein and of the requirements for
obtaining the expression thereof, when so desired, in a specific
cell or cell-type under the conditions desired. In particular, it
will be known to those skilled in the art that the genetic
manipulations required to perform the present invention may require
the propagation of a genetic construct described herein or a
derivative thereof in a prokaryotic cell such as an E. coli cell or
a plant cell or an animal cell.
[0209] The synthetic genes of the present invention may be
introduced to a suitable cell, tissue or organ without modification
as linear DNA in the form of a genetic construct, optionally
contained within a suitable carrier, such as a cell, virus particle
or liposome, amongst others. To produce a genetic construct, the
synthetic gene of the invention is inserted into a suitable vector
or episome molecule, such as a bacteriophage vector, viral vector
or a plasmid, cosmid or artificial chromosome vector which is
capable of being maintained and/or replicated and/or expressed in
the host cell, tissue or organ into which it is subsequently
introduced.
[0210] Accordingly a further aspect of the invention provides a
genetic construct which at least comprises the synthetic gene
according to any one or more of the embodiments described herein
and one or more origins of replication and/or selectable marker
gene sequences.
[0211] Genetic constructs are particularly suitable for the
transformation of a eukaryotic cell to introduce novel genetic
traits thereto, in addition to the provision of resistance
characteristics to viral pathogens. Such additional novel traits
may be introduced in a separate genetic construct or, alternatively
on the same genetic construct which comprises the synthetic genes
described herein. Those skilled in the art will recognise the
significant advantages, in particular in terms of reduced genetic
manipulations and tissue culture requirements and increased
cost-effectiveness, of including genetic sequences which encode
such additional traits and the synthetic genes described herein in
a single genetic construct.
[0212] Usually, an origin of replication or a selectable marker
gene suitable for use in bacteria is physically-separated from
those genetic sequences contained in the genetic construct which
are intended to be expressed or transferred to a eukaryotic cell,
or integrated into the genome of a eukaryotic cell.
[0213] In a particularly preferred embodiment, the origin of
replication is functional in a bacterial cell and comprises the pUC
or the ColEl origin or alternatively the origin of replication is
operable in a eukaryotic cell, tissue and more preferably comprises
the 2 micron (2.multidot.m) origin of replication or the SV40
origin of replication.
[0214] As used herein, the term "selectable marker gene" includes
any gene which confers a phenotype on a cell in which it is
expressed to facilitate the identification and/or selection of
cells which are transfected or transformed with a genetic construct
of the invention or a derivative thereof.
[0215] Suitable selectable marker genes contemplated herein include
the ampicillin-resistance gene (Amp.sup.r), tetracycline-resistance
gene (Tc.sup.r), bacterial kanamycin-resistance gene (Kan.sup.r),
the zeocin resistance gene (Zeocin is a drug of bleomycin family
which is trademark of InVitrogen Corporation), the AURI-C gene
which confers resistance to the antibiotic aureobasidin A,
phosphinothricin-resistance gene, neomycin phosphotransferase gene
(nptil), hygromycin-resistance gene, .multidot.-glucuronidase (GUS)
gene, chloramphenicol acetyltransferase (CAT) gene, green
fluorescent protein-encoding gene or the luciferase gene, amongst
others.
[0216] Preferably, the selectable marker gene is the nptil gene or
Kan.sup.r gene or green fluorescent protein (GFP)-encoding
gene.
[0217] Those skilled in the art will be aware of other selectable
marker genes useful in the performance of the present invention and
the subject invention is not limited by the nature of the
selectable marker gene.
[0218] The present invention extends to all genetic constructs
essentially as described herein, which include further genetic
sequences intended for the maintenance and/or replication of said
genetic construct in prokaryotes or eukaryotes and/or the
integration of said genetic construct or a part thereof into the
genome of a eukaryotic cell or organism.
[0219] As with dispersed or foreign nucleic acid molecules,
standard methods described supra may be used to introduce synthetic
genes and genetic constructs into the cell, tissue or organ for the
purposes of modulating the expression of the target gene, for
example liposome-mediated transfection or transformation,
transformation of cells with attenuated virus particles or
bacterial cells, cell mating, transformation or transfection
procedures known to those skilled in the art or described by
Ausubel et al (1992).
[0220] Additional means for introducing recombinant DNA into plant
tissue or cells include, but are not limited to, transformation
using CaCI.sub.2 and variations thereof, in particular the method
described by Hanahan (1983), direct DNA uptake into protoplasts
(Krens et al, 1982; Paszkowski et al, 1984), PEG-mediated uptake to
protoplasts (Armstrong et al, 1990) microparticle bombardment,
electroporation (Fromm et al., 1985), microinjection of DNA
(Crossway et a., 1986), microparticle bombardment of tissue
explants or cells (Christou et at, 1988; Sanford, 1988),
vacuum-infiltration of tissue with nucleic acid, or in the case of
plants, T-DNA-mediated transfer from Agrobacterium to the plant
tissue as described essentially by An et al. (1985),
Herrera-Estrella et a. (1983a, 1983b, 1985).
[0221] For microparticle bombardment of cells, a microparticle is
propelled into a cell to produce a transformed cell. Any suitable
ballistic cell transformation methodology and apparatus can be used
in performing the present invention. Exemplary apparatus and
procedures are disclosed by Stomp et al. (U.S. Pat. No. 5,122,466)
and Sanford and Wolf (U.S. Pat. No. 4,945,050). When using
ballistic transformation procedures, the genetic construct may
incorporate a plasmid capable of replicating in the cell to be
transformed.
[0222] Examples of microparticles suitable for use in such systems
include 1 to 5 .mu.m gold spheres. The DNA construct may be
deposited on the microparticle by any suitable technique, such as
by precipitation.
[0223] In a further embodiment of the present invention, the
synthetic genes and genetic constructs described herein are adapted
for integration into the genome of a cell in which it is expressed.
Those skilled in the art will be aware that, in order to achieve
integration of a genetic sequence or genetic construct into the
genome of a host cell, certain additional genetic sequences may be
required. In the case of plants, left and right border sequences
from the T-DNA of the Agrobacterium tumefaciens Ti plasmid will
generally be required.
[0224] The present invention further extends to an isolated cell,
tissue or organ comprising the synthetic gene described herein or a
genetic construct comprising same. The present invention extends
further to regenerated tissues, organs and whole organisms derived
from said cells, tissues and organs and to propagules and progeny
thereof.
[0225] For example, plants may be regenerated from transformed
plant cells or tissues or organs on hormone-containing media and
the regenerated plants may take a variety of forms, such as
chimeras of transformed cells and non-transformed cells; clonal
transformants (e.g., all cells transformed to contain the
expression cassette), grafts of transformed and untransformed
tissues (e.g., a transformed root stock grafted to an untransformed
scion in citrus species). Transformed plants may be propagated by a
variety of means, such as by clonal propagation or classical
breeding techniques. For example, a first generation (or T1)
transformed plants may be selfed to give homozygous second
generation (or T2) transformed plants, and the T2 plants further
propagated through classical breeding techniques.
[0226] The present invention is further described with reference to
the following non-limiting Examples.
EXAMPLE 1
[0227] Generic techniques
[0228] 1, Tissue culture manipulations
[0229] (a) Adherent cell lines
[0230] Adherent cell monolayers were grown in medium consisting of
either DMEM (Life Technologies) supplemented with 10% v/v FBS
(TRACE Biosciences or Life Technologies) or RPMI 1640 Medium (Life
Technologies) supplemented with 10% v/v FBS. Cells were grown in
incubators at 37.degree. C. in an atmosphere containing 5% v/v
CO.sub.2.
[0231] During the course of these experiments it was frequently
necessary to passage the cell monolayer. To achieve this, the
monolayers were washed twice with 1 x PBS (Sigma) and then treated
with trypsin-EDTA (Life Technologies) for 5 min at 37.degree. C.
The volumes of trypsin-EDTA used for such manipulations were
typically 20 .mu.l, 1100 .mu.l, 500 ,.mu.l, 1 ml and 2 ml for
96-well, 48-well, 6-well, T25 and T75 vessels, respectively. The
action of the trypsin-EDTA was stopped with an equal volume of
growth medium. The cells were suspended by trituration. A 1/5volume
of the cell suspension was then transferred to a new vessel
containing growth medium. Tissue culture medium volumes were
typically 192 .mu.l/well for 96-well tissue culture plates, 360
.mu.llwell for 48-well tissue culture plates, 3.8 ml/well for
6-well tissue culture plates, 9.6 ml for T25 and 39.2 ml for T75
tissue culture vessels.
[0232] Cell suspensions were counted after resuspending in an
appropriate volume of DMEM, 10% v/v FBS. An aliquot, typically 100
.mu.l, was transferred to a haemocytometer (Hawksley) and cell
numbers counted microscopically.
[0233] (b) Non-adherent cells
[0234] Non-adherent cells were grown in growth medium similarly to
adherent cell lines.
[0235] As in the case of adherent monolayers, frequent changes of
tissue culture vessels were necessary. For T25 and T75 vessels, the
cell suspension was removed to 50 ml sterile plastic tubes (Falcon)
and centrifuged for 5 min at 500 x g and 4.degree. C. The
supernatant was then discarded and the cell pellet suspended in
growth medium. The cell suspension was then placed into a new
tissue culture vessel. For 96-well, 48-well, and 6-well vessels,
the vessels were centrifuged for 5 min at 500 x g and 4.degree. C.
The supernatant was then aspirated away from the cell pellet and
the cells suspended in growth medium. The cells were then
transferred to a new tissue culture vessel. Tissue culture media
volumes were typically 200 .mu.l/well for 96-well tissue culture
plates, 400 .mu.l/well for 48-well tissue culture plates, 4 ml/well
for 6-well tissue culture plates, 11 ml for T25 and 40 ml for T75
tissue culture vessels.
[0236] Passaging the cell suspensions was achieved in the following
manner: Cells were centrifuged for 5 min at 500 x g and 4.degree.
C. and suspended in 5 ml growth medium. Then 0.5 ml (T25) or 1.0 ml
(T75) of the cell suspension was transferred to a new vessel
containing growth medium. For cells in 96-well, 48-well, and 6-well
plates, a 1/5volume of cells was transferred to the corresponding
wells of a new vessel containing 4/5volume of growth medium.
[0237] Cells were counted as described for adherent cells.
[0238] 2. Protocol for Freezing and Thawing Cells
[0239] Adherent monolayers were washed twice with 1 x PBS and then
treated with trypsin-EDTA for 5 min at 37.degree. C. Non-adherent
cells were centrifuged for 5 min at 500 x g and 4.degree. C. The
cells were suspended by trituration and transferred to storage
medium consisting of DMEM or RPMI 1640 supplemented with 20% v/v
FBS and 10% v/v dimethylsulfoxide (Sigma). The concentration of
cells was determined by haemocytometer counting and diluted to
10.sup.5 cells per ml. Aliquots were transferred to 1.5 ml
cryotubes (Nunc) and the tubes were placed in a Cryo 1.degree. C.
Freezing Container (Nalgene) containing propan-2-ol (BDH) and
cooled slowly to -70.degree. C. The tubes of cells were then stored
at -70.degree. C. Reanimation of frozen cells was achieved by
warming the tubes to 0.degree. C. on ice then transferring the
cells to a T25 flask containing DMEM and 20% v/v FBS and incubating
at 37.degree. C. in an atmosphere of 5% v/v CO.sub.2.
[0240] 3. Cloning of Cell Lines
[0241] Adherent and non-adherent mammalian cell lines were
transfected with plasmid vectors containing expression constructs
to target specific genes of interest. Stable, transformed colonies
were selected over a period of 2-3 weeks using cell growth medium
(either DMEM, 10% v/v FBS or RPMI 1640, 10% v/v FBS) supplemented
with geneticin. Individual colonies were cloned to establish clonal
lines of transfected cells.
[0242] (a) Conditioned Medium
[0243] Conditioned media were prepared by overlaying
20-30%-confluent monolayers of cells grown in a T75 vessel with 40
ml of DMEM, 10% v/v FBS. Vessels were incubated at 37.degree. C. in
5% v/v CO.sub.2 for 24 hr, after which the growth medium was
transferred to a sterile 50 ml tube (Falcon) and centrifuged at 500
x g. The medium was passed through a 0.45 .mu.m filter and decanted
to a fresh sterile tube for use as conditioned medium.
[0244] (b) Adherent Cells
[0245] Individual lines were cloned from discrete colonies as
follows: First, the medium was removed from an individual well of a
6-well tissue culture vessel and the cell colonies washed twice
with 2 ml of 1 x PBS. Individual colonies were then detached from
the plastic culture vessel with a sterile plastic pipette tip and
moved to a 96-well plate containing 200 .mu.l of conditioned medium
supplemented with geneticin. The vessel was incubated at 37.degree.
C. in 5% v/v CO.sub.2 for approximately 72 hr. Individual wells
were examined microscopically for growing colonies and the medium
replaced with fresh growth medium. When the monolayer of each
stable line had reached about 90% confluency it was transferred in
successive steps until the stable, transformed line was housed in a
T25 tissue culture vessel. At this point, aliquots of each stable
cell line were frozen for long term maintenance.
[0246] (c) Non-adherent Cells
[0247] Non-adherent cells were cloned by dilution cloning. Cell
concentration was determined microscopically using a haemocytometer
slide and cells were diluted to 10 cells per ml in conditioned
medium. Single wells of 96-well tissue culture vessels were seeded
with 200 .mu.l of the diluted cells and the plates were incubated
at 37.degree. C. in 5% v/v CO.sub.2 for 48 hr. Wells were inspected
microscopically and those containing a single colony, arising from
a single cell, were defined as clonal cell lines. The medium was
removed and replaced with 200 .mu.l of fresh conditioned medium and
cells incubated at 37.degree. C. in 5% v/v CO.sub.2 for 48 hr.
After this time, conditioned medium was replaced with 200 .mu.l of
DMEM, 10% v/v FBS and 1.5 mg/l genetecin and cells again incubated
at 37.degree. C. in 5% v/v CO.sub.2. Colonies were allowed to
expand in successive steps, with medium changes every 48 hr, until
the stable, transformed lines were housed in T25 tissue culture
vessels. At this point, aliquots of each stable cell line were
frozen for long term maintenance
[0248] 4. Southern Blot Analysis of Mammalian Genomic DNA
[0249] For all subsequent examples, Southern blot analyses of
genomic DNA were carried out according to the following
protocol.
[0250] A T75 tissue culture vessel containing 40 ml of DMEM or RPMI
1640, 10% v/v FBS was seeded with 4.times.10.sup.6 cells and
incubated at 37.degree. C. in 5% v/v CO.sub.2 for 24 hr.
[0251] (a) Adherent Cells
[0252] Medium was decanted and 5 ml of 1 x PBS was added to the T75
flask to wash the monolayer by gentle rocking then the PBS was
decanted. The wash was repeated and the monolayer overlaid with 2
ml of 1 x PBS/1 x Trypsin-EDTA, ensuring even coverage of the
monolayer by gentle rocking. The flask was incubated at 37.degree.
C. in 5% v/v CO.sub.2 until the monolayer separated from the flask,
then 2 ml of medium with 10% v/v FBS was added. The suspended cells
were transferred into a 10 ml capped tube to which was added 3 ml
of ice-cold 1 x PBS. The tubewas inverted several times to mix and
the cells were collected by centrifugation at 500 x g for 10 min at
4.degree. C. The supernatant was decanted and the pellet suspended
in 5 ml of ice-cold 1 x PBS by gentle vortexing and a sample was
counted (.apprxeq.2.times.10.sup.8). The cells were collected by
centrifugation at 500 x g for 10 min at 4.degree. C. and the
supernatant decanted.
[0253] (b) Non-adherent Cells
[0254] The cell suspension is transferred into a 50 ml Falcon tube,
centrifuged at 500 x g for 10 min at 4.degree. C. and the
supernatant decanted. The pellet was suspended in 5 ml ice-cold 1 x
PBS by gentle vortexing and the cells collected by centrifugation
at 500 x g for 10 min at 4.degree. C. The supernatant was decanted
and the pellet was resuspended in 5 ml of ice-cold 1 x PBS by
gentle vortexing and a sample counted (.apprxeq.2.times.10.sup.8).
The cells were collected by centrifugation at 500 x g for 10 min at
4.degree. C. and the supernatant decanted.
[0255] (c) DNA Extraction and Analysis
[0256] Genomic DNA, for both adherent and non-adherent cell lines,
was extracted using the Qiagen Genomic DNA extraction kit (Cat No.
10243) according to the supplier's instructions. The concentration
of genomic DNA was determined from absorbance at 260 nm using a
Beckman model DU64 photospectrometer.
[0257] Genomic DNA (10 .mu.g) was digested with appropriate
restriction endonucleases and buffer in a volume of 200 .mu.l at
37.degree. C. for approximately 16 hr. Following digestion, 20
.mu.l of 3M sodium acetate, pH 5.2, and 500.mu.l of absolute
ethanol were added to the digest and the solution mixed by
vortexing and chilled at -20.degree. C. for 2 hr. DNA was recovered
by centrifugation at 10,000 x g for 30 min at 4.degree. C. The
supernatant was removed and the DNA pellet rinsed with 500 .mu.l of
70% v/v ethanol, the pellet air-dried and the DNA dissolved in 20
.mu.l of water.
[0258] Gel loading buffer (0.25% w/v bromophenol blue (Sigma),
0.25% w/v xylene cyanol FF (Sigma), 15% w/v Ficoll Type 400
(Pharmacia)) (5 .mu.l) was added to the digested DNA and the
mixture transferred to a well of a 0.7% w/v agarose/TAE gel
containing 0.5 .mu.g/ml of ethidium bromide. The DNA fragments were
electrophoresed through the gel at 14 volts for approximately 16
hr. An appropriate DNA size marker was included in a parallel
lane.
[0259] The gel was soaked in 1.5 M NaCI, 0.5 M NaOH then in 1.5 M
NaCI, 0.5 M Tris-HOCI, pH 7.0. The DNA fragments were then
capillary blotted to Hybond NX (Amersham) membrane and fixed by UV
cross-linking (Bio Rad GS Gene Linker).
[0260] The Hybond membrane was rinsed in sterile water and stained
in 0.4% v/v methylene blue in 300mM sodium acetate, pH 5.2, for 5
min to visualize the transferred genomic DNA. The membrane was then
rinsed twice in sterile water, destained in 40% v/v ethanol then
rinsed in sterile water.
[0261] The membrane was placed in a Hybaid bottle with 5 ml of
pre-hybridization solution (6 x SSPE, 5 x Denhardt's reagent, 0.5%
w/v SDS, 100 .mu.g/ml denatured, fragmented herring sperm DNA) and
pre-hybridized at 60.degree. C. for approximately 14 hr in a
hybridization oven with constant rotation (6 rpm).
[0262] Probe (25 ng) was labelled with [.alpha..sup.32P]-dCTP
(specific activity 3000 Ci/mmol) using the Megaprime DNA labelling
system as per the supplier's instructions (Amersham Cat. No.
RPN1606). Labelled probe was passed through a G50 Sephadex Quick
Spin (trademark) column (Roche, Cat. No. 1273973) to remove
unincorporated nucleotides as per the supplier's instructions.
[0263] The heat-denatured labelled probe was added to 2 ml of
hybridization buffer (6 x SSPE, 0.5% w/v SDS, 100 .mu.g/ml
denatured, fragmented herring sperm DNA) pre-warmed to 60.degree.
C. The pre-hybridization buffer was decanted and replaced with 2 ml
of pre-warmed hybridization buffer containing the labelled probe.
The membrane was hybridized at 60.degree. C. for approximately 16
hr in a hybridization oven with constant rotation (6 rpm).
[0264] The hybridization buffer containing the probe was decanted
and the membrane subjected to sequential washes as follows:
[0265] 2 x SSC, 0.5% w/v SDS for 5 min at room temperature;
[0266] 2 x SSC, 0.1% w/v SDS for 15 min at room temperature;
[0267] 0.1 x SSC, 0.5% w/v SDS for 30 min at 37.degree. C. with
gentle agitation;
[0268] 0.1 x SSC, 0.5% w/v SDS for 1 hour at 68.degree. C. with
gentle agitation; and
[0269] 0.1 x SSC for 5 min at room temperature with gentle
agitation.
[0270] Washing duration at 68.degree. C. varied based on the amount
of radioactivity detected with a hand-held Geiger counter.
[0271] The damp membrane was wrapped in plastic wrap and exposed to
X-ray film (Curix Blue HC-S Plus, AGFA) for 24-48 hr and the film
developed to visualize bands of probe hybridized to genomic
DNA.
[0272] 5. Immunofluorescent Labelling of Cultured Cells
[0273] Glass microscope cover slips (12 mm .times.12 mm) were
flamed with ethanol then submerged in 2 ml growth medium, two per
well, in six-well plates. Cells were added to wells in 1-2 ml
medium to give a density after 16 hr growth such that cells remain
isolated (200,000 to 500,000 per well depending on size and growth
rate). Without removing the cover slips from the wells, the medium
was aspirated and cells were washed with PBS. For fixation, cells
were treated for 1 hr with 4% w/v paraformaldehyde (Sigma) in PBS
then washed three times with PBS. Fixed cells were permeabilized
with 0.1% v/v Triton X-100 (Sigma) in PBS for 5 min then washed
three times with PBS. Cells on cover slips were blocked on one drop
(about 100 .mu.l) of 0.5% w/v bovine serum albumin Fraction V (BSA,
Sigma) for 10 min. Cover slips were then placed for at least 1 h on
25 .mu.l drops of primary mouse monoclonal antibody which had been
diluted {fraction (1/100)}in 0.5% v/v BSA in PBS. Cells on cover
slips were then washed three times with 100 .mu.l of 0.5% v/v BSA
in PBS for about 3 min each before being placed for 30 min to 1 hr
on 25 .mu.l drops of Alexa Fluor (registered trademark) 488 goat
anti-mouse IgG conjugate (Molecular Probes) secondary antibody
diluted {fraction (1/100)}in 0.5% v/v BSA in PBS. Cells on cover
slips were then washed three times with PBS. Cover slips were
mounted on glass microscope slides, three to a slide, in
glycerol/DABCO (25 mg/ml DABCO (1,4-diazabicyclo (2.2.2)octane
(Sigma D 2522)) in 80% v/v glycerol in PBS) and examined with a
100x oil immersion objective under UV illumination at 500-550
nm.
EXAMPLE 2
[0274] Genetic Constructs Comprising BEV Polymerase Gene Sequences
Linked to the CMV Promoter Sequence and/or the SV40L Promoter
Sequence
[0275] 1. Commercial Plasmids
[0276] Plasmid pBluescriptII (SK+)
[0277] Plasmid pBluescriptII (SK+) is commercially available from
Stratagene and comprises the LacZ promoter sequence and lacZ-alpha
transcription terminator, with a multiple cloning site for the
insertion of structural gene sequences therein. The plasmid further
comprises the ColE1 and f1 origins of replication and
ampicillin-resistance gene.
[0278] Plasmid pSVL
[0279] Plasmid pSVL is commercially-obtainable from Pharmacia and
serves as a source of the SV40 late promoter sequence. The
nucleotide sequence of pSVL is also publicly available as GenBank
Accession Number U13868.
[0280] Plasmid pCR2.1
[0281] Plasmid pCR2.1 is commercially available from Invitrogen and
comprises the LacZ promoter sequence and lacZ-.alpha. transcription
terminator, with a cloning site for the insertion of structural
gene sequences there between. Plasmid pCR2.1 is designed to clone
nucleic acid. fragments by virtue of the A-overhang frequently
synthesized by Taq polymerase during the polymerase chain reaction.
PCR fragments cloned in this fashion are flanked by two EcoRI
sites. The plasmid further comprises the ColE1 and f1 origins of
replication and kanamycin-resistance and ampicillin-resistance
genes.
[0282] Plasmid pEGFP-N1 MCS
[0283] Plasmid pEGFP-N1 MCS (FIG. 1; Olontech) contains the CMV IE
promoter operably connected to an open reading frame encoding a
red-shifted variant of wild-type green fluorescent protein (GFP;
Prasher et al, 1992; Chalfie et aL, 1994; Inouye and Tsuji, 1994),
which has been optimised for brighter fluorescence. The specific
GFP variant encoded by pEGFP-N1 MCS has been disclosed by Cormack
et al. (1996). Plasmid pEGFP-N1 MCS contains a multiple cloning
site comprising Bg/ll and BamHl sites and many other restriction
endonuclease cleavage sites, located between the CMV IE promoter
and the GFP open reading frame. Structural genes cloned into the
multiple cloning site will be expressed at the transcriptional
level if they lack a functional translation start site, however
such structural gene sequences will not be expressed at the protein
level (i.e. translated). Structural gene sequences inserted into
the multiple cloning site which comprise a functional translation
start site will be expressed as GFP fusion polypeptides if they are
cloned in-frame with the GFP-encoding sequence. The plasmid further
comprises an SV40 polyadenylation signal downstream of the GFP open
reading frame to direct proper processing of the 3'-end of mRNA
transcribed from the CMV-IE promoter sequence. The plasmid further
comprises the SV40origin of replication functional in animal cells;
the neomycin-resistance gene comprising SV40 early promoter (SV40
EP in FIG. 1) operably connected to the
neomycin/kanamycin-resistance gene derived from Tn5 (Kan/neo in
FIG. 1) and the HSV thymidine kinase polyadenylation signal (HSV TK
poly (A) in FIG. 1), for selection of transformed cells on
kamanycin, neomycin or G418; the pUC19 origin of replication which
is functional in bacterial cells (pUC Ori in FIG. 1), and the f1
origin of replication for single-stranded DNA production (f1 Ori in
FIG. 1).
[0284] 2. Expression Cassettes
[0285] Plasmid pCMV.cass
[0286] Plasmid pCMV.cass (FIG. 2) is an expression cassette for
driving expression of a structural gene sequence under control of
the CMV-IE promoter sequence. Plasmid pCMV.cass was derived from
pEGFP-N1 MCS by deletion of the GFP open reading frame as follows:
Plasmid pEGFP-N1 MCS was digested with PinAl and Notl blunt-ended
using Pful polymerase and then re-ligated. Structural gene
sequences are cloned into pCMV.cass using the multiple cloning
site, which is identical to the multiple cloning site of pEGFP-N1
MCS, except it lacks the PinAl site.
[0287] Plasmid pCMV.SV40L.cass
[0288] Plasmid pCMV.SV40L.cass (FIG. 3) comprises the synthetic
poly A site and the SV40 late promoter sequence from plasmid
pCR.SV40L (FIG. 4), sub-cloned as a Sal I fragment, into the Sal I
site of plasmid pCMV.cass (FIG. 2), such that the CMV-IE promoter
and SV40 late promoter sequences are capable of directing
transcription in the same direction. Accordingly, the synthetic
poly(A) site at the 5'end of the SV40 promoter sequence is used as
a transcription terminator for structural genes expressed from the
CMV IE promoter in this plasmid, which also provides for the
insertion of said structural gene via the multiple cloning site
present between the SV40 late promoter and the synthetic poly(A)
site (FIG. 5). The multiple cloning sites are located behind the
CMV-IE and SV40 late promoters, including BamHl and BgAl sites.
[0289] Plasmid pCMV.SV40LR.cass
[0290] Plasmid pCMV.SV40LR.cass (FIG. 4) comprises the SV40 late
promoter sequence derived from plasmid pCR.SV40L, sub-cloned as a
SaAl fragment into the SaA site of the plasmid pCMV.cass (FIG. 2),
such that the CMV-IE or the SV40 late promoter may drive
transcription of a structural gene or a multiple structural gene
unit, in the sense or antisense orientation, as desired. A multiple
cloning site is positioned between the opposing CMV-IE and SV40
late promoter sequences in this plasmid to facilitate the
introduction of a structural gene sequence. In order for expression
of a structural gene sequence to occur from this plasmid, it must
be introduced with its own transcription termination sequence
located at the 3'end, because there are no transcription
termination sequences located between the opposing CMV-IE and SV40
late promoter sequences in this plasmid. Preferably, the structural
gene sequence or multiple structural gene unit which is to be
introduced into pCMV,SV40LR.cass will comprise both a 5'and a
3'polyadenylation signal as follows:
[0291] (i) where the structural gene sequence or multiple
structural gene unit is to be expressed in the sense orientation
from the CMV IE promoter sequence and/or in the antisense
orientation from the SV40 late promoter, the 5'polyadenylation
signal will be in the antisense orientation and the
3'polyadenylation signal will be in the sense orientation; and
[0292] (ii) where the structural gene sequence or multiple
structural gene unit is to be expressed in the antisense
orientation from the CMV IE promoter sequence and/or in the sense
orientation from the SV40 late promoter, the 5'polyadenylation
signal will be in the sense orientation and the 3'polyadenylation
signal will be in the antisense orientation.
[0293] Alternatively or in addition, suitably-oriented terminator
sequences may be placed at the 5'-end of the CMV and SV40L
promoters, as shown in FIG. 4.
[0294] Alternatively, plasmid pCMV.SV40LR.cass is further modified
to produce a derivative plasmid which comprises two polyadenyfation
signals located between the CMV IE and SV40 late promoter
sequences, in approriate orientations to facilitate expression of
any structural gene located therebetween in the sense or antisense
orientation from either the CMV IE promoter or the SV40 promoter
sequence. The present invention clearly encompasses such
derivatives.
[0295] Alternatively appropriately oriented terminators could be
placed upstream of the CMV and SV40L promoters such that
transcriptional termination could occur after readthrough of each
of the two promoters in the antisense orientation.
[0296] 3. Intermediate Constructs
[0297] Plasmid pCR-Bgl-GFP-Bam
[0298] Plasmid pCR.Bgt-GFP-Bam (FIG. 5) comprises an internal
region of the GFP open reading frame derived from plasmid pEGFP-N1
MCS (FIG. 1) placed operably under the control of the lacZ
promoter. To produce this plasmid, a region of the GFP open reading
frame was amplified from pEGFP-N1 MCS using the amplification
primers Bgl-GFP (SEQ ID NO:1) and GFP-Bam (SEQ ID NO:2) and cloned
into plasmid pCR2.1. The internal GFP-encoding region in plasmid
pCR.Bgl-GFP-Bam lacks functional translational start and stop
codons.
[0299] Plasmid pBSlI(SK+).EGFP
[0300] Plasmid pBSII(SK+).EGFP (FIG. 6) comprises the EGFP open
reading frame derived from plasmid pEGFP-N1 MCS (FIG. 1) placed
operably under the control of the lacZ promoter. To produce this
plasmid, the EGFP encoding region of pEGFP-N1 MCS was excised as a
NotllXhol fragment and cloned into the Notl/Xhol cloning sites of
plasmid pBluescript 11 (SK+).
[0301] Plasmid pCMV.EGFP
[0302] Plasmid pCMV.EGFP (FIG. 7) is capable of expressing the EGFP
structural gene under the control of the CMV-IE promoter sequence.
To produce this plasmid the EGFP sequence from plasmid
pBSll(SK+).EGFP was excised as BamHI/Sacl fragment and cloned into
the Bgil/Sacl sites of plasmid pCMV.cass (FIG. 2).
[0303] Plasmid pCR.SV40L
[0304] Plasmid pCR.SV40L (FIG. 8) comprises the SV40 late promoter
derived from plasmid pSVL (GenBank Accession No. U13868;
Pharmacia), cloned into pCR2.1 (Stratagene). To produce this
plasmid, the SV40 late promoter was amplified using the primers
SV40-1 (SEQ ID NO:3) and SV40-2 (SEQ ID NO:4) which comprise Sal I
cloning sites to facilitate sub-cloning of the amplified DNA
fragment into pCMV.cass. The primer also contains a synthetic poly
(A) site at the 5'end, such that the amplicification product
comprises the synthetic poly(A) site at the 5'end of the SV40
promoter sequence.
[0305] Plasmid pCR.BEV.1
[0306] The BEV RNA-dependent RNA polymerase coding region was
amplified as a 1,385 bp DNA fragment from a full-length cDNA clone
encoding same, using primers designated BEV-1 (SEQ ID NO:5) and
BEV-2 (SEQ ID NO:6), under standard amplification conditions. The
amplified DNA contained a 5'-Bgl ll restriction enzyme site,
derived from the BEV-1 primer sequence and a 3'BamHl restriction
enzyme site, derived from the BEV-2 primer sequence. Additionally,
as the BEV-1 primer sequence contains a translation start signal
5'-ATG-3'engineered at positions 15-17, the amplified BEV
polymerase structural gene comprises the start site in-frame with
BEV polymerase-encoding nucleotide sequences, Thus, the amplified
BEV polymerase structural gene comprises the ATG start codon
immediately upstream (ie. juxtaposed) to the BEV
polymerase-encoding sequence. There is no translation stop codon in
the amplified DNA. This plasmid is present as FIG. 9.
[0307] Plasmid pCR.BEV.2
[0308] The complete BEV polymerase coding region was amplified from
a full-length CDNA clone encoding same, using primers BEV-1 and
BEV-3. Primer BEV-3 comprises a BamHI restriction enzyme site at
positions 5 to 10 inclusive and the complement of a translation
stop signal at positions 11 to 13. As a consequence, an open
reading frame comprising a translation start signal and translation
stop signal, contained between the Bgl II and BamHl restriction
sites. The amplified fragment was cloned into pCR2.1 (Stratagene)
to produce plasmid pCR2.BEV.2 (FIG. 10).
[0309] Plasmid pCR.BEV.3
[0310] A non-translatable BEV polymerase structural gene was
amplified from a full-length BEV polymerase CDNA clone using the
amplification primers BEV-3 (SEQ ID NO:7) and BEV-4 (SEQ ID NO:8).
Primer BEV-4 comprises a BglI cloning site at positions 5-10 and
sequences downstream of this Bg/II site are homologous to
nucleotide sequences of the BEV polymerase gene. There is no
functional ATG start codon in the amplified DNA product of primers
BEV-3 and BEV-4. The BEV polymerase is expressed as part of a
polyprotein and, as a consequence, there is no ATG translation
start site in this gene. The amplified DNA was cloned into plasmid
pCR2.1 (Stratagene) to yield plasmid pCR.BEV.3 (FIG. 11).
[0311] Plasmid pCMV.EGFP.BEV2
[0312] Plasmid pCMV.EGFP.BEV2 (FIG. 12) was produced by cloning the
BEV polymerase sequence from pCR.BEV.2 as a Bglll/BamHl fragment
into the BamHl site of pCMV.EGFP.
[0313] 4. Control Plasmids
[0314] Plasmid pCMV.BEV-2
[0315] Plasmid pCMV.BEV.2 (FIG. 13) is capable of expressing the
entire BEV polymerase open reading frame under the control of
CMV-IE promoter sequence. To produce pCMV.BEV.2, the BEV polymerase
sequence from pCR.BEV.2 was sub-cloned in the sense orientation as
a Bg/ll-to-BamHl fragment into Bg/ll/BamHl-digested pCMV.cass (FIG.
2).
[0316] Plasmid pCMVBEEV.3
[0317] Plasmid pCMV.BEV.3 (FIG. 14) expresses a non-translatable
BEV polymerase structural gene in the sense orientation under the
control of the CMV-IE promoter sequence. To produce pCMV.BEVnt, the
BEV polymerase sequence from pCR.BEV.3 was sub-cloned in the sense
orientation as a Bg/ll-to-BamHl fragment into Bg/ll/BamHl-digested
pCMV.cass (FIG. 2).
[0318] Plasmid pCMV.VEB
[0319] Plasmid pCMV.VEB (FIG. 15) expresses an antisense BEV
polymerase mRNA under the control of the CMV-lE promoter sequence.
To produce plasmid pCMV.VEB, the BEV polymerase sequence from
pCR.BEV.2 was sub-cloned in the antisense orientation as a
Bg/ll-to-BamrHI fragment into Bg/ll-BamHl-digested 'pCMV.cass (FIG.
2).
[0320] Plasmid pCMV.BEV.GFP
[0321] Plasmid pCMV.BEV.GFP (FIG. 16) was constructed by cloning
the GFP fragment from pCR.Bgl-GFP-Bam as a Bg/ll/BamHl fragment
into BamHl-digested pCMV.BEV.2. This plasmid serves as a control in
some experiments and also as an intermediate construct.
[0322] Plasmid pCMV.BEV-SV40L.0
[0323] Plasmid pCMV.BEV.SV40L.0 (FIG. 17) comprises a translatable
BEV polymerase structural gene derived from plasmid pCR.BEV.2
inserted in the sense orientation between the CMV-lE promoter and
the SV40 late promoter sequences of plasmid pCMV.SV40L.cass. To
produce plasmid pCMV.BEV.SV40L-O, the BEV polymerase structural
gene was sub-cloned as a BgAl-to-BamHl fragment into Bg/ll-digested
pCMV.SV40L.cass DNA.
[0324] Plasmid pCMV.O.SV40L-BEV
[0325] Plasmid pCMV.O.SV40L.BEV (FIG. 18) comprises a translatable
BEV polymerase structural gene derived from plasmid pCR.BEV.2
cloned downstream of tandem CMV-lE promoter and SV40 late promoter
sequences present in plasmid pCMV.SV40L.cass. To produce plasmid
pCMV.O.SV40L.BEV, the BEV polymerase structural gene was sub-cloned
in the sense orientation as a Bg/ll-to-BamHI fragment into
BamHl-digested pCMV.SV40L.cass DNA.
[0326] Plasmid pCMV.O.SV40L.VEB
[0327] Plasmid pCMV.O.SV40L.VEB (FIG. 19) comprises an antisense
BEV polymerase structural gene derived from plasmid pCR.BEV.2
cloned downstream of tandem CMV-lE promoter and SV40 late promoter
sequences present in plasmid pCMV.SV40L.cass. To produce plasmid
pCMV.O.SV40L.VEB, the BEV polymerase structural gene was sub-cloned
in the antisense orientation as a Bg/ll-to-BamHI fragment into
BamHl-digested pCMV.SV40L.cass DNA.
[0328] 5. Test Plasmids
[0329] Plasmid pCMV.BEVx2
[0330] Plasmid pCMV.BEVx2 (FIG. 20) comprises a direct repeat of a
complete BEV polymerase open reading frame under the control of the
CMV-lE promoter sequence. In eukaryotic cells at least, the open
reading frame located nearer the CMV-lE promoter is translatable.
To produce pCMV.BEVx2, the BEV polymerase structural gene from
plasmid pCR.BEV.2 was sub-cloned in the sense orientation as a
BgAl-to-BamHl fragment into BamHi-digested pCMV.BEV.2, immediately
downstream of the BEV polymerase structural gene already present
therein.
[0331] Plasmid pCMV.BEVx3
[0332] Plasmid pCMV.BEVx3 (FIG. 21) comprises a direct repeat of
three complete BEV polymerase open reading frames under the control
of the CMV-1 E promoter. To produce pCMV.BEVx3, the BEV polymerase
fragment from pCR.BEV.2 was cloned in the sense orientation as a
BgAilBamHl fragment into the BamHl site of pCMV.BEVx2, immediately
downstream of the BEV polymerase sequences already present
therein.
[0333] Plasmid pCMV.BEVx4
[0334] Plasmid pCMV.BEVx4 (FIG. 22) comprises a direct repeat of
four complete BEV polymerase open reading frames under the control
of the CMV-1 E promoter. To produce pCMV.BEVx4, the BEV polymerase
fragment from pCR.BEV.2 was cloned in the sense orientation as a
BgIll/BamHf fragment into the BamHI site of pCMV.BEVx3, immediately
downstream of the BEV polymerase sequences already present
therein.
[0335] Plasmid pCMV.BEV.SV40L.BEV
[0336] Plasmid pCMV.BEV.SV40L.BEV(FIG. 23) comprises a multiple
structural gene unit comprising two BEV polymerase structural genes
placed operably and separately under control of the CMV-lE promoter
and SV40 late promoter sequences. To produce plasmid
pCMV.BEV.SV40L.BEV, the translatable BEV polymerase structural gene
present in pCR.BEV.2 was sub-cloned in the sense orientation as a
BgIll-to-BamHI fragment behind the SV40 late promoter sequence
present in BamHi-digested pCMV.BEV.SV40L-O.
[0337] Plasmid pCMV.BEV.SV40L.VEB
[0338] Plasmid pCMV.BEV.SV40L.VEB (FIG. 24) comprises a multiple
structural gene unit comprising two BEV polymerase structural genes
placed operably and separately under control of the CMV-lE promoter
and SV40 late promoter sequences. To produce plasmid
pCMV.BEV.SV40L.VEB, the translatable BEV polymerase structural gene
present in pCR.BEV.2 was sub-cloned in the antisense orientation as
a Bg/ll-to-BamHI fragment behind the SV40 late promoter sequence
present in BamHl-digested pCMV.BEV.SV4OL-O. In this plasmid, the
BEV polymerase structural gene is expressed in the sense
orientation under control of the CMV-lE promoter to produce a
translatable mRNA, whilst the BEV polymerase structural gene is
also expressed under control of the SV40 promoter to produce an
antisense mRNA species.
[0339] Plasmid pCMV.BEV.GFP.VEB
[0340] Plasmid pCMV.BEV.GFP.VEB (FIG. 25) comprises a BEV
structural gene inverted repeat or palindrome, interrupted by the
insertion of a GFP open reading frame (stuffer fragment) between
each BEV structural gene sequence in the inverted repeat. To
produce plasmid pCMV.BEV.GFP.VEB, the GFP stuffer fragment from
pCR.BgI-GFP-Bam was first sub-cloned in the sense orientation as a
BgAl-to-BamHl fragment into BamHi-digested pCMV.BEV.2 to produce an
intermediate plasmid pCMV.BEV.GFP wherein the BEV
polymerase-encoding and GFP-encoding sequences are contained within
the same 5'-Bg/ll-to-BamHI-3'fragment. The BEV polymerase
structural gene from pCMV.BEV.2 was then cloned in the antisense
orientation as a Bg/ll-to-BamHI fragment into BamHl-digested
pCMV.BEV.GFP. The BEV polymerase structural gene nearer the CMV-lE
promoter sequence in plasmid pCMV.BEV.GFP.VEB is capable of being
translated, at least in eukaryotic cells.
[0341] Plasmid pCMV.EGFP.BEV2.PFG
[0342] Plasmid pCMV.EGFP.BEV2.PFG (FIG. 26) comprise a GFP
palindrome, interrupted by the insertion of a BEV polymerase
sequence between each GFP structural gene in the inverted repeat.
To produce this plasmid the GFP fragment from pCR.BgI-GFP-Bam was
cioned as a Bg/ll/BamHI fragment into the BamHI site of
pCMV.EGFP.BEV2 in the antisense orientation relative to the CMV
promoter.
[0343] Plasmid pCMV.BEV.SV40LR
[0344] Plasmid pCMV.BEV.SV40LR (FIG. 27) comprises a structural
gene comprising the entire BEV polymerase open reading frame placed
operably and separately under control of opposing CMV-lE promoter
and SV40 late promoter sequences, thereby potentially producing BEV
polymerase transcripts at least from both strands of the fun-length
BEV polymerase structural gene. To produce plasmid pCMV.BEV.SV40LR,
the translatable BEV polymerase structural gene present in
pCR.BEV.2 was sub-cloned, as a BgAl-to-BamHl fragment, into the
unique Bgni site of plasmid pCMV.SV40LR.cass, such that the BEV
open reading frame is present in the sense orientation relative to
the CMV-lE promoter sequence.
[0345] Those skilled in the art will recognise that it is possible
to generate a plasmid wherein the BEV polymerase fragment from
pCR.BEV.2 is inserted in the antisense orientation, relative to the
CMV IE promoter sequence, using this cloning strategy. The present
invention further encompasses such a genetic construct.
EXAMPLE 3
[0346] Genetic constructs comprising the porcine .alpha.-1
,3-galactosyltransferase (Gait) structural gene sequence or
sequences operably connected to the CMV promoter sequence and/or
the SV40L promoter sequence
[0347] 1. Commercial Plasmids
[0348] Plasmid pcDNA3
[0349] Plasmid pcDNA3 is commercially available from Invitrogen and
comprises the CMV-lE promoter and BGHpA transcription terminator,
with multiple cloning sites for the insertion of structural gene
sequences there between. The plasmid further comprises the ColE1
and fl origins of replication and neomycin-resistance and
ampicillin-resistance genes.
[0350] 2. Intermediate Plasmids
[0351] Plasmid pcDNA3.Galt
[0352] Plasmid pcDNA3.Galt (BresaGen Limited, South Australia,
Australia; FIG. 28) is plasmid pcDNA3 (Invitrogen) and comprises
the cDNA sequence encoding porcine gene
alpha-1,3-galactosyltransferase (Galt) operably under the control
of the CMV-lE promoter sequence such that it is capable of being
expressed therefrom. To produce plasmid pcDNA3.Galt, the porcine
gene alpha-1,3-galactosyltransferase cDNA was cloned as an EcoRl
fragment into the EcoRl cloning site of pcDNA3. The plasmid further
comprises the ColE1 and fl origins of replication and the neomycin
and ampicillin-resistance genes.
[0353] 3. Control Plasmids
[0354] Plasmid pCMV.Galt
[0355] Plasmid pCMV.Galt (FIG. 29) is capable of expressing the
Galt structural gene under the control of the CMV-lE promoter
sequence. To produce plasmid pCMV.Galt, the Galt sequence from
plasmid pcDNA3.Galt was excised as an EcoRl fragment and cloned in
the sense orientation into the EcoRi site of plasmid pCMV.cass
(FIG. 2).
[0356] Plasmid pCMV.EGFP.Galt
[0357] Plasmid pCMV.EGFP.Galt (FIG. 30) is capable of expressing
the Galt structural gene as a Galt fusion polypeptide under the
control of the CMV-lE promoter sequence. To produce plasmid
pCMV.EGFP.Galt, the Galt sequence from pCMV.GaIt (FIG. 29) was
excised as a Bg/lllBamHl fragment and cloned into the BamHl site of
pCMV.EGFP.
[0358] Plasmid pCMV.Galt.GFP
[0359] Plasmid pCMV.Galt.GFP (FIG. 31) was produced by cloning the
Galt cDNA as an EcORI fragment from pCDNA3 into EcoRi-digested
pCMV.EGFP in the sense orientation. This plasmid serves as both a
control and construct intermediate. Plasmid pCMV.Galt-SV40L.0
[0360] The plasmid pCMV.Galt.SV40L0 (FIG. 32) comprises a Galt
structural gene cloned downstream of the CMV promoter present in
pCMV.SV40L.cass. To produce the plasmid the Galt cDNA fragment from
pCMV.GaIt was cloned as a 'Bg/ll/BamHl into Bg/ll-digested
pCMV.SV40L.cass in the sense orientation.
[0361] Plasmid pCMV.O.SV40L.tlaG
[0362] The plasmid pCMV.O.SV40L.tlaG (FIG. 33) comprises a Galt
structural gene clones in an antisense orientation downstream of
the SV40L promoter present in pCMV.SV40L.cass. To produce this
plasmid the Galt CDNA fragment from pCMV.Galt was cloned as a
Bg/ll/BamHl into BamHl-digested pCMV.SV40L.cass in the antisense
orientation.
[0363] Plasmid pCMV.OSV40L.GaIt
[0364] The plasmid pCMV.O.SV40L.Galt (FIG. 34) comprises a Galt
structural gene cloned downstream of the SV40L promoter present in
pCMV.SV40L.cass. To produce the plasmid the Galt cDNA fragment from
pCMV.Galt was cloned as a Bg/ll/BamHl into BamHi-digested
pCMV.SV40L.cass in the sense orientation.
[0365] 4. Test Plasmids
[0366] Plasmid pCMV.Galtx2
[0367] Plasmid pCMV.Galtx2 (FIG. 35) comprises a direct repeat of a
Galt open reading frame under the control of the CMV-lE promoter
sequence. In eukaryotes cells at least, the open reading frame
located nearer the CMV-lE promoter is translatable. To produce
pCMV.Galtx2, the Galt structural gene from pCMV.Galt was excised as
a Bg/ll/BamHl fragment and cloned in the sense orientation into the
BamHl cloning site of pCMV.Galt.
[0368] Plasmid pCMV.Galtx4
[0369] Plasmid pCMV.Galtx4 (FIG. 36) comprises a quadruple direct
repeat of a Galt open reading frame under the control of the CMV-lE
promoter sequence. In eukaryotes cells at least, the open reading
frame located nearer the CMV-lE promoter is translatable. To
produce pCMV.Galtx4, the Galtx2 sequence from pCMV.Galtx2 was
excised as a Bg/ll/BamHl fragment and cloned in the sense
orientation into the BamHI cloning site of pCMV.Galtx2.
[0370] Plasmid pCMV.Galt.SV40L.Galt
[0371] The plasmid pCMV.Galt.SV40L.Galt (FIG. 37) is designed to
express two sense transcripts of Galt, one driven by the CMV
promoter, the other by the SV40L promoter. To produce the plasmid a
Galt cDNA fragment from pCMV.Galt was cloned as a Bg/ll/BamHl
fragment into Bg/ll-digested pCMV.O.SV40.Galt in the sense
orientation.
[0372] Plasmid pCMV.Galt.SV40L-tlaG
[0373] The plasmid pCMV.Galt.SV40.tlaG (FIG. 38) is designed to
express a sense transcript of Galt driven by the CMV promoter and
an antisense transcript driven by the SV40L promoter. To produce
the piasmid a Galt cDNA fragment from pCMV.Galt was cloned as a
Bglll/BamHl fragment into Bglll-digested pCMV.O.SV40.taIG in the
sense orientation.
[0374] Plasmid pCMV.Galt.GFP.tlaG
[0375] Plasmid pCMV.Galt.GFP.tiaG (FIG. 39) comprise a Galt
palindrome, interrupted by the insertion of a GFP sequence between
each Galt structural gene in the inverted repeat. To produce this
plasmid the Bg/ll/BarnHl Galt cDNA fragment from pCMV.Galt was
cloned into the BamHl site of pCMV.GaIt.GFP in the antisense
relative to the CMV promoter.
[0376] Plasmid pCMV.EGFP.Galt.PFG
[0377] The plasmid pCMV.EGFP.Galt.PFG (FIG. 40) comprises a GFP
palindrome, interrupted by the insertion of a Galt sequence between
each GFP structural gene of the inverted repeat, expression of
which is driven by the CMV promoter. To produce this plasmid the
Galt sequences from pCMV.Galt were cloned as a Bglll/BamHl fragment
into BamHl-digested pCMV.EGFP in the sense orientation to produce
the intermediate pCMV.EGFP.Galt (not shown); following this further
GFP sequences from pCR.Bgl-pCMV.EGFP.Galt in the antisense
orientation.
[0378] Plasmid pCMV.Galt.SV40LR
[0379] The plasmid pCMV.Galt.SV4GLR (FIG. 41) is designed to
express Galt cDNA sequences cloned between the opposing CMV and
SV40L promoters in the expression cassette pCMV.SV40LR.cass. To
produce this plasmid Galt sequences from pCMV.Galt were cloned as a
Bgll/BamHl fragment in Bglll-digested pCMV.SV40LR.cass in the sense
orientation relative to the 35S promoter.
EXAMPLE 4
[0380] Genetic constructs comprising PVY Nia sequences operably
linked to the35S promoter sequence and/or the SCBV promoter
sequence
[0381] 1. Binary Vector
[0382] Plasmid pART27
[0383] Plasmid pART27 is a binary vector, specifically designed to
be compatible with the pART7 expression cassette. It contains
bacterial origins of replication for both E. coil and Agrobacterium
tumefaciens, a spectinomycin resistance gene for bacterial
selection, left and right T-DNA borders for transfer of DNA from
Agrobacterium to plant cells and a kanamycin resistance cassette to
permit selection of transformed plant cells. The kanamycin
resistance cassette is located between the T-DNA borders, pART27
also contains a unique Notl restriction site which permits cloning
of constructs prepared in vectors such as pART7 to be cloned
between the T-DNA borders. Construction of pART27 is described in
Gleave, AP (1992).
[0384] When cloning NotI inserts into this vector, two insert
orientations can be obtained. In all the following examples the
same insert orientation, relative to the direction of the 35S
promoter in the described pART7 constructs was chosen; this was
done to minimise any experimental artefacts that may arise from
comparing different constructs with different insert
orientations.
[0385] 2. Commercial Plasmids
[0386] Plasmid pBC (KS-)
[0387] Plasmid pBC (KS-) is commercially available from Stratagene
and comprises the
[0388] LacZ promoter sequence and lacZ-alpha transcription
terminator, with a multiple cloning site for the insertion of
structural gene sequences therein. The plasmid further comprises
the ColEl arid fl origins of replication and a
chloroamphenicol-resistance gene.
[0389] Plasmid pSP72
[0390] Plasmid pSP72 is commercially available from Promega and
contains a multiple cloning site for the insertion of structural
gene sequences therein. The plasmid further comprises the ColE1
origin of replication and an ampicillin-resistance gene.
[0391] 3. Expression Cassettes
[0392] Plasmid pART7
[0393] Plasmid pART7 is an expression cassette designed to drive
expression of sequences cloned behind the 35S promoter. It contains
a polylinker to assist cloning and a region of the octipine
synthase terminator. The 35S expression cassette is flanked by two
Not I restriction sites which permits cloning into binary
expression vectors, such as pART27 which contains a unique NotI
site. Its construction as described in Gleave, AP (1992), a map is
shown in FIG. 42,
[0394] Plasmid pART7.35S.SCBV.cass
[0395] Plasmid p35S.CMV.cass was designed to express two separate
gene sequences cloned into a single plasmid. To create this
plasmid, sequences corresponding to the nos terminator and the SCBV
promoter were amplified by PCR then cloned in the polylinker of
pART7 between the 35S promoter and OCS.
[0396] The resulting plasmid has the following arrangement of
elements:
[0397] 35S promoter -- polylinker 1 -- NOS terminator -- SCBV
promoter - polylinker 2-OCS terminator.
[0398] Expression of sequences cloned into polytinker 1 is
controlled by the 35S promoter, expression of sequences cloned into
polylinker 2 is controlled by the SCBV promoter.
[0399] The NOS terminator sequences were amplified from the plasmid
pAHC27 (Christensen and Quail, 1996) using the two
oligonucleotides;
[0400] NOS 5'(forward primer; SEQ ID 9)
[0401] 5'-GGATTCCCGGGACGTCGCGAATTTCCCCCGATCGTTC-3'; and
[0402] NOS 3'(reverse primer; SEQ ID 10)
[0403] 5'-CCATGGCCATATAGGCCCGATCTAGTAACATAG-3'
[0404] Nucleotide residues 1 to 17 for NOS 5'and 1 to 15 for NOS
3'represent additional nucleotides designed to assist in construct
preparation by adding additional restriction sites. For NOS 5'these
are BamHl, Smal, Aatll and the first 4 bases of an Nrul site, for
NOS 3'these are Ncol and Sfil sites. The remaining sequences for
each oligonucleotide are homologous to the 5'and 3'ends
respectively of NOS sequences in pAHC 27.
[0405] The SCBV promoter sequences were amplified from the plasmid
pScBV-20 (Tzafir et at, 1998) using the two oligonucleotides:
[0406] SCBV 5': 5'-CCATGGCCTATATGGCCATTCCCCACATTCAAG-3'(SEQ ID NO:1
1); and
[0407] SCBV 3': 5'-AACGTTAACTTCTACCCAGTTCCAGAG-3'(SEQ ID NQ:12)
[0408] Nucleotide residues 1 to 17 of SCBV 5'encode Ncol and Sfil
restriction sites designed to assist in construct preparation, the
remaining sequences are homologous to upstream sequences of the
SCMV promoter region. Nucleotide residues 1 to 9 of SCBV 3'encode
Psp10461 and Hpal restriction sites designed to assist in construct
preparation, the remaining sequences are homologous to the reverse
and complement of sequences near the transcription initiation site
of SCBV.
[0409] Sequences amplified from pScBV-20 using PCt and cloned into
pCR2.1 (Invitrogen) to produce pCR.NOS and pCR.SCBV respectively.
Smal l/Sfil cut pCR.NOS and Sfil/Hpal cut pCR.SCBV were ligated
into Sma I cut pART7 and a plasmid with a suitable orientation was
chosen and designated pART7.35S.SCBV.cass, a map of this construct
is shown in FIG. 70.
[0410] 4. Intermediate Constructs
[0411] Plasmid pBC.PVY
[0412] A region of the PVY genome was amplified by PCR using
reverse-transcribed RNA isolated from PVY-infected tobacco as a
template using standard protocols and cloned into a plasmid pGEM 3
(Stratagene), to create pGEM.PVY. A Sall/Hindlll fragment from
pGEM.PVY, corresponding to a Sall/Hindlll fragment positions
1536-2270 of the PVY strain O sequence (Acc. No D12539, Genbank),
was then subcloned into the plasmid pBC (Stratagene Inc.) to create
pBC.PVY (FIG. 44).
[0413] Plasmid pSP72.PVY
[0414] Plasmid pSP72.PVY was prepared by inserting an EcoRl/Sall
fragment from pBC.PVY into EcoRi/Sall cut pSP72 (Promega). This
construct contains additional restriction sites flanking the PVY
insert which were used to assist subsequent manipulations. A map of
this construct is shown in FIG. 45.
[0415] Plasmid ClapBC.PVY
[0416] Plasmid ClapBC.PVY was prepared by inserting a Clal/Sall
fragment from pSP72.PVY into Clal/Sal I cutpBC (Stratagene). This
construct contains additional restriction sites flanking the PVY
insert which were used to assist subsequent manipulations. A map of
this construct is shown in FIG. 46.
[0417] Plasmid pBC.PVYx2
[0418] Plasmid pBC.PVYx2 contains two direct head-to-tail repeats
of the PVY sequences derived from pBC.PVY. The plasmid was
generated by cloning an Accf/Clal PVY fragment from pSP72.PVY into
Acci cut pBC.PVY and is shown in FIG. 47.
[0419] Plasmid pSP72.PVYx2
[0420] Plasmid pSP72.PVYx2 contains two direct head-to-tail repeats
of the PVY sequences derived from pBC.PVY. The plasmid was
generated by cloning an Accl/Clal PVY fragment from pBc.PVY into
Accl cut pSP72.PVY and is shown in FIG. 48.
[0421] Plasmid pBC.PVYx3
[0422] Plasmid pBC.PVYx3 contains three direct head-to-tail repeats
of the PVY sequences derived from pBC.PVY. The plasmid was prepared
by cloning an Accl/Clal PVY fragment from pSP72.PVY into Accl cut
pBC.PVYx2 and is shown in FIG. 49.
[0423] Plasmid pBC.PVYx4
[0424] Plasmid pBC.PVYx4 contains four direct head-to-tail repeats
of the PVY sequences derived from pBC.PVY. The plasmid was prepared
by cloning the direct repeat of PVY sequences from pSP72.PVYx2 as
an Accl/Clal fragment into Acci cut pBC.PVYx2 and is shown in FIG.
50.
[0425] Plasmid PC.PPVY.LNYV
[0426] All attempts to create direct palindromes of PVY sequences
failed, presumably such sequence arrangements are unstable in
commonly used E. coli cloning hosts. Interrupted palindromes
however proved stable.
[0427] To create interrupted palindromes of PVY sequences a
"stuffer" fragment of approximately 360 bp was inserted into Cla
pBV.PVY downstream of the PVY sequences. The stuffer fragment was
made as follows:
[0428] A clone obtained initially from a CDNA library prepared from
lettuce necrotic yellows virus (LNYV) genomic RNA (Deitzgen et al,
1989), known to contain the 4b gene of the virus, was amplified by
PCR using the primers:
[0429] LNYV 1:5'-ATGGGATCCGTTATGCCAAGAAGAAGGA-3'(SEQ ID NO:13);
and
[0430] LNYV 2:5'-TGTGGATCCCTAACGGACCCGATG-3'(SEQ ID NO:14)
[0431] The first 9 nucleotide of these primers encode a BamHl site,
the remaining nucleotides are homologous to sequences of the LNYV
4b gene. Following amplification, the fragment was cloned into the
EcoRl site of pCR2.1 (Stratagene). This EcoRl fragment was cloned
into the EcoRl site of Cia pBC.PVY to create the intermediate
plasmid pBC.PVY.LNYV which is shown in FIG. 51.
[0432] Plasmid pBC.PVY.LNYV.PVY
[0433] The plasmid pBC.PVY.LNYV.YVP contains an interrupted direct
repeat of PVY sequences. to create this plasmid a Hpal/Hincil
fragment from pSP72 was cloned into Smal-digested pBC.PVY.LNYV and
a plasmid containing the sense orientation isolated, a map of this
construct is shown in FIG. 52.
[0434] Plasmid pBC-PVY.LNYV.YVPn
[0435] The plasmid pBV.PVY.LNYV.YVPL contains a partial interrupted
palindrome of PVY sequences. One arm of the palindrome contains all
the PVY sequences from pBC.PVY, the other arm contains part of the
sequences from PVY, corresponding to sequences between the EcoRV
and Hincli sites of pSP72.PVY. To create this plasmid an
EcoRV/Hincli fragment from pSP72.PVY was cloned into Smal-digested
pBC.PVY.LNYV and a plasmid containing the desired orientation
isolated, a map of this construct is shown in FIG. 53.
[0436] Plasmid pBC,PVY.LNYV.YVP
[0437] The plasmid pBC.PVY.LNYV.YVP contains an interrupted
palindrome of PVY sequences. To create this plasmid a Hpal/Hincil
fragment from pSP72. was cloned into Sma-digested pBC.PVY.LNYV and
a plasmid containing the antisense orientation isolated, a map of
this construct is shown in FIG. 54.
[0438] 5. Control Plasmids
[0439] Plasmids pART7.PVY & pART7.PVY
[0440] Plasmid pART7.PVY (FIG. 55) was designed to express PVY
sequences driven by the 35S promoter. This plasmid serves as a
control construct in these experiments, against which all other
constructs was compared. To generate this plasmid a Clal/Accl
fragment from ClapBC.PVY was cloned into Clal-digested pART7 and a
plasmid with expected to express a sense PVY sequence with respect
to the PVY genome, was selected. Sequences consisting of the 35S
promoter, PVY-sequences and the OCS terminator were excised as a,
Notl fragment and cloned into Not[-digested pART27, a plasmid with
the desired insert orientation was selected and designated
pART27.
[0441] Plasmids pART7.35S.PVY.SCBV.O &
pART27.35S.PVY.SCBV.O
[0442] Plasmid pART7.35S.PVY.SCBV.O (FIG. 56) was designed to act
as a control for co-expression of multiple constructs from a single
plasmid in transgenic plants. The 35S promoter was designed to
express PVY sense sequences, whilst the SCBV promoter was empty. To
generate this plasmid, the PVY fragment from Cla pBC.PVY was cloned
as a Xhol/EcoRl fragment into Xhol/EcoRl-digested
pART7.35S.SCBV.cass to create p35S.PVY.SCBV.O. Sequences consisting
of the 35S promoter driving sense PVY sequences and the NOS
terminator and the SCBV promoter and OCS terminator were excised as
a Notl fragment and cloned into pART27, a plasmid with the desired
insert orientation was isolated and designated pART27.35S
.PVY.SCBV.0.
[0443] Plasmids pART7-35S.O.SCBV.PVY &
pART27.35S.O.SCBV.PVY
[0444] Plasmid pART27.35S.O.SCBV.PVY (FIG. 57) was designed to act
as an additional control for co-expression of multiple constructs
from a single plasmid in transgenic plants. No expressible
sequences were cloned behind the 35S promoter, whilst the SCBV
promoter drove expression of a PVY sense fragment. To generate this
plasmid, the PVY fragment from Cla pBC.PVY was cloned as a CIal
fragment into Clal-digested pART7.35S.SCBV.cass, a plasmid
containing PVY sequences in a sense orientation was isolated and
designated p35S.O.SCBV.PVY. Sequences, consisting of the 35S
promoter and NOS terminator, the SCBV promoter driving sense PVY
sequences and the OCS terminator were excised as a NotI fragment
and cloned into pART27, a plasmid with the desired insert
orientation was isolated and designated pART27.35S.O.SCBV.PVY.
[0445] Plasmids pART7.35S.O.SCBV.YVP & pART7.35S.O.SCBV.YVP
[0446] Plasmid pART7.35S.O.SCBV.YVP (FIG. 58) was designed to act
as an additional control for co-expression of multiple constructs
from a single plasmid in transgenic plants. No expressible
sequences were cloned behind the 35S promoter, whilst the SCBV
promoter drove expression of a PVY antisense fragment. To generate
this plasmid, the PVY fragment from Cla pBC.PVY was cloned as a
Clal fragment into Clai-digested p35S.SCBV.cass, a plasmid
containing PCY sequences in an antisense orientation was isolated
and designated p35S.O.SCBV.YVP. Sequences, consisting of the 35S
promoter and NOS terminator, the SCBV promoter driving sense PVY
sequences and the OCS terminator were excised as a Notl fragment
and cloned into pART27, a plasmid with the desired insert
orientation was isolated and designated pART27.35S.O.SCBV.YVP.
[0447] 6. Test Plasmids
[0448] Plasrmids pART7.PVYx2 & pART27.PVYx2
[0449] Plasmid pART7.PVYx2 (FIG. 59) was designed to express a
direct repeat of PVY sequences driven by the 35S promoter in
transgenic plants. To generate this plasmid, direct repeats from
pBC.PVYx2 were cloned as a Xhol/BamHl fragment into Xhol/BamHl cut
pART7. Sequences consisting of the 35 S promoter, direct repeats of
PVY and the OCS terminator were excised as a Notl fragment from
pART7.PVYx2 and cloned into Notl-digested pART27, a plasmid with
the desired insert orientation was selected and designated
pART27.PVYx2.
[0450] Plasmids pART7.PVYx3 & pART27.PVYx3
[0451] Plasmid pART7.PVYx3 (FIG. 60) was designed to express a
direct repeat of three PVY sequences driven by the 35S promoter in
transgenic plants. To generate this plasmid, direct repeats from
pBC.PVYx3 were cloned as a Xhol/BamHl fragment into XhoflBamHl cut
pART7. Sequences consisting of the 35S promoter, direct repeats of
PVY and OCS terminator were excised as a Notl fragment from
pART.PVYx3 and cloned into Notl-digested pART27, a plasmid with the
desired insert orientation was selected and designated
pART27.PVYx3.
[0452] Plasmids pART7.PVYx4 & pART27.PVYx4
[0453] Plasmid pART7.PVYx4 (FIG. 61) was designed to express a
direct repeat of four PVY sequences driven by the 355 promoter in
transgenic plants. To generate this plasmid, direct repeats from
pBC.PVYx4 were cloned as a Xhol/BamHl fragment into xhol/BamHi cut
pART7. Sequences consisting of the 35S promoter, direct repeats of
PVY and the OCS terminator were excised as a Notl fragment from
pART7.PVYx3 and cloned into Notl-digested pART27, a plasmid with
the desired insert orientation was selected and designated
pART27.PVYx3.
[0454] Plasmids pART7.PVY.LNYV.PVY & pART27.PVY.LNYV.PVY
[0455] Plasmid pART7.PVY.LNYV.PVY (FIG. 62) was designed to express
the interrupted direct repeat of PVY sequences driven by the 35S
promoter in transgenic plants. This construct was prepared by
cloning the interrupted direct repeat of PVY from pBC.PVY.LNYV.PVY
as a Xhol/Xbal fragment into pART7 digested with Xhol/Xbal.
Sequences consisting of the 35S promoter, the interrupted direct
repeat of PVY sequences and the OCS terminator were excised from
pART7.PVY.LNYV.PVY as a Notl fragment and cloned into Notl-digested
pART27, a plasmid with the desired insert orientation was selected
and designated pART27.PVY.LNYV,PVY.
[0456] Plasmids pART7.PVY.LNYV.YVPo &
pART27.PVY.LNYV.YVP.DELTA.
[0457] Plasmid pART7.PVY.LNYV.YVPa (FIG. 63) was designed to
express the partial interrupted palindrome of PVY sequences driven
by the 35S promoter in transgenic plants. This construct was
prepared by cloning the partial interrupted palindrome of PVY
sequences from pBC.PVY.LNYV.YVP.DELTA. as a Xhol/Xbal fragment into
pART7 digested with Xhol/Xbal. Sequences consisting of the 35S
promoter, the partial interrupted palindrome of PVY sequences and
the OCS terminator were excised from pART7.PVY.LNYV.YVP.DELTA. as a
Notl fragment and cloned into Notl-digested pART27, a plasmid with
the desired insert orientation was selected and designated
pART27.PVY.LNYV.YVP.
[0458] Plasmlds pART7.PVY.LNYV.YVP & pART27.PVY.LNYV-YVP
[0459] Plasmid pART7.PVY.LNYV.YVP (FIG. 64) was designed to express
the interrupted palindrome of PVY sequences driven by the 35S
promoter in transgenic plants. This construct was prepared by
cloning the interrupted palindrome of PVY sequences from
pBC.PVY.LNYV.YVP.DELTA. as a Xhol/Xbal fragment into pART7 digested
with Xho)lIXbal. Sequences consisting of the 35S promoter, the
interrupted palindrome of PVY sequences and the OCS terminator were
excised from pART7.PVY.LNYV.YVP as a Noti fragment and cloned into
pART27, a plasmid with the desired insert orientation was selected
and designated pART27.PVY.NYV.YVP.
[0460] Plasmids pART7.35S.PVY-SCBV.YVP &
pART27.35S.PVY.SCBV.YVP
[0461] Plasmid pART7.35S.PVY.SCBV.YVP (FIG. 65) was designed to
co-express sense and antisense constructs in transgenic plants. To
generate this plasmid the PVY fragment from Cla pBC.PVY was cloned
as a Xhol/EcoRl fragment into xhol/EcoRl-digested
p35S.SCBV.O.SCBV.YVP. Sequences, consisting of the 35S promoter
driving sense PVY sequences and the NOS terminator and the SCBV
promoter driving antisense PVY and the OCS terminator were excised
as a Notl fragment and cloned into pART27, a plasmid with the
desired insert orientation was isolated and designated
pART27.353.PVY.SCBV.YVP. Plasmids pART7.35S.PVYx3.SCBV.YVPx3 &
pART27.35S.PVYx3.SCBV.YVPx3
[0462] Plasmid pART7.35S.PVYx3.SCBV.YVPx3 (FIG. 66) was designed to
co-express sense and antisense repeats of PVY in transgenic plants.
to generate this plasmid, the intermediate pART7.35S.O.SCBV.YVPx3
was constructed by cloning the triple direct PVY repeat from
ClapBC.PVYx3 as a Clal/Accl fragment into Cla-digested
p35S.SCBV.cass and isolating a plasmid with an antisense
orientation. for p35S.PVYx3.SCBV.YVPx3 the triple direct PVY repeat
from Cia pBC.PVYx3 was cloned as a Kpnl/Smal fragment into
Kpnl/Smal-digested p35S.O.SCBV.YVPx3 to create
p35S.PVYx3.SCBV.YVPx3. Sequences including both promoters,
terminators and direct PVY repeats were isolated as a Notl fragment
and cloned into pART27. A plasmid with an appropriate orientation
was chosen and designated pART27.35S.PVYx3.SCBV.
[0463] Plasmids pART7.PVYx3.LNYV.YVPx3 &
pART27.PVYx3.LNYV.YVPx3
[0464] Plasmid pART7.PVYx3.LNYV.YVPx3 (FIG. 67) was designed to
express triple repeats of PVY sequences as an interrupted
palindrome. To generate this plasmid an intermediate,
pART7x3.PVY.LNYV.YV was constructed by cloning a PVY.LNYV.YVP
fragment from pBC.PVY.LNYV.YVP as an Accl/Clal fragment into the
plasmid pART7.PVYx2. pART7.35S.PVYx3.LNYV.YVPx3, was made by
cloning an additional PVY direct repeat from pBC.PVYx2 as an
Accl/Clal fragment into Clal digested pART7x3.PVY.LNYV.YVP.
Sequences from pART7.35S.PVYx3.LNYV.YVPx3, including the 35S
promoter, all PVY sequences and the OCS terminator were excised as
a Noti fragment and cloned into Noti-digested pART27, a plasmid
with an appropriate orientation was chosen and designated
pART27.35S.PVYx3.LNYV.
[0465] Plasmids pART7.PVY multi & pART27.PVY multi
[0466] Plasmid pART7.35S.PVY multi (FIG. 68) was designed to
express higher order direct repeats of regions of PVY sequences in
transgenic plants. Higher order direct repeats of a 72 bp of the
PVY Nia region from PVY were prepared by annealing two partially
complementary oligonucleotides as follows:
2 (SEQ ID NO:15) 5'-TAATGAGGATGATGTCCCTACCTTTAATTGGCAGAAATTT-
CTGTGGA AAGACAGGGAAATCTTTCGGCATTT-3'; and
[0467]
3 (SEQ ID NO:16) 5'-TTCTGCCAATTAAAGGTAGGGACATCATCCTCATTAAAAT-
GCCGAAA GATTTCCCTGTCTTTCCACAGAAAT-3'
[0468] The oligonucleotides were phosphorylated with T4
pofynucleotide kinase, heated and cooled slowly to permit
self-annealing, ligated with T4 DNA ligase, end-filled with Klenow
polymerase and cloned into pCR2.1 (Invitrogen). Plasmids containing
multiple repeats were isolated and sequences were cloned as EcoRi
fragments in a sense orientation into EcoRl-digested pART7, to
create the intermediate pART7.PVY multi. to create pART27.PVY
multi, the 35S promoter, PVY sequences and the OCS terminator were
excised as a Noti fragment and cloned into Notl-digested pART27. A
plasmid with an appropriate insert orientation was isolated and
designated pART27.PVY multi.
EXAMPLE5
[0469] InactivatIon of virus gene expression in mammals
[0470] Viral immune lines are created by expressing viral sequences
in stably transformed cell lines.
[0471] In particular, lytic viruses are used for this approach
since cell lysis provides very simple screens and also offer the
ability to directly select for potentially rare transformation
events which might create viral immunity. Sub-genomic fragments
derived from a simple single stranded RNA virus (Bovine enterovirus
--BEV) or a complex double stranded DNA virus, Herpes Simplex Virus
I (HSV I) are cloned into a suitable vector and expressed in
transformed cells. Mammalian cell lines are transformed with
genetic constructs designed to express viral sequences driven by
the strong cytomegalovirus (CMV-lE) promoter. Sequences utilised
include specific viral replicase genes. Random "shotgun" libraries
comprising representative viral gene sequences, may also be used
and the introduced dispersed nucleic acid molecule, to target the
expression of virus sequences.
[0472] Exemplary genetic constructs for use in this procedure,
comprising nucleotide sequences derived from the BEV RNA-dependent
RNA polymerase gene, are presented herein.
[0473] For viral polymerase constructs, large numbers
(approximately 100) of transformed cell lines are generated and
infected with the respective virus. For cells transformed with
shotgun libraries very large numbers (hundreds) of transformed
lines are generated and screened in bulk for viral immunity.
Following virus challenge, resistant cell lines are selected and
analysed further to determine the sequences conferring immunity
thereon.
[0474] Resistant cell lines are supportive of the ability of the
introduced nucleotide sequences to inactivate viral gene expression
in a mammalian system.
[0475] Additionally, resistant lines obtained from such experiments
are used to more precisely define molecular and biochemical
characteristics of the modulation which is observed.
EXAMPLE 6
[0476] Induction of virus resistance in transgenic plants
[0477] Agrobacterium tumefaciens, strain LBA4404, was transformed
independently with the constructs
[0478] pART27.PVY
[0479] pART27.PVYx2
[0480] pART27.PVYx3
[0481] pART27. PVYx4
[0482] pART27,PVY.LNYV.PVY
[0483] pART27.PVY.LNYV.YVP.DELTA.
[0484] pART27.PVY.LNYV.YVP
[0485] pART27.35S . PVY.SCBV.O
[0486] pART27.35S.O.SCBV.PVY
[0487] pART27.35S.O.SCBV.YVP
[0488] pART27.35S. PVY.SCBV.YVP
[0489] pART27.35S.PVYx3.SCBV.YPVx3
[0490] pART27,PVYx3.LNYV.YVPx3
[0491] pART27.PVYx10
[0492] using tri-parental matings. DNA mini-preps from these
strains were prepared and examined by restriction with Notl to
ensure they contained the appropriate binary vectors.
[0493] Nicotiana tabaccum (cultivar W38) were transformed with
these Agrobacterium strains using standard procedures. Putative
transformed shoots were excised and rooted on media containing
kanamycin. Under these conditions we have consistently observed
that only transgenic shoots will root on kanamycin plates. Rooted
shoots were transferred to soil and allowed to establish. After two
to three weeks, vigorous plants with at least three sets of leaves
were chosen and infected With PVY.
[0494] Viral inoculum was prepared from W38 tobacco previously
infected with the virus, approximately 2 g of leaf material,
showing obvious viral symptoms were ground with carbarundum in 10
ml of 100mM Na phosphate buffer (pH 7.5). the inoculum was diluted
to 200 ml with additional Na phosphate buffer. Two leaves from each
transgenic plant were sprinkled with carbarundum, then 0.4 ml of
inoculum was applied to each leaf and leaves rubbed fairly
vigorously with fingers. Using this procedure 100% of
non-transgenic control plants were infected with PVY.
[0495] To assay for viral resistance and immunity transgenic plants
are monitored for symptom development. The PVY strain (PVY-D, an
Australian PVY isolate) gives obvious symptoms on W38 tobacco,
a-vein clearing symptom is readily observed on the two leaves above
the inoculated leaves, subsequent leaves show uniform chlorotic
lesions. Symptom development was monitored over a six week
period.
[0496] Transgenic lines were described as resistant if they showed
reduced viral symptoms, which manifests as a reduction in the leaf
are showing chlorotic lesions. Resistance ranges from very strong
resistance where only a few viral lesions are observed on a plant
to weak resistance which manifects as reduced symptoms on leaves
that develop late in plant growth.
[0497] Transgenic plants which showed absolutely no evidence of
viral symptoms were classified as immune. To ensure these plants
were immune they were re-inoculated with virus, most plants
remained immune, the few that showed symptoms were re-classified as
resistant.
[0498] For plant lines generated Southern blots are performed,
resistance in subsequent generations is monitored to determine that
resistance/immunity is transmissable. Additionally, the breadth of
viral resistance is monitored by challenging lines with other PVY
strains, to determine whether host range susceptibility is
modified.
[0499] Results from these experiments are described in Table 2.
These data indicate that constructs comprising tandem repeats of
target gene sequence, either in the configuration of palindromes,
interrupted palindromes as direct repeat sequences, are capable of
conferring viral resistance and/or immunity in transgenic
plants.
[0500] Accordingly, such inverted and/or direct repeat sequences
modulate expression of the virus target gene in the transgenic
plant.
[0501] Constructs combining the use of direct and inverted repeat
sequences, namely pART27.35S.PVYx3.SCBV.YVPx3 and
pART27.PVYx3.LNYV.YVPx3- , are also useful in modulating gene
expression.
EXAMPLE 7
[0502] Inactivation of Galt in animal cells
[0503] To assay for Galt inactivation, porcine PK2 cells were
transformed with the relevant constructs. PK2 cells constitutively
express Galt enzyme, the activity of which results in the addition
of a variety of a:-1,3-galactosyl groups to a range of proteins
expressed on the cell surface of these cells. Cells were
transformed using lipofectin and stably transformed lines were
selected using genetecin.
[0504] As an initial assay cell lines were probed for the presence
of the Galt-encoded epitope, i.e, .alpha.-1,3-galactosyl moieties
decorating cell surface proteins, using the lectin 1B4. IB4 binding
was assayed either in situ or by FACS sorting.
[0505] For in situ binding, cells were fixed to solid supports with
cold methanol for 5 mins, cells were rinsed in PBS (phosphate
buffered saline) and non-specific 164 binding was blocked with 1%
BSA in PBS for 10 mins. Fixed cells were probed using 20 ugiml
IB4-biotin (Sigma) in 1% BSA, PBS for 30 mins at room temperature,
cells were washed in PBS then probed with a 1:200 dilution of
ExtrAvidin-FITC (Sigma) in PBS for 30 mins followed by further
rinses in PBS. Cells were then examined using fluorescence
microscopy, under these conditions the outer surface of PK2 control
cells uniformly stained green.
[0506] For FACS analysis, cells were suspended after treatment with
trypsin, washed in HBSS/Hepes (Hank's buffered saline solution with
20 mM Hepes, pH7.4) and probed with 10 ug/ml IB4-biotin (Sigma) in
HBSS/Hepes for 45 mins at 4.degree. C. Cells were washed in
HBSS/Hepes, probed with a 1:200 dilution of ExtrAvidin-FITC (Sigma)
in HBSSYHepes for 45 mins at 4.degree. C. at and rinsed in cold
HBSS/Hepes prior to FACS sorting.
[0507] Using this approach transformed cell lines are assayed for
Galt inactivation and quantitative assessment of construct
effectiveness is determined. Moreover cell lines showing Galt
inactivation are isolated and subject to further molecular analyses
to determine the mechanism of gene inactivation.
EXAMPLE8
[0508] Preparation of plasmld construct cassettes for use in
achieving co-suppression
[0509] 1. Generic RNA Isolation, cDNA synthesis and PCR
protocol
[0510] Total RNA was purified from the indicated cell lines using
an RNeasy Mini Kit according to the supplier's protocol (Qiagen).
To prepare cDNA, this RNA was reverse-transcribed using Omniscript
Reverse Transcriptase (Qiagen). Two micrograms of total RNA was
reverse-transcribed using 1iM oligo dT (Sigma) as a primer in a 20
.mu.l reaction according to the supplier's protocol (Qiagen).
[0511] To amplify specific products, 2 .mu.l of this mixture was
used as a substrate for PCR amplification, which was performed
using HotStarTaq DNA polymerase according to the supplier's
protocol (Qiagen). PCR amplification conditions involved an initial
activation step at 950C for 15 min, followed by 35 amplification
cycles of 94.degree. C for 30 sec, 60.degree. C. for 30 sec and
72.degree. C. for 60 sec. with a final elongation step at
72.degree. C. for 4 min.
[0512] PCR products to be cloned were usually purified using a
QlAquick PCR Purification Kit (Qiagen); in instances where multiple
fragments were generated by PCR, the fragment of the correct size
was purified from agarose gels using a QlAquick Gel Purification
Kit (Qiagen) according to the supplier's protocol.
[0513] Amplification products were then cloned into pCR (registered
trademark) 2.1-TOPO (Invitrogen) according to the supplier's
protocol.
[0514] 2. Generic Cloning Techniques
[0515] To prepare the constructs described below, insert fragments
were excised from intermediate vectors using restriction enzymes
according to the supplier's protocols (Roche) and fragments
purified from agarose gels using QlAquick Gel Purification Kits
(Qiagen) according to the supplier's protocol. Vectors were usually
prepared by restriction digestion and treated with Shrimp Alkaline
Phosphatase according to the supplier's protocol (Amersham). Vector
and inserts were ligated using T4 DNA ligase according to the
supplier's protocols (Roche) and transformed into competent
Escherichia coli strain DH5o using standard procedures (Sambrook,
Fritsch et al. 1989).
[0516] 3 Constructs
[0517] (a) Commercial Plasmids
[0518] Plasmid pEGFP-N1
[0519] Plasmid pEGFP-N1 (Clontech) contains the CMV IE promoter
operably connected to an open reading frame encoding a red-shifted
variant of the wild-type GFP which has been optimized for brighter
fluorescence. The specific GFP variant encoded by pEGFP-N1 has been
disclosed by (Cormack, Valdivia et al. 1996). Plasmid pEGFP-N1
contains a multiple cloning site comprising BgIll and BamHl sites
and many other restriction endonuclease cleavage sites, located
between the CMV IE promoter and the EGFP open reading frame, The
plasmid pEGFP-N1 will express the EGFP protein in mammalian cells.
In addition, structural genes cloned into the multiple cloning site
will be expressed as EGFP fusion polypeptides if they are in-frame
with the EGFP-encoding sequence and lack a functional translation
stop codon. The plasmid further comprises an SV40 polyadenylation
signal downstream of the EGFP open reading frame to direct proper
processing of the 3'-end of mRNA transcribed from the CMV IE
promoter sequence (SV40 pA). The plasmid further comprises the SV40
origin of replication functional in animal cells; the
neomycin-resistance gene comprising the SV40 E (early) promoter
operably connected to the neomycin/kanamycin-resistance gene
derived from Tn5 and the HSV thymidine kinase polyadenylation
signal, for selection of transformed cells on kanamycin, neomycin
or geneticin; the pUC19 origin of replication which is functional
in bacterial cells and the f1 origin of replication for
single-stranded DNA production.
[0520] Plasmid pBluescript If SK+
[0521] Plasmid pBluescript 11 SK+(Stratagene) comprises the lacZ
promoter sequence and lacz-a transcription terminator, with
multiple restriction endonuclease cloning sites located there
between. Plasmid pBluescript ll SK.sup.+is designed to clone
nucleic acid fragments by virtue of the multiple restriction
endonuclease cloning sites. The plasmid further comprises the ColEl
and f1 origins of replication and the ampicillin-resistance gene
(.alpha.-lactamase).
[0522] Plasmid pCR (registered trademark) 2.1
[0523] Plasmid pCR (registered trademark) 2.1 (Invitrogen ) is a
T-tailed vector comprising the lacZ promoter sequence and
lacZ-.alpha. transcription terminator, with a cloning site for the
insertion of structural gene sequences there between. Plasmid pCR
(registered trademark) 2.1 is designed to clone nucleic acid
fragments by virtue of the A-overhang frequently synthesized by Taq
polymerase during the polymerase chain reaction. The plasmid
further comprises the ColEl and f1 origins of replication and
kanamycin-resistance and ampicillin-resistance genes.
[0524] Plasmid pCR (registered trademark) 2.1-TOPO
[0525] Plasmid pCR (registered trademark) 2.1-TOPO (Invitrogen) is
a T-tailed vector comprising the lacz promoter sequence and
lacZ-.alpha. transcription terminator, with multiple restriction
endonuclease cloning sites located there between. Plasmid pCR
(registered trademark) 2.1-TOPO is provided with covalently bound
topoisomerase I enzyme for fast cloning. The plasmid further
comprises the ColE] and f1 origins of replication and the kanamycin
and ampicillin-resistance genes.
[0526] (b) Intermediate cassettes
[0527] Plasmid TOPO.BGI2
[0528] Plasmid TOPO-BGI2 comprises the human P-globin intron number
2 (BGI2) placed in the multiple cloning region of plasmid pCR
(registered trademark) 2.1-TOPO. To produce this prasmid, the human
P-globin intron number 2 (BGI2) was amplified from human genomic
DNA using the amplification primers:
[0529] GD1 GAG CTC TTC AGG GTG AGT CTA TGG GAC CC [SEQ ID NO:17
]and
[0530] GAl CTG CAG GAG CTG TGG GAG GAA GAT AAG AG [SEQ ID
NO:18]
[0531] and cloned into plasmid pCR (registered trademark) 2.1-TOPO.
BGI2 is a functional intron sequence that is capable of being
post-transcriptionally cleaved from RNA transcripts containing it
in mammalian cells.
[0532] (c) Plasmid cassettes
[0533] Plasmid pCMV.cass
[0534] Plasmid pCMV.cass is an expression cassette for driving
expression of a structural gene sequence under control of the
CMV-lE promoter sequence. Plasmid pCMV.cass was derived from
pEGFP-N1 by deletion of the EGFP open reading frame as follows:
Plasmid pEGFP-N1 was digested with PinAl and Nod, blunt-ended using
Pful DNA polymerase and then religated. Structural gene sequences
are cloned into pCMV.cass using the multiple cloning site, which is
identical to the multiple cloning site of pEGFP-N1, except it lacks
the PinAl site.
[0535] Plasmid pCMV.BG[2.cass
[0536] To create pCMV.BGI2.cass, the human 0-globin intron 2
sequence was isolated as a Sacl/Pstl fragment from TOPO,BGI2 and
cloned between the Sacl and Pst! sites of pCMV.cass. In
pCMV.BGI2.cass, any RNAs transcribed from the CMV promoter will
include the human P-globin intron 2 sequences; these intron
sequences will presumably be excised from transcripts as part of
the normal intron processing machinery, since the intron sequences
include both the splice donor and splice acceptor sequences
necessary for normal intron processing.
EXAMPLE 9
[0537] Co-suppression of Green Fluorescent Protein in Porcine
Kidney Type 1 cells in vitro
[0538] 1. Culturing of Cell Lines
[0539] PK-1 cells (derived from porcine kidney epithelial cells)
were grown as adherent monolayers using DMEM supplemented with 10%
v/v FBS.
[0540] 2. Preparation of Genetic Constructs
[0541] (a) Interim Plasmids
[0542] Plasmid pBluescript.EGFP
[0543] Plasmid pBluescript.EGFP comprises the EGFP open reading
frame derived from plasmid pEGFP-Nl placed in the multiple cloning
region of plasmid pBluescript ll SK.sup.+(Stratagene). To produce
this plasmid, the EGFP open reading frame was excised from pEGFP-Nl
by restriction endonuclease digestion using the enzymes Nod and
Xhol and ligated into Nodl/Xhol-digested pBluescript ll SK;.
[0544] (b) Test Plasmids
[0545] Plasmid pCMV.EGFP
[0546] Plasmid pCMV.EGFP is capable of expressing the entire EGFP
open reading frame under the control of CMV-lE promoter sequence.
To produce pCMV.EGFP, the EGFP sequence from pBluescript.EGFP was
sub-cloned in the sense orientation as a BamHl-to-Sad fragment into
Bgll/Sacl-digested pCMV.cass.
[0547] 3. Detection of Co-suppression Phenotype
[0548] (a) Insertion of EGFP-expressing transgene into PK-1
cells
[0549] Transformations were performed in 6-well tissue culture
vessels (Nunc). Individual wells were seeded with 4.times.10.sup.4
PK-1 cells in 2 ml of DMEM, 10% v/v FBS and incubated at 37.degree.
C. in 5% v/v CO.sub.2 until the monolayer was 60-90% confluent,
typically 16 to 24 hr.
[0550] To transform a single plate (six wells), 12 .mu.g of
pCMV.EGFP plasmid DNA and 108 .mu.l of GenePORTER2 (trademark)
(Gene Therapy Systems) were diluted into OPTI-MEM I (registered
trademark) medium (Life Technologies) to obtain a final volume of 6
ml and incubated at room temperature for 45 min.
[0551] The tissue growth medium was removed from each well and the
monolayer therein was washed with 1 ml of 1 x PBS (Sigma). The
monolayers were overlayed with 1 ml of the plasmid DNA/GenePORTER2
(trademark) conjugate for each well and incubated at 37.degree. C.
in 5% V/V CO.sub.2 for 4.5 hr.
[0552] OPTT-MEM-I (registered trademark) (1 ml) supplemented with
20% vIv FBS was added to each well and the vessel incubated for a
further 24 hr, at which time the monolayers were washed with 1 x
PBS and medium was replaced with 2 ml of fresh DMEM including 10%
v/v FBS. Cells transformed with pCMV.EGFP were examined after 24-48
hr for transient EGFP expression using fluorescence microscopy at a
wavelength of 500-550 nm.
[0553] Forty-eight hr after transfection the medium was removed,
the cell monolayer washed with 1 x PBS and 4 ml of fresh DMEM
containing 10% v/v FBS supplemented with 1.5 mg/ml genetecin (Life
Technologies) was added to each well. Genetecin was included in the
medium to select for stably transformed cell lines. The DMEM, 10%
v/v FBS, 1.5 mg/ml genetecin medium was changed every 48-72 hr.
After 21 days of selection, stable, EGFP-expressing PK-1 colonies
were apparent.
[0554] Individual colonies of stably transfected PK-1 cells were
cloned, maintained and stored as described in Example 5, above.
[0555] A number of parental cell lines were transformed with
pCMV.EGFP. Following continuous culture, in many of these lines GFP
expression was either extremely low or completely undetectable as
listed in Table 3 and shown in FIG. 42.
4TABLE 3 Number of cell lines Number of cloned lines with extremely
low or Parental Cell line examined undetectable GFP PK-1 (pig) 59 2
MM96L (human) 12 4 B16 (mouse) 12 10 MDAMB468 (human) 11 1
[0556] These data demonstrate that silencing of GFP expression
occurred frequently in different types of cell lines, established
from three different species.
[0557] 4. Southern analysis
[0558] Individual transgenic PK-1 cell lines (transfected and
co-transfected) are analyzed by Southern blot analysis to confirm
integration and determine copy number of the transgenes. The
procedure is carried out according to the protocol set forth in
Example 1, above. An example is illustrated in FIG. 70.
EXAMPLE 10
[0559] Co-suppression of Bovine Enterovirus in Madin Darby Bovine
Kidney Type CRIB-1 cells In vitro
[0560] 1. Culturing of cell lines
[0561] CRIB-1 cells (derived from bovine kidney epithelial cells)
were grown as adherent monolayers using DMEM supplemented with 10%
v/v Donor Calf Serum (DCS; Life Technologies), as described in
Example 1.
[0562] 2. Preparation of genetic constructs
[0563] (a) Interim plasmid
[0564] Plasmid pCR.BEV2
[0565] The complete Bovine enterovirus (BEV) RNA polymerase coding
region was amplified from a full-length cDNA clone encoding same,
using primers:
5 BEV-1 CGG CAG ATC CTA ACA ATG GCA [SEQ ID NO:19] GGA CAA ATC GAG
TAC ATC
[0566] and
[0567] BEV-3 GGG CGG ATC CTT AGA AAG AAT CGT ACC AC [SEQ ID
NO:20].
[0568] Primer BEV-1 comprises a Bglll restriction endonuclease site
at positions 4-9, inclusive, and an ATG start site at positions
16-18, inclusive. Primer BEV-3 comprises a BamHl restriction enzyme
site at positions 5-10, inclusive, and the complement of a TAA
translation stop signal at positions 11-13, inclusive. As a
consequence, an open reading frame comprising a translation start
signal and a translation stop signal is contained between the Bg/ll
and BamHl restriction sites.
[0569] The amplified fragment was cloned into pCR2.1 to produce
plasmid pCR.BEV2.
[0570] Plasmid pBS.PFGE
[0571] Plasmid pBS.PFGE contains the,EGFP coding sequences,from
pEGFP-N1 cloned in antisense orientation into the polylinker of
pBluescript 11 SK+. To generate this plasmid, the EGFP coding
sequences from pEGFP-Nl was cloned as a Notl-to-Sacf fragment into
Notg/Sacl-digested pBluescript 11 SK+.
[0572] (b) Test Plasmids
[0573] Plasmid pCMV.BEV2.EIG12.2VEB
[0574] Plasmid pCMV.BEV2.BGI2.2VEB contains an inverted repeat or
palindrome of the BEV polymerase coding region that is interrupted
by the insertion of the human .beta.-globin intron 2 sequence
therein. Plasmid pCMV.BEV2.BG[2.2VEB was constructed in successive
steps: (i) the BEV2 sequence from plasmid pCR.BEV2 was sub-cloned
in the sense orientation as a Bgfll-to-BamHI fragment into
Bg/ll-digested pCMV.BGI2.cass to make plasmid pCMV.BEV2.BGI2, and
(ii) the BEV2 sequence from plasmid pCR.BEV2 was sub-cloned in the
antisense orientation as a Bg/ll-to-BamHl fragment into
BamHl-digested pCMV.BEV2.BGI2 to make plasmid
pCMV.BEV2.BGI2.2VEB.
[0575] Plasmid pCMV.BEV.EGFP.VEB
[0576] Plasmid pCMV.BEV.EGFP.VEB contains an inverted repeat or
palindrome of the BEV polymerase coding region that is interupted
by EGFP coding sequences which act as a stuffer fragment To
generate this plasmid, the EGFP coding sequence from pBS.PFGE was
isolated as an EcoRl fragment and cloned into EcoRl-digested
pCMV.cass in the sense orientation relative to the CMV promoter to
generate pCMV.EGFP.cass. Plasmid pCMV.BEV.EGFP.VEB was constructed
in successive steps: (i) the BEV polymerase sequence from plasmid
pCR.BEV2 was sub-cloned in the sense orientation as a
13gIl-to-BamHI fragment into Bg/ll-digested pCMV.EGFP.cass to make
plasmid pCMV.BEV.EGFP, and (ii) the BEV polymerase sequence from
plasmid pCR.BEV2 was sub-cloned in the antisense orientation as a
Bg/ll-to-BamHl fragment into BamHl-digested pCMV.BEV.EGFP to make
plasmid pCMV. BEV.EGFP.VEB.
[0577] 3, Detection of cosuppression phenotype
[0578] (a) Insertion of Bovine enterovirus RNA
polyrnerase-expressing transgene into CRIB-1 cells
[0579] Transformations were performed in 6-well tissue culture
vessels. Individual wells were seeded with 2.times.10.sup.5 CRIB-1
cells in 2 ml of DMEM, 10% v/v DCS and incubated at 37.degree. C.
in 5% v/v CO.sub.2 until the monolayer was 60-90% confluent,
typically 16-24 hr.
[0580] The following solutions were prepared in 10 ml sterile
tubes:
[0581] Solution A: For each transfection, 1 .mu.g of DNA
(pCMV.BEV2.BGI2.2VEB or pCMV.EGFP) was diluted into 100 .mu.l of
OPTI-MEM-l (registered trademark) and;
[0582] Solution B: For each transfection, 10 .mu.l of LIPOFECTAMINE
(trademark) Reagent (Life Technologies) was diluted into 100 .mu.l
OPTI-MEM-1 (registered trademark).
[0583] The two solutions were combined and mixed gently, and
incubated at room temperature for 45 min to allow DNA-liposome
complexes to form. While complexes formed, the CRIB-1 cells were
rinsed once with 2 ml of OPTI-MEM l (registered trademark).
[0584] For each transfection, 0.8 ml of OPTr-MEM l (registered
trademark) was added to the tube containing the compfexes, the tube
mixed gently, and the diluted complex solution overlaid onto the
rinsed CRIB-1 cells. Cells were then incubated with the complexes
at 37.degree. C. in 5% v/v CO.sub.2 for 16-24 hr.
[0585] Transfection mixture was then removed and the CRIB-1
monolayers overlaid with 2 ml of DMEM, 10% v/v DCS. Cells were
incubated at 37.degree. C. in 5% V/V CO.sub.2 for approximately 48
hr. To select for stable transformants, the medium was replaced
every 72 hr with 4 ml of DMEM, 10% v/v DOCS, 0.6 mg/ml
geneticin.
[0586] Cells transformed with the transfection control pCMV.EGFP
were examined after 24-48 hr for transient EGFP expression using
fluorescence microscopy at a wavelength of 500-50 nm. After 21 days
of selection, stably transformed CRIB-1 colonies were apparent.
[0587] Individual colonies of stably transfected CRIB-1 cells were
cloned, maintained and stored as described in Example 1.
[0588] (b) Determination of Bovine Enterovirus titre
[0589] The BEV isolate used in these experiments was a cloned
isolate, K2577. To amplify BEV virus from this stock, cells were
infected with 5 .mu.l of viral stock per well and the virus allowed
to replicate for 48 hr, as described below, Culture medium was
harvested at this time and transferred to a screw-capped tube. Dead
cells and debris were removed by centrifugation at 3,500 rpm for 15
min at 4.degree. C. in a Sigma 3K18 centrifuge. The supernatant was
decanted into a fresh tube and centrifuged at 20,000 rpm for 30 min
at 40.degree. C. in a Beckman J2-M1 centrifuge to remove remaining
debris. The supernatant was decanted and this new BEV stock titred
as described below and stored at 4.degree. C.
[0590] Absolute.
[0591] In a 6-well tissue culture plate, 2.5.times.10.sup.5 CRIB-1
cells were seeded per well in 2 ml DMEM, 10% v/v DCS. The cells
were incubated at 37.degree. C. in 5% v/v CO.sub.2 until 90-100%
confluent.
[0592] BEV was diluted in serum-free DMEM at dilutions of 10.sup.-1
to 10.sup.-9. Medium was aspirated from the CRIB-1 monolayers and
the cells overlaid with 2 ml of 1 x PBS and the vessels rocked
gently to wash the monolayer. PBS was aspirated from the monolayer
and the wash repeated.
[0593] One ml of diluted virus solutions (10.sup.-4 to 10.sup.-9)
was added directly onto the rinsed CRIB-1 cells, using one dilution
per well in duplicate. The cells were incubated with BEV for 1 hr
at 37.degree. C. in 5% v/v CO.sub.2 with gentle agitation. Medium
was aspirated and the infected cells overlaid with 3 ml of nutrient
agar (1% Noble Agar in DMEM).
[0594] The agar overlay was allowed to set and the plates incubated
(inverted) at 37.degree. C. in 5% v/v CO.sub.2 for 18-24 hr.
Following incubation, each well was overlaid with 3 ml of Neutral
Red Agar (1.7 ml Neutral Red Solution (Life Technologies) in 100 ml
Nutrient Agar). The overlay was allowed to set and the plates
incubated (inverted) in the dark at 37.degree. C. in 5% V/V
CO.sub.2 for 18-24 hr. Plaques were counted to determine the titre
of the BEV viral stock.
[0595] Empirical:
[0596] In a 24-well tissue culture plate, 4.times.10.sup.4 CRIB-1
cells were seeded per well in 800 .mu.l DM EM, 10% v/v DCS. The
cells were incubated at 37.degree. C. in 5% v/v C02 until 90-100%
confluent.
[0597] From concentrated BEV viral stock, BEV was diluted in
serum-free DMEM at dilutions of 10.sup.-1 to 10.sup.-9. The medium
was aspirated from the CRIB-1 monolayers and the monolayers
overlaid with 800 .mu.l of 1 x PBS and washed by gently rocking the
tissue culture vessel. PBS was aspirated from the monolayers and
the wash repeated.
[0598] 200 .mu.l of the diluted virus solutions (10.sup.-3 to
10.sup.-9) was added immediately directly onto the rinsed CRIB-1
cells using one dilution per well in duplicate. The CRIB-1 cells
were incubated with BEV for 24 hr at 37.degree. C. in 5% v/v
CO.sub.2 and each well inspected microscopically for cell lysis. A
further 600 .mu.l of serum-free DMEM was then added to each well.
After a further 24 hr, each well was inspected microscopically for
cell lysis. The working dilution is the minimum viral concentration
that kills most of the CRIB-1 cells after 24 hr and all cells after
48 hr.
[0599] (c) Bovine enterovirus challenge of CRIB-1 cells transformed
with pC4V. BEV2. BGI2.2 VEB
[0600] In a 24-well tissue culture plate, 4.times.10.sup.4 CRIB-1
cells per well were seeded in triplicate, in 800 .mu.l DMEM, 10%
v/v DCS. The cells were incubated at 37CC in 5% V/V CO.sub.2 until
90-100% confluent.
[0601] From concentrated BEV viral stock, BEV virus was diluted in
serum-free DMEM at an appropriate dilution. In addition, the BEV
viral stock was diluted to 10x and 0.1x the working dilution
(typically 10.sup.-4 to 10.sup.-6 pfu).
[0602] Medium was aspirated from the CRIB-1 monolayers and the
monolayers overlaid with 800 .mu.l of 1 x PBS and washed gently by
rocking the tissue culture vessel. PBS was aspirated from the
monolayers and the wash repeated.
[0603] 200 .mu.l of the diluted virus solutions (one dilution per
replicate) was added immediately, directly onto the rinsed CRIB-1
cells. The cells were incubated with BEV for 24 hr at 37.degree. C.
in 5% vlv CO.sub.2, and each well inspected microscopically for
cell lysis. A further 600 .mu.l of serum-free DMEM was added to
each well. After a further 24 hr, each well was inspected
microscopically for cell lysis.
[0604] d) Generation of CRIB-1 viral tolerant cell lines
[0605] To determine whether cells transformed with
pCMV.BEV.EGFP.VEB or pCMV.BEV2.BGI2.2VEB became tolerant to BEV
infection, transformed cell lines were challenged with dilutions of
BEV and monitored for survival. To overcome inherent variation in
these assays, multiple challenges were performed and lines
consistently showing viral tolerance were isolated for further
examination. Results of these experiments are shown below in Tables
4 and 5.
6TABLE 4 CRIB-1 cells transfected with pCMV.BEV.EGFP.VEB (CRIB-1
EGFP) Challenge Challenge Challenge Challenge 1 2 3 4 Cell line
10.sup.-4 10.sup.-6 10.sup.-4 10.sup.-6 10.sup.-4 10.sup.-6
10.sup.-4 10.sup.-6 CRIB-1 nd nd - - - - - - CRIB-1 - - - - - - + -
EGFP # 1 CRIB-1 - - + ++ - - nd nd EGFP # 3 CRIB-1 - - - - - - ++ -
EGFP # 4 CRIB-1 - - + +++ - - nd nd EGFP # 5 CRIB-1 - + - - - - - -
EGFP # 6 CRIB-1 + + - + + + nd nd EGFP # 7 CRIB-1 + +++ + + + +++ -
++ EGFP # 8 CRIB-1 - - - + + + nd nd EGFP # 9 CRIB-1 - + - + + ++
nd nd EGFP # 10 CRIB-1 + ++ - - + +++ nd nd EGFP # 11 CRIB-1 - + +
++ + + nd nd EGFP # 12 CRIB-1 - - + + - - nd nd EGFP # 13 CRIB-1 ++
++ + ++ ++ + + + EGFP # 14 CRIB-1 - + ++ ++ + ++ nd nd EGFP # 15
CRIB-1 - + - ++ + ++ nd nd EGFP # 16 CRIB-1 - - + + - - nd nd EGFP
# 17 CRIB-1 + + ++ + ++ ++ nd nd EGFP # 18 CRIB-1 - - - - + +++ nd
nd EGFP # 20 CRIB-1 - ++ + ++ + + nd nd EGFP # 21 CRIB-1 - + + + +
+ nd nd EGFP # 22 CRIB-1 - - - +++ - ++ - - EGFP # 23 CRIB-1 - - +
++ - + EGFP # 24 CRIB-1 - + - +++ - - nd nd EGFP # 25 CRIB-1 + ++
++ +++ ++ +++ - - EGFP # 26 - no cells surviving + 1-10% of cells
surviving. ++ 10-90% of cells surviving. +++ 90%+ of cells
surviving nd not done.
[0606]
7TABLE 5 CRIB-1 cells transfected with pCMV.BEV2.BGI2.2VEB (CRIB-1
BGI2) Challenge Challenge Challenge Challenge 1 2 3 4 Cell line
10.sup.-4 10.sup.-6 10.sup.-4 10.sup.-6 10.sup.-4 10.sup.-6
10.sup.-4 10.sup.-6 CRIB-1 nd nd - - - - - - CRIB-1 - - - - - - nd
nd BGI2 # 1 CRIB-1 - - - + - - - - BGI2 # 2 CRIB-1 - - ++ ++ + ++
nd nd BGI2 # 3 CRIB-1 - - - + - - nd nd BGI2 # 4 CRIB-1 - - - ++ -
- nd nd BGI2 # 5 CRIB-1 + + +++ ++ + + nd nd BGI2 # 6 CRIB-1 + + -
+++ - - nd nd BGI2 # 7 CRIB-1 - + +++ ++ - + nd nd BGI2 # 8 CRIB-1
- + - ++ + ++ - ++ BGI2 # 9 CRIB-1 ++ ++ ++ +++ + + - - BGI2 # 10
CRIB-1 + ++ + + - + nd nd BGI2 # 11 CRIB-1 + + + +++ - - nd nd BGI2
# 12 CRIB-1 - - +++ +++ - - nd nd BGI2 # 13 CRIB-1 + ++ + ++ + + nd
nd BGI2 # 14 CRIB-1 + + + ++ + ++ - - BGI2 # 15 CRIB-1 - - - - - -
nd nd BGI2 # 16 CRIB-1 - + - ++ - - nd nd BGI2 # 17 CRIB-1 - - -
+++ - - nd nd BGI2 # 18 CRIB-1 - - - ++ + +++ + +++ BGI2 # 19
CRIB-1 + + + +++ + + nd nd BGI2 # 20 CRIB-1 - - - - - - - - BGI2 #
21 CRIB-1 - - - - - - - - BGI2 # 22 CRIB-1 - + +++ +++ + + nd nd
BGI2 # 23 CRIB-1 - ++ +++ + - - nd nd BGI2 # 24 - no cells
surviving + 1-10% of cells surviving. ++ 10-90% of cells surviving.
+++ 90%+ of cells surviving nd not done.
[0607] These data showed that viral-tolerant cell lines could be
defined in this fashion. In addition, cells which survived this
viral challenge could be grown up for further analyses.
[0608] To further define the degree of viral tolerance in such cell
lines, the cell line CRIB-1 BG12 #19, and viral-tolerant cells
grown from cells that survived the initial challenge (line CRIB-1
BGI2 # 19(tof)), were further analyzed using finer scale (3-fold)
serial dilutions of BEV in triplicate. The results of these
experiments are shown in Table 6.
8 TABLE 6 Dilution of viral stool Cell line 3.3 .times. 10 1.1
.times. 10 3.7 .times. 10 1.2 .times. 10 4.1 .times. 10 1.3 .times.
10 CRIB-1 Replicate 1 - - - - - +++ CRIB-1 Replicate 1 - - - - - +
CRIB-1 Replicate 1 - - - - - +++ CRIB-1 BGI2 #19 - - + + ++ +++
Replicate 1 CRIB-1 BGI2 #19 - - - - ++ +++ Replicate 2 CRIB-1 BGI2
#19 - - - + +++ +++ Replicate 3 CRIB-1 BGI2 #19 (tol) - - + + +++
+++ Replicate 1 CRIB-1 BGI2 #19 (tol) - - + + ++ +++ Replicate 2
CRIB-1 BGI2 #19 (tol) - - + + +++ +++ Replicate 3 - no cells
surviving 48 hr post-infection + 1-10% of cells surviving 48 hr
post-infection. ++ 10-90% of cells surviving 48 hr post-infection.
+++ 90%+ of cells surviving 48 hr post-infection.
[0609] These data showed that the cell lines CRIB-1 BGI2 ft9 and
CRIB-1 BGI2 # 19(tol) were tolerant to higher titres of BEV than
the parental CRIB-1 line. FIG. 44 shows micrographs comparing
CRIB-1 and CRIB-i BG]2 # 19(tol) cells before and 48 hr after BEV
infection.
EXAMPLE 11
[0610] Co-suppresSion of Tyrosinase in Murine Type B16 cells In
vitro
[0611] 1. Culturing of cell lines
[0612] B16 cells derived from murine melanoma (ATCC CRL-6322) were
grown as adherent monolayers in RPMI 1640 supplemented with 10% v/v
FBS, as described in Example 1.
[0613] 2. Preparation of Genetic Constructs
[0614] (a) Interim Plasmid
[0615] Plasmid TOPO.TYR
[0616] Total RNA was purified from cultured murine 816 melanoma
cells and cDNA prepared as described in Example 8.
[0617] To amplify a region of the murine tyrosinase gene, 2 .mu.l
of this mixture was used as a substrate for PCR amplification using
the primers:
[0618] TYR-F: GTT TCC AGA TCT CTG ATG GC [SEQ ID NO:21]
[0619] and
[0620] TYR-R: AGT CCA CTC TGG ATC CTA GG [SEQ ID NO:22],
[0621] The PCR amplification was performed using HotStarTaq DNA
polymerase according to the supplier's protocol (Qiagen). PCR
amplification conditions involved an initial activation step at
95.degree. C. for 15 mins, followed by 35 amplification cycles of
94.degree. C. for 30 sec, 55.degree. C. for 30 sec and 72.degree.
C. for 60 sec, with a final elongation step at 72.degree. C. for 4
min.
[0622] The PCR amplified region of tyrosinase was column-purified
(PCR purification column, Qiagen) and then cloned into pCR
(registered trademark) 2.1-TOPO according to the supplier's
instructions (Invitrogen) to make plasmid TOPO.TYR.
[0623] (b) Test Plasmids
[0624] Plasmid pCMV.TYR.BGI2.RYT
[0625] Plasmid pCMV.TYR.BGI2.RYT contains an inverted repeat, or
palindrome, of a region of the murine tyrosinase gene that is
interrupted by the insertion of the human .beta.-globin intron 2
(BGI2) sequence therein. Plasmid pCMV.TYR.BGI2.RYT was constructed
in successive steps: (i) the TYR sequence from plasmid TOPO.TYR was
sub-cloned in the sense orientation as a Bg/ll-to-BamHl fragment
into Bg/ll-digested pCMV.BGI2 to make plasmid pCMV.TYR.BG)2, and
(ii) the TYR sequence from plasmid TOPO.TYR was sub-cloned in the
antisense orientation as a Bg/ll-to-BamHl fragment into
BamHl-digested pCMV.TYR.BGI2 to make plasmid pCMV.TYR.BGI2.RYT.
[0626] Plasmid pCMV.TYR
[0627] Plasmid pCMV.TYR contains a single copy of mouse tyrosinase
CDNA sequence, expression of which is driven by the CMV promoter.
Plasmid pCMV.TYR was constructed by cloning the TYR sequence from
plasmid TOPO.TYR as a BamHl-to-Bg/ll fragment into BamHl-digested
pCMV.cass and selecting plasmids containing the TYR sequence in a
sense orientation relative to the CMV promoter.
[0628] Plasmid pCMV.TYR.TYR
[0629] Plasmid pCMV.TYR.TYR contains a direct repeat of the mouse
tyrosinase cDNA sequence, expression of which is driven by the CMV
promoter. Plasmid pCMV.TYR.TYR was constructed by cloning the TYR
sequence from plasmid TOPO.TYR as a BarnHl-to-BgAl fragment into
BamHi-digested pCMV.TYR and selecting plasmids containing the
second TYR sequence in a sense orientation relative to the CMV
promoter.
[0630] 3. Detection of co-suppression phenotype
[0631] (a) Reduction of melanin pigmentation through PTGS of
tyrosinase by insertion of a region of the tyrosinase gene into
murine melanoma B 16 cells
[0632] Tyrosinase is the major enzyme controlling pigmentation in
mammals. If the gene is inactivated, melanin will no longer be
produced by the pigmented B16 melanoma cells. This is essentially
the same process that occurs in albino animals.
[0633] Transformations were performed in 6-well tissue culture
vessels. Individual wells were seeded with 1.times.10.sup.5 cells
in 2 ml of RPMI 1640, 10% v/V FBS and incubated at 37.degree. C. in
5% V/V CO.sub.2 until the monolayer was 60-90% confluent, typically
16-24 hr.
[0634] Subsequent procedures were as described above in Example 8,
except that B16 cells were incubated with the DNA-liposome
complexes at 37.degree. C. in 5% V/V CO.sub.2 for 34 hr only.
[0635] Individual colonies of stably transfected B16 cells were
cloned, maintained and stored as described in Example 1.
[0636] Thirty six clones stably transformed with pCMV.TYR.BGI2.RYT,
34 clones stably transformed with pCMV.TYR and 37 clones stably
transformed with pCMV.TYR.TYR were selected for subsequent
analyses.
[0637] When the endogenous tyrosinase gene is
post-transcriptionally silenced, melanin production in the B16
cells is reduced. B66 cells that wNould normally appear to contain
a dark brown pigment will now appear lightly pigmented or
unpigmented.
[0638] (b) Visual monitoring of melanin production in transformed
B16 cell lines
[0639] To monitor melanin content of transformed cell lines, cells
were trypsinized and transferred to media containing FBS to inhibit
trypsin activity. Cells were then counted with a haemocytometer and
2.times.10.sup.8 cells transferred to a microfuge tube. Cells were
collected by centrifugation at 2,500 rpm for 3 min at room
temperature and pellets examined visually.
[0640] Five clones transformed with pCMV.TYR.BGI2.RYT, namely B16.2
1.11, B16 3.1.4, 816 31.15, B16 4.12.2 and B16 4.12.3, were
considerably paler than the B16 controls (FIG. 45). Four clones
transformed with pCMV.TYR (B16+Tyr 2.3, B16+Tyr 2.9, B16+Tyr 3.3,
816+Tyr 3.7 and 816+Tyr 4.10) and five clones transformed with
pCMV.TYR.TYR (816+TyrTyr 1,1, 816+TyrTyr 2.9, B16+TyrTyr 3.7,
616+TyrTyr 3.13 and B16+TyrTyr 4.4) were also significantly paler
than the 8 16 controls.
[0641] (c) Identification of melanin by staining according to
Schmori
[0642] Specific diagnosis for the presence of cellular melanin can
be achieved using a modified Schmorl's melanin staining (Koss
1979). Using this method, the presence of melanin in the cell is
detected by a specific staining procedure that converts melanin to
a greenish-black pigment.
[0643] Cell populations to be stained were resuspended at a
concentration of 500,000 cells per ml in RPMI 1640 medium. Volumes
of 200 .mu.l were dropped onto surface-sterilized microscope slides
and slides were incubated at 37.degree. C. in a humidified
atmosphere in 100 mm TC dishes until cells had adhered firmly. The
medium was removed and cells were fixed by air drying on a heating
block at 37.degree. C. for 30 min then post-fixed with 4% w/v
paratormaldehyde (Sigma) in PBS for 1 hr. Fixed cells were hydrated
by dipping in 96% v/v ethanol in distilled water, 70% v/v ethanol,
50% v/v ethanol then distilled water. Slides with adherent cells
were left for 1 hr in a ferrous sulfate solution (2.5% w/v ferrous
sulfate in water) then rinsed in four changes of distilled
water.sub., 1 min each. Slides were left for 30 min in a solution
of potassium ferricyanide (1% w/v potassium ferricyanide in 10% v/v
acetic acid in distilled water). Slides were dipped in 1% v/v
acetic acid (15 dips) then dipped in distilled water (15 dips).
[0644] Cells were stained for 1-2 min in a Nuclear Fast Red
preparation (0.1% w/v Nuclear Fast Red (C.i. 60760 Sigma N 8002)
dissolved with heating in 5% w/v ammonium sulfate in water). Fixed
and stained cells on slides were washed by dipping in distilled
water (15 dips). Cover slips were mounted on slides in
glycerol/DABCO (25 mg/ml DABCO (1,4-diazabicyclo(2.2.2)octane
(Sigma D 2522)) in 80% v/v glycerol in PBS). Cells were examined by
bright field microscopy using a 100x oil immersion objective.
[0645] The results of staining with Schmorl's stain correlated with
the simple visual data illustrated in FIG. 45 for all cell lines.
When B16 cells were stained with the above procedure, melanin was
obvious in most cells. In contrast, fewer cells stained for melanin
in the transformed lines B16 2.1.11, B16 3.1.4, B16 3.1.15, B16
4.12.2, B16 4.12.3, B16 Tyr 2.3, B16 Tyr 2.9, B16 Tyr 4.10, B16
TyrTyr 1.1, B16 TyrTyr 2.9 and B16 TyrTyr 3.7, consistent with the
reduced gross pigmentation observed in these cell lines.
[0646] (d) Assaying tyrosinase enzyme activity in transformed cell
lines
[0647] Tyrosinase catalyzes the first two steps of melanin
synthesis: the hydroxylation of tyrosine to dopa
(dihydroxyphenylalanine) and the oxidation of dopa to dopaquinone.
Tyrosinase can be measured as its dopa oxidase activity. This assay
uses Besthorn's hydrazone (3-methyl-2-benzothiazolinonehydrazone
hydrochloride, MBTH) to trap dopaquinone formed by the oxidation of
L-dopa. Presence of a low concentration of N,N'-dimethylformamide
in the assay mixture renders the MBTH soluble and the method can be
used over a range of pH values. MBTH reacts with dopaquinone by a
Michael addition reaction and forms a dark pink product whose
presence is monitored using a spectrophotometer or plate reader. It
is assumed that the reaction of the MBTH with dopaquinone is very
rapid relative to the enzyme-catalyzed oxidation of L-dopa. The
rate of production of the pink pigment can be used as a
quantitative measure of enzyme (Winder and Harris 1991; Dutkiewicz,
Albert et al, 2000).
[0648] B16 cells and transformed B16 cell lines were plated into
individual wells of a 96-well plate in triplicate. Constant numbers
of cells (25,000) were transferred into individual wells and cells
were incubated overnight. Tyrosinase assays were performed as
described below after either 24 or 48 hr incubation.
[0649] Individual wells were washed with 200 .mu.l PBS and 20 .mu.l
of 0.5% v/v Triton X-100 in 50mM sodium phosphate buffer (pH 6.9)
was added to each well. Cell lysis and solubilisation was achieved
by freeze-thawing plates at -70.degree. C. for 30 min, followed by
incubating at room temperature for 25 min and 37.degree. C. for 5
min.
[0650] Tyrosinase activity was assayed by adding 190 ul
freshly-prepared assay buffer (6.3mM MBTH, 1.mM L-dopa, 4% v/v
N,N'-dimethylformamide in 48mM sodium phosphate buffer (pH 7.1)) to
each well. Colour formation was monitored at 505 nm in a Tecan
plate reader and data collected using X/Scan Software. Readings
were taken at constant time intervals and reactions monitored at
room temperature, typically 22.degree. C. Results were calculated
as the average of enzyme activities as measured for the triplicate
samples. Data were analyzed and tyrosinase activity estimated at
early time-points when product formation was linear, typically
between 2 and 12 min. Results from these experiments are shown
below in Tables 7 and 8.
9TABLE 7 Tyrosinase activity Relative tyrosinase (.DELTA. OD 505
nm/min/ activity compared with Cell Line 25,000 cells) B16 cells
(%) B16 0.0123 100 B16 2.1.6 (Tyr.BGI2.ryT) 0.0108 87.8 B16 2.1.11
(Tyr.BGI2.ryT) 0.0007 5.7 B16 3.1.4 (Tyr.BGI2.ryT) 0.0033 26.8 B16
3.1.15 (Tyr.BGI2.ryT) 0.0011 8.9 B16 4.12.2 (Tyr.BGI2.ryT) 0.0013
10.6 B16 4.12.3 (Tyr.BGI2.ryT) 0.0011 8.9 B16 Tyr Tyr 1.1 0.0043 34
B16 Tyr Tyr 2.9 0.0042 34.1 B16 Tyr Tyr 3.7 0.0087 70.7
[0651]
10 TABLE 8 Tyrosinase activity Relative tyrosinase (.DELTA. OD 505
nm/min/ activity compared with Cell Line 25,000 cells) B16 cells
(%) B16 0.0200 100 B16 Tyr 2.3 0.0036 18.2 B16 Tyr 2.9 0.0017 8.7
B16 Tyr 4.10 0.0034 17.2
[0652] These data showed that tyrosinase enzyme activity was
reduced in lines transformed with the constructs pCMV.TYR.BGI2.RYT,
pCMV.TYR and pCMV.TYR.TYR
[0653] 4. Southern Analysis
[0654] Individual transgenic B16 cell lines were analyzed by
Southern blot analysis to confirm integration of the transgene,
according to the protocol set forth in Example 1.
EXAMPLE 12
[0655] Co-suppression of HER-2 in MDA-MB-468 cells in vitro
[0656] HER-2 (also designated neu and erbB-2) encodes a 185 kDa
transmembrane receptor tyrosine kinase that is constitutively
activated at low levels and displays potent oncogenic activity when
over-expressed. HER-2 protein over-expression occurs in about 30%
of invasive human breast cancers. The biological function of HER-2
is not well understood. It shares a common structural organisation
with other members of the epidermal growth factor receptor family
and may participate in similar signal transduction pathways leading
to changes in cytoskeleton reorganisation, cell motility, protease
expression and cell adhesion. Over-expression of HEFR-2 in breast
cancer cells leads to increased tumorigenicity, invasiveness and
metastatic potential (Slamon, Clark et al. 1987).
[0657] 1. Culturing of Cell Lines
[0658] Human MDA-MB-468 cells were cultured in RPMI 1640
supplemented with 10% v/v FBS. Cells were passaged twice a week by
treating with trypsin to release cells and transferring a
proportion of the culture to fresh medium, as described in Example
1.
[0659] 2. Preparation of Genetic Constructs
[0660] (a) Interim Plasmid
[0661] Plasmid TOPO.HER-2
[0662] A region of the human HER-2 gene was amplified by PCR using
human cDNA as a template. The cDNA was prepared from total RNA
isolated from a human breast tumour line, SK-BR-3. Total RNA was
purified as described in Example 8. Human HER-2 sequences were
amplified using the primers:
[0663] Hi: CTC GAG AAG TGT GCA CCG GCA CAG ACA TG [SEQ ID
NO:23]
[0664] and
[0665] H3: GTC GAC TGT GTT CCA TCC TCT GOCT GTC AC [SEQ ID
NO:241].
[0666] The amplification product was cloned into pCR (registered
trademark) 2.1-TOPO to create the intermediate clone
TOPO.HER-2.
[0667] (b) Test Plasmid
[0668] Plasmid pCMV.HER2.BGl2-2REH
[0669] Plasmid pCMV.HER2.BGl2.2REH contains an inverted repeat or
palindrome of the HER-2 coding region that is interrupted by the
insertion of the human .beta.-globin intron 2 (BGI2) sequence
therein. Plasmid pCMV.HER2.BGl2.2REH was constructed in successive
steps: (i) the HER-2 sequence from plasmid TOPO.HER2 was sub-cloned
in the sense orientation as a SaAlXhol fragment into Sat-digested
pCMV.BGI2.cass (Example 6) to make plasmid pCMV.HER2.BGI2, and (ii)
the HER2 sequence from plasmid TOPO.HER2 was sub-cloned in the
antisense orientation as a SaAlXhol fragment into Xhol-digested
pCMV.HER2.BGI2 to make plasmid pCMV.HER2.BGI2.2REH.
[0670] 3. Determination of Onset of Co-suppression
[0671] (a) Transfection of HER-2 Constructs
[0672] Transformations were performed in 6-well tissue culture
vessels. Individual wells were seeded with 4.times.10.sup.5
MDA-MB-468 cells in 2 ml of RPMI 1640 medium, 10% v/v FIBS and
incubated at 37.degree. C. in 5% v/v CO.sub.2 until the monolayer
was 60-90% confluent, typically 16-24 hr.
[0673] Subsequent procedures were as described above in Example 10,
except that MDA-MB-468 cells were incubated with the DNA-liposome
complexes at 37.degree. C. in 5% v/v CO.sub.2 for 34 hr only.
Thirty-six transformed cell lines were isolated for subsequent
analysis.
[0674] (b) Post-transcriptional Silencing of HER-2 in MDA-MB-468
Cells
[0675] MDA-MB-468 cells over-express HER-2 and PTGS of the gene in
geneticin-selected clones derived from this cell line were tested
by immunofluorescence labelling of clones (see Example 1) with a
primary murine monoclonal antibody directed against the
extracellular domain of HER-2 protein. The primary antibody was a
mouse Anti-erbB2 monoclonal antibody (Transduction Laboratories,
Cat. No. E19420, an IgG2b isotype) used at 11400 dilution; the
secondary antibody was Alexa Fluor 488 goat anti-mouse IgG (H+L)
conjugate (Molecular Probes, Cat. No. A-11001) used at 11100
dilution. As a negative control, MDA-MB-468 cells (parental and
transformed lines) were probed with Alexa Fluor 488 goat anti-mouse
IgG (H+L) conjugate only.
[0676] Several MDA-MB-468 cell lines transformed with
pCMV.HER2.BGI2.2REH were found to have reduced immunofluorescence,
examples of which are illustrated in FIG. 46.
[0677] (c) FACS Analysis to Define Cell Lines Showing Reduced
Expression of Her-2
[0678] To determine the level of expression of HER-2 in transformed
cell lines, approximately 500,000 cells grown in a 6-well plate
were washed twice with I x PBS then dissociated with 500 .mu.l cell
dissociation solution (Sigma C 5789) according to the suppliers
instructions (Sigma). Cells were transferred to medium in a
microcentrifuge tube and collected by centifugation at 2,500 rpm
for 3 min. The supernatant was removed and cells resuspended in 1
ml 1 x PBS.
[0679] For fixation, cells were collected by centrifugation as
above and suspended in 50 .mu.l PBA (1 x PBS, 0.1 % wlv BSA
fraction V (Trace) and 0.1% w/v sodium azide) followed by the
addition of 250 .mu.l of 4% w/v paraformaldehyde in 1 x PBS. and
incubated at 40.degree. C. for 10 min. To permeabilize cells, cells
were collected by centrifugation at 10,000 rpm for 30 sec, the
supernatant removed and cells suspended in 50 .mu.l 0.25% w/v
saponin (Sigma S 4521) in PBA and incubated at 40.degree. C. for 10
min. To block cells, cells were collected by centrifugation at
10,000 rpm for 30 sec, the supernatant removed and cells suspended
in 50 .mu.l PBA, 1% v/v FBS and incubated at 4.degree. C. for 10
min.
[0680] To quantify HER-2 protein, fixed, permeabilized cells were
probed with Anti-erbB2 monoclonal antibody at {fraction
(1/100)}dilution followed by Alexa Fluor 488 goat anti-mouse IgG
conjugate at {fraction (1/100)}dilution. Cells were then analysed
by FACS using a Becton Dickinson FACSCalibur and Cellquest software
(Becton Dickinson). True background fluorescence values were
established with unstained MDA-MB-468 cells and cells probed with
an irrelevant primary antibody (MART-1, an IgG2b antibody
(NeoMarkers)) and the Alexa Fluor 488 secondary antibody, both at
{fraction (1/100)}dilutions. Examples of FACS data are shown in
FIG. 74. Results of analyses of all cell lines are compiled in
Table 10.
11TABLE 10 Geometric Mean mean Median Cell line Fluorescence
Fluorescence Fluorescence MDA-MB-468 control.1 5.07 4.72 4.78
MDA-MB-468 control.2 137.24 121.68 117.57 MDA-MB-468 1224.90
1086.47 1175.74 MDA-MB-468 1.1 1167.94 1056.17 1124.04 MDA-MB-468
1.4 781.72 664.67 673.17 MDA-MB-468 1.5 828.34 673.82 710.50
MDA-MB-468 1.6 925.16 807.09 850.53 MDA-MB-468 1.7 870.81 749.27
791.48 MDA-MB-468 1.8 1173.92 938.72 1124.04 MDA-MB-468 1.10 701.24
601.84 604.30 MDA-MB-468 1.11 1103.18 980.10 1064.99 MDA-MB-468
1.12 817.39 666.61 710.50 MDA-MB-468 2.5 966.72 862.76 905.80
MDA-MB-468 2.6 752.70 633.49 649.38 MDA-MB-468 2.7 842.00 677.15
716.92 MDA-MB-468 2.8 986.05 792.13 881.68 MDA-MB-468 2.9 802.36
686.06 716.92 MDA-MB-468 2.10 1061.79 944.49 1009.04 MDA-MB-468
2.12 931.63 790.81 820.47 MDA-MB-468 2.13 894.47 792.46 827.88
MDA-MB-468 2.15 1052.87 946.79 1009.04 MDA-MB-468 3.1 1049.88
931.96 991.05 MDA-MB-468 3.2 897.00 802.43 842.91 MDA-MB-468 3.4
981.63 858.95 913.98 MDA-MB-468 3.5 1072.00 930.17 982.17
MDA-MB-468 3.7 1098.95 993.26 1036.63 MDA-MB-468 3.8 1133.86
1026.31 1074.61 MDA-MB-468 3.9 831.73 729.32 763.51 MDA-MB-468 3.12
1120.82 998.67 1064.99 MDA-MB-468 3.13 1039.41 963.71 1036.63
MDA-MB-468 4.5 770.93 681.01 697.83 MDA-MB-468 4.7 838.16 752.74
784.39 MDA-MB-468 4.8 860.76 769.51 813.12 MDA-MB-468 4.10 1016.21
904.69 947.46 MDA-MB-468 4.11 870.10 776.73 813.12 MDA-MB-468 4.12
986.93 857.20 913.98 MDA-MB-468 4.13 790.41 712.25 743.18
MDA-MB-468 4.14 942.36 842.34 873.79 MDA-MB-468 4.16 771.81 677.69
697.83 "MDA-MB-468 control.1" is MDA-MB-468 cells without staining
- neither primary nor secondary antibody. "MDA-MB-468 control.2" is
MDA-MB-468 cells stained with irrelevant primary antibody MART-1
and the Alexa Fluor 488 secondary antibody.
[0681] All other cells, as described, were stained with Anti-erbB2
primary antibody and Alexa Fluor 488 secondary antibody.
[0682] These data show that MDA-MB-468 cells transformed with
pCMV.HER2.BG(2.2REH have significant reduced expression of HER-2
protein.
[0683] 4. Southern Analysis
[0684] Individual transgenic NIH/3T3 cell lines were analyzed by
Southern blot to confirm integration of the transgene, according to
the protocol set forth in Example 1.
[0685] S. Western Blot Analysis
[0686] Selected clones and control MDA-MB-468 cells were grown
overnight to near-confluence on 100 mm TC plates (10.sup.7 cells).
Cells in plates were first washed with buffer containing
phosphatase inhibitors (50mM Tris-HCI, pH 6.8, 1mM
Na.sub.4P.sub.2O.sub.7, 10mM NaF, 20.mu.M Na.sub.2MoO.sub.4, 1mM
Na.sub.3VO.sub.4), and then scraped from the plate in 600 .mu.l of
lysis buffer (50mM Tris-HCI, pH 6.8, 1mM Na.sub.4P.sub.2O.sub.7,
10mM NaF, 20.mu.M Na.sub.2MoO.sub.4, 1mM Na.sub.3VO.sub.4, 2% w/v
SDS) which had been heated to 100.degree. C. Suspensions were
incubated in screw-capped tubes at 100.degree. C. for 15 min. Tubes
with lysed cells were centrifuged at 13,000 rpm for 10 min and
supernatant extracts were removed and stored at -20.degree. C.
[0687] SDS-PAGE 10% v/v separating and 5% v/v stacking gels (0.75
mm) were prepared in a Protean apparatus (BioRad) using 29:1
acrylarnide:bisacrylamide (Bio-Rad) and Tris-HCI buffers at pH 8.8
and 6.8, respectively. Volumes of 60 .mu.l from extracts were
combined with 20 .mu.l of 4x loading buffer (50mM Tris-HCI, pH 6.8,
2% w/v SDS, 40% v/v glycerol, bromophenol blue and 400mM
dithiothreitol added before use), heated to 100.degree. C. for 5
min, cooled then loaded into wells before the gel was run in the
cold room at 120V until protein samples entered the separating gel,
then at 240V. Separated proteins were transferred to Hybond-ECL
nitrocellulose membranes (Amersham) using an electroblotter
(Bio-Rad), according to the supplier's instructions.
[0688] Membranes were rinsed in TBST buffer (10mM Tris-HCI, pH 8.0,
150mM NaCI, 0.05% v/v Tween 20) then blocked in a dish in TBST with
5% w/v skim milk powder plus phosphatase inhibitors (1mM
Na.sub.4P.sub.2O.sub.7, 10mM NaF, 20,uM Na.sub.2MoO.sub.4, 1 mM
Na.sub.3VO.sub.4). Membranes were incubated in a small volume in
TBST with 2.5% w/v skim milk powder plus phosphatase inhibitors
containing a mouse monoclonal antibody against the ECD of HER-2
(Transduction Laboratories, NeoMarkers) diluted 1:4000. Membranes
were washed three times for 10 min in TBST with 2.5% w/v skim milk
powder plus phosphatase inhibitors. Membranes were incubated in a
small volume in TBST with 2.5% w/v skim milk powder plus
phosphatase inhibitors containing the horseradish
peroxidase-conjugated secondary antibody diluted 1:1000. Membranes
were washed three times for 10 min in TBST with 2.5% w/v skim milk
powder plus phosphatase inhibitors.
[0689] The presence of HER-2 protein was detected using the ECL
luminol-based system (Amersham), according to manufacturer's
instructions. Several cell lines transformed with
pCMV.HER2.BGI2.2REH showed greatly reduced or no detectable HER-2
protein.
EXAMPLE 13
[0690] Co-suppresslon of YB-1 and p53 in Murine Type B10.2 and Pam
212 cells in vitro
[0691] 1. Culturing of Cell Lines
[0692] BIO.2 calls (Immunex) derived from murine fibrosarcoma and
Pam 212 cells (Auckland Medical School) derived from sponstaneously
transformed murine epidermal keratinocytes were grown as adherent
monolayers in cDMEM (DMEM with 0.77mM asparagine, 160.mu.M
penicillin G, 70.mu.M dihydrostreptomycin sulfate) supplemented
with 5% v/v FBS (B10.2) or 5% v/v equine serum (Pam 212), as
described in Example 1, above.
[0693] 2. Preparation of Genetic Constructs
[0694] (a) Interim Plasrnids
[0695] Plasmid TOPO.YS-1
[0696] To amplify a region of the mouse YB-1 gene, 25 ng of a
plasmid clone containing a mouse YB-1 cDNA (obtained from Genesis
Research & Development Corporation, Auckland NZ) was used as a
substrate for PCR amplification using the primers:
[0697] Y1: AGA TCT GCA GCA GAC CGT AAC CAT TAT AGG [SEQ ID
NO:25].
[0698] and
[0699] Y4: GGA TCC ACC TTT ATT AAC AGG TGC TTG CAG (SEQ ID
NO:261.
[0700] The PCR amplification was performed using HotStarTaq DNA
polymerase according to the supplier's protocol (Qiagen). PCR
amplification conditions involved an initial activation step at
95.degree. C. for 15 min, followed by 35 amplification cycles of
94.degree. C. for 30 sec, 55.degree. C. for 30 sec and 72.degree.
C. for 60 sec, with a final elongation step at 72.degree. C. for 4
min.
[0701] The PCR-amplified region of YB-1 was column-purified (PCR
purification column, Qiagen) and then cloned into pCR (registered
trademark) 2.1-TOPO (Invitrogen) according to the supplier's
instructions, to make plasmid TOPO.YB-1.
[0702] Plasmid TOPO.p53
[0703] To amplify a region of the mouse p53 gene, 25 ng of a
plasmid clone containing a mouse p53 cDNA (obtained from Genesis
Research & Development Corporation, Auckland, NZ) was used as a
substrate for PCR amplification using the primers:
[0704] P2: AGA TCT AGA TAT CCT GCC ATC ACC TCA CTG [SEQ ID
NO:27]
[0705] and
[0706] P4: GGA TCC CAG GCC CCA CTT TCT TGA CCA TTG [SEQ ID
NO;28].
[0707] The PCR amplification was performed using HotStarTaq DNA
polymerase according to the supplier's protocol (Qiagen). PCR
amplification conditions involved an initial activation step at
95.degree. C. for 15 min, followed by 35 amplification cycles of
94.degree. C. for 30 sec, 55.degree. C. for 30 sec and 720C for 60
sec, with a final elongation step at 72.degree.C. for 4 min.
[0708] The PCR-amplified region of p53 was column-purified (PCR
purification column, Qiagen) and then cloned into pCR (registered
trademark) 2.1-TOPO (Invitrogen) according to the manufacturer's
instructions, to make plasmid TOPO.p53.
[0709] Plasmid TOPO.YB1.p53
[0710] To create a construct fusing YB-1 and p53 cDNA sequences,
the murine YB-1 sequence from TOPO.YB-1 was isolated as a
Bg/ll-to-BamHl fragment and cloned into the BamHl site of TOPO.p53.
A clone in which the YB-1 insert was oriented in the same sense as
the p53 sequence was selected and designated TOPO.YB1.p53.
[0711] (b) Test Plasmids
[0712] Plasmid pCMV.YB1.BGI2.1 BY
[0713] Plasmid pCMV.YB1 .BGI2.1 BY is capable of transcribing a
region of the murine YB-1 gene as an inverted repeat or palindrome
that is interrupted by the human .beta.-globin intron 2 (BGI2)
sequence therein. Plasmid pCMV.YBI.BGI2.1BY was constructed in
successive steps: (i) the YB-1 sequence from plasmid TOPO.YB-1 was
sub-cloned in the sense orientation as a Bg/ll-to-BamHl fragment
into Bg/ll-digested pCMV.BGI2 to make plasmid pCMV.YBI.BGI2, and
(ii) the YB-1 sequence from plasmid TOPO.YB-1 was sub-cloned in the
antisense orientation as a Bg/ll-to-BamHl fragment into
BamHl-digested pCMV.YB1.BGI2 to make plasmid pCMV.YB1.BGI2.1
BY.
[0714] PIasmld pCMV.YB1.p53.BGI2.35p.1BY
[0715] Plasmid pCMV.YB1 p53.BGI2.35p.lBY is capable of expressing
fused regions of the murine YB-1 and p53 genes as an inverted
repeat or palindrome that is interrupted by the human .beta.-globin
intron 2 (BGI2) sequence therein. Plasmid pCMV.YBi.p53.BG[2.35p.1BY
was constructed in successive steps: (i) the YB-1.p53 fusion
sequence from plasmid TOPO.YB1.p53 was sub-cloned in the sense
orientation as a Bg/ll-to-BamHl fragment into Bg/ll-digested
pCMV.BGI2 to make plasmid pCMV.YB1.p53.BGI2, and (ii) the YB-i.p53
fusion sequence from plasmid TOPO.YBI.p53 was sub-cloned in the
antisense orientation as a Bg/ll-to-BamHl fragment into
BamHi-digested pCMV.YB1.p53,BGI2 to make plasmid pCMV.YB1
.p53.BGI2.35p.1 BY.
[0716] 3. Detection of Co-suppression Phenotypes
[0717] (a) Post-transcriptional Gene Silencing of YB-1 by Insertion
of a Region of the YB-1 Gene into Murine Fibrosarcoma B10.2 Cells
and Murine Epidermal Keratinocyte Pam 212 Cells
[0718] YB-1 (Y-box DNA/RNA-binding factor 1) is a transcription
factor that binds, inter alia, to the promoter region of the p53
gene and in so doing represses its expression. In cancer cells that
express normal p53 protein at normal levels (some 50% of all human
cancers), the expression of p53 is under the control of YB-i, such
that diminution of YB-1 expression results in increased levels of
p53 protein and consequent apoptosis. The murine cell lines Bi0.2
and Pam 212 are two such tumorigenic cell lines with normal p53
expression. The expected phenotype for co-suppression of YB-1 in
these two cell lines is apoptosis.
[0719] Transformations with pCMV.YB1.BGI2.1BY were performed in
6-well tissue culture vessels. Individual wells were seeded with
3.5.times.10.sup.4 cells (B10.2 or Pam 212) in 3 ml of cDMEM, 5%
v/v FBS (B10.2) or equine serum (Pam 212) and incubated at
37.degree. C. in 5% v/v CO.sub.2 for 24 hr prior to
transfection.
[0720] The two mixes used to prepare transfection medium were:
[0721] Mix A: 1.5 .mu.l of LIPOFECTAMINE 2000 (trademark) Reagent
in 100 .mu.l of OPTI-MEM I (registered trademark), incubated at
room temperature for 5 min;
[0722] Mix B: 1 .mu.l (400 ng) of pCMV.YBI.BGI2.1BY DNA in 100
.mu.l of OPTI-MEM I (registered trademark) medium.
[0723] After preliminary incubation, Mix A was added to Mix B and
the mixture incubated at room temperature for a further 20 min.
[0724] Medium overlaying each cell culture was replaced with 800
.mu.l of fresh medium and 200 .mu.l of transfection mix added.
Cells were incubated at 37.degree. C. in 5% V/V CO.sub.2 for 72
hr.
[0725] Duplicate cultures of both cell types (B10.2 and Pam 212)
were transfected.
[0726] Cells were suspended with trypsin, centrifuged and
resuspended in PBS according to the protocol described in Example
1.
[0727] Live and dead cell numbers were determined by trypan blue
staining (0.2%) and counting in quadruplicate on a haemocytometer
slide. Results are presented in FIG. 75.
[0728] (b) Post-transcriptional Gene Silencing of YB-1 and p53 by
Co-insertion of Regions of the YB-1 and p53 Genes into Murine
Fibrosarcoma B 10.2 Cells and Murine Epidermal Keratinocyte Pam 212
Cells
[0729] The data presented in FIG. 75 show that cell death is
increased in B10.2 and Pam 212 cells following insertion of a YB-1
construct designed to induce co-suppression of YB-1, consistent
with induction of co-suppression.
[0730] Simultaneous co-suppression of p53, which is responsible for
initiating the apoptotic response in these cells, would be expected
to eliminate excess cell death by apoptosis.
[0731] Transformations with pCMV.YBI .p53.BGI2.35p.1 BY were
performed in 6-well tissue culture vessels. Individual wells were
seeded with 3.5.times.10.sup.4 cells (B10.2 or Pam 212) in 3 ml of
cDM EM, 5% v/v FBS (B10.2) or equine serum (Pam 212) and incubated
at 37.degree. C. in 5% v/V CO.sub.2 for 24 hr prior to
transfection.
[0732] The two mixes used to prepare transfection medium were:
[0733] Mix A: 1.5 .mu.l of LIPOFECTAMINE 2000 (trademark) Reagent
in 100 .mu.l of OPTI-MEM I (registered trademark) medium, incubated
at room temperature for 5 min;
[0734] Mix E: 1 .mu.l (400 ng) of pCMV.YB1.p53.BGI2.35p.1BY DNA in
100 .mu.l of OPTI-MEM I (registered trademark) medium.
[0735] After preliminary incubation, Mix A was added to Mix B and
the mixture incubated at room temperature for a further 20 min.
[0736] Medium overlaying each cell culture was replaced with 800
.mu.l of fresh medium and 200 .mu.l of transfection mix added.
Cells were incubated at 37.degree. C. in 5% v/v C02 for 72 hr.
[0737] Cells were suspended with trypsin, centrifuged and
resuspended in PBS according to the protocol described in Example
1.
[0738] Live and dead cell numbers were determined by trypan blue
staining (0.2%) and counting in quadruplicate on a haemocytometer
slide. Results are presented in FIG. 75.
[0739] (c) Control: Insertion of EGFP into Murine Fibrosarcoma
810.2 Cells and Murine Epidermal Keratinocyte Pam 212 Cells
[0740] Transformations with pCMV.EGFP were performed in 6-well
tissue culture vessels. Individual wells were seeded with
3.5.times.10.sup.4 cells (B1O.2 or Pam 212) in 3 ml of cDMEM, 5%
v/v FBS (B1O.2) or equine serum (Pam 212) and incubated at
37.degree. C. in 5% V/V CO.sub.2 for 24 hr prior to
transfection.
[0741] The two mixes used to prepare transfection medium were:
[0742] Mix A: 1.5 .mu.l of LiPOFECTAMINE 2000 (trademark) Reagent
in 100 .mu.l of OPTI-MEM i (registered trademark) medium, incubated
at room temperature for 5 min;
[0743] Mix B: 1 .mu.l (400 ng) of pCMV.EGFP DNA in 100 .mu.l of
OPTI-MEM I (registered trademark) medium.
[0744] After preliminary incubation, Mix A was added to Mix B and
the mixture incubated at room temperature for a further 20 min.
[0745] Medium overlaying each cell culture was replaced with 800
.mu.l of fresh medium and 200 .mu.l of transfection mix added.
Cells were incubated at 37.degree. C. in 5% v/v CO.sub.2 for 72
hr.
[0746] Cells were suspended with trypsin, centrifuged and
resuspended in PBS according to the protocol described in Example
1.
[0747] Live and dead cell numbers were determined by trypan blue
staining (0.2%) and counting in quadruplicate on a haemocytometer
slide. Results are presented in FIG. 75.
[0748] (d) Control: Attenuation of YB-1 Phenotype by Insertion of a
Decoy Y-box Oligonucleotide into Murine Fibrosarcoma B10.2 Cells
and Murine Epidermal Keratinocyte Pam 212 Cells
[0749] The role of YB-lin repressing p53-initiated apoptosis in
B10.2 and Pam 212 cells has been demonstrated by relieving the
repression in two ways: (i) transfection with YB-1 antisense
oligonucleotides; (ii) transfection with a decoy oligonucleotide
that corresponds to the YB-1 cis element derived from the fas
silencer region (-1035 to -1008 of the 5'-flanking sequence of the
human fas gene). The latter was used as a positive control in the
present example.
[0750] The double-stranded ofigonucleotides used were:
12 [SEQ ID NO:29] YB1 decoy: GAA CCT GAA TTT GGA TGC AGT TCC AGA C
CTT GGA CTT AAA CCT ACG TCA AGG TCT G [SEQ ID NO:30] YB1 control:
GCG GAT AAC AAT TTC ACA CAG G CGC CTA TTG TTA AAG TGT GTC C
[0751] Transformations with YB1 decoy and a control (non-specific)
oligonucleotide were performed in 24 well tissue culture vessels,
Individual wells were seeded with 3.5.times.10.sup.4 cells (610.2
or Pam 212) in 3 ml of cDMEM, 5% v/v FBS (B10.2) or equine serum
(Pam 212) and incubated at 37.degree. C., 5% VIV CO.sub.2 for 24 hr
prior to transfection.
[0752] The two mixes used to prepare transfection medium were:
[0753] Mix A: 1.5 .mu.l of Lipofectin (trademark) Reagent (Life
Technologies) in 100 .mu.l of OPTi-MEM I (registered trademark)
medium, incubated at room temperature for 30 min;
[0754] Mix B: 0.4 pi (40 pmol) of oligonucleotide (YB1 decoy or
control) in 100 .mu.l of OPTI-MEM I (registered trademark)
medium.
[0755] After preliminary incubation, Mix A was added to Mix B and
the mixture incubated at room temperature for a further 15 min.
[0756] A no-oligonucleotide (Lipofectin (trademark) only) control
was also prepared.
[0757] Cells were washed in serum-free medium (OPTI-MEM I
(registered trademark)) and transfection mix added. Cells were
incubated at 37.degree. C. in 5% v/v CO.sub.2 for 4 hr, after which
medium was replaced with 1 ml of cDMEM containing 5% v/v serum and
incubation continued overnight (18 hr).
[0758] Cells were suspended with trypsin, centrifuged and
resuspended in PBS according to the protocol described in Example
1.
[0759] Live and dead cell numbers were determined by trypan blue
staining (0.2%) and counting in quadruplicate on a haemocytometer
slide. Results are presented in FIG. 75.
13 TABLE 2 Percentage of plants showing No of specified phenotype
plants Suscep- Plasmid construct tested tible Immune Resistant
pART27.PVY 19 16 1 2 pART27.PVY .times. 2 13 5 4 4 pART27.PVY
.times. 3 21 2 5 14 pART27.PVY .times. 4 21 5 7 9
pART27.35S.PVY.SCBC.0 25 8 0 17 pART27.35S.O.SCBV.PVY 22 8 0 14
pART27.35S.O.SCBV.YVP 18 14 0 4 pART27.35S.PVY.SCBV.YVP 17 3 8 6
pART27.PVY.LNYV.PVY 26 18 2 6 pART27.PVY.LNYV.YVP 20 6 10 4
pART27.PVY.LNYV.YVP.DELTA. 18 7 11 0
[0760] Post-transcriptional Effect
[0761] The experiment was carried out to demonstrate the effect
that the silencing was at least, in part, post-transcriptional.
[0762] Referring to FIG. 76, there is shown a Northem blot as
explained in the description of FIG. 76 set out above. This blot
shows that clone #18 may be regarded as a "positive control", in
that there was no significant silencing. However, clones #3 and #9
do show silencing as can be seen from the absence of a band in
their respective lanes in the Northern blot of FIG. 76.
[0763] FIG. 77 graphs the results of Real-Time RT-PCR analysis of
these cell lines, as explained in the description of FIG. 77 set
out above. While it would be apparent to one skilled in the art the
method used to generate these graphs, the method is set out in more
detail in U.S. Provisional Patent application Ser. No.
60/316,308.
[0764] The results of these graphs are tabulated for reference
purposes in the table set out in FIG. 78. Referring to FIG. 78, it
can be seen that the results for clone #18 show that the mRNA level
for EGFP is set as the standard (1.000) relative to which other
mRNA levels are measured. It can be seen that there is also
significant mRNA present for GAPD (little under half the amount for
EGFP). Under the heading "Transcription", it can be seen that there
is a measurable rate of mRNA transcription for EGFP and a lower
rate of transcription measurable for GAPD. The results for clone #3
are shown on the second line. Consistent with the results of FIG.
76, there is no mRNA for EGFP although there is still mRNA for
GAPD. Further, the rate transcription for EGFP is negligible but
significant for GAPD. This demonstrates that EGFP is silenced at
the transcription point, with GAPO expression showing that other
genes are not silenced. Finally, the last line shows the results
for clone #9. Again, there is no detectable mRNA for EGFP, which is
again consistent with the EGFP having been silenced as shown in
FIG. 76. The mRNA level for the GAPD again shows that other genes
are being transcribed into mRNA. However, the significance of these
results is that there is a significant level of EGFP transcription
measured (as if course there is for GAPD as well). Thus, in clone
#9, EGFP is being transcribed but the lack of mRNA for EGFP shows
that the silencing must be occurring post-transcriptionally,
REFERENCES
[0765] 1. An etal. (1985) EMBOJ4.277-284,
[0766] 2. Armstrong, et al.Plant Cell Reports 9: 335-339, 1990.
[0767] 3. Ausubel, F. M. et al.(1987) In: Current Protocols in
Molecular Biology, Wiley lnterscience (ISBN 047140338)..
[0768] 4. Chalfie, M. et al (1 994) Science 253: 802-805.
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Sequence CWU 1
1
30 1 26 DNA Jellyfish 1 agatctgtaa acggccacaa gttcag 26 2 26 DNA
jellyfish 2 ggatccttgt acagctcgtc catgcc 26 3 74 DNA virus 3
gtcgacaata aaatatcttt attttcatta catctgtgtg ttggtttttt gtgtgatttt
60 tgcaaaagcc tagg 74 4 31 DNA virus 4 gtcgacgttt agagcagaag
taacacttcc g 31 5 38 DNA virus 5 cggcagatct aacaatggca ggacaaatcg
agtacatc 38 6 31 DNA virus 6 cccgggatcc tcgaaagaat cgtaccactt c 31
7 29 DNA virus 7 gggcggatcc ttagaaagaa tcgtaccac 29 8 28 DNA virus
8 cggcagatct ggacaaatcg agtacatc 28 9 37 DNA Agrobacterium 9
ggattcccgg gacgtcgcga atttcccccg atcgttc 37 10 33 DNA Agrobacterium
10 ccatggccat ataggcccga tctagtaaca tag 33 11 33 DNA virus 11
ccatggccta tatggccatt ccccacattc aag 33 12 27 DNA virus 12
aacgttaact tctacccagt tccagag 27 13 28 DNA virus 13 atgggatccg
ttatgccaag aagaagga 28 14 24 DNA virus 14 tgtggatccc taacggaccc
gatg 24 15 72 DNA virus 15 taatgaggat gatgtcccta cctttaattg
gcagaaattt ctgtggaaag acagggaaat 60 ctttcggcat tt 72 16 72 DNA
virus 16 ttctgccaat taaaggtagg gacatcatcc tcattaaaat gccgaaagat
ttccctgtct 60 ttccacagaa at 72 17 29 DNA primer 17 gagctcttca
gggtgagtct atgggaccc 29 18 29 DNA primer 18 ctgcaggagc tgtgggagga
agataagag 29 19 39 DNA primer 19 cggcagatcc taacaatggc aggacaaatc
gagtacatc 39 20 29 DNA primer 20 gggcggatcc ttagaaagaa tcgtaccac 29
21 20 DNA primer 21 gtttccagat ctctgatggc 20 22 20 DNA virus 22
agtccactct ggatcctagg 20 23 29 DNA primer 23 ctcgagaagt gtgcaccggc
acagacatg 29 24 29 DNA primer 24 gtcgactgtg ttccatcctc tgctgtcac 29
25 30 DNA primer 25 agatctgcag cagaccgtaa ccattatagg 30 26 30 DNA
primer 26 ggatccacct ttattaacag gtgcttgcag 30 27 30 DNA primer 27
agatctagat atcctgccat cacctcactg 30 28 30 DNA primer 28 ggatcccagg
ccccactttc ttgaccattg 30 29 28 DNA double-stranded 29 gaacctgaat
ttggatgcag ttccagac 28 30 22 DNA double-stranded 30 gcggataaca
atttcacaca gg 22
* * * * *