U.S. patent application number 13/311511 was filed with the patent office on 2012-08-02 for znf206: a novel regulator of embryonic stem cell self-renewal and pluripotency.
This patent application is currently assigned to SANFORD-BURNHAM MEDICAL RESEARCH INSTITUTE. Invention is credited to Rodolfo GONZALEZ, Evan Yale SNYDER.
Application Number | 20120196922 13/311511 |
Document ID | / |
Family ID | 40341597 |
Filed Date | 2012-08-02 |
United States Patent
Application |
20120196922 |
Kind Code |
A1 |
SNYDER; Evan Yale ; et
al. |
August 2, 2012 |
ZNF206: A NOVEL REGULATOR OF EMBRYONIC STEM CELL SELF-RENEWAL AND
PLURIPOTENCY
Abstract
We have identified ZNF206, a novel repressor of human embryonic
stem cell (hESC) differentiation. Repressing extra-embryonic
endoderm development preserves the pluripotent state of human
embryonic stem cells, and, conversely down-regulating expression of
ZNF206 in hESCs causes them to upregulate the expression of genes
associated with the extra-embryonic endodermal lineage,
down-regulate genes associated with the pluripotent state, and may
lead to the further emergence of genes associated with even more
differentiated lineages and phenotypes.
Inventors: |
SNYDER; Evan Yale; (La
Jolla, CA) ; GONZALEZ; Rodolfo; (La Jolla,
CA) |
Assignee: |
SANFORD-BURNHAM MEDICAL RESEARCH
INSTITUTE
La Jolla
CA
|
Family ID: |
40341597 |
Appl. No.: |
13/311511 |
Filed: |
December 5, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12221824 |
Aug 6, 2008 |
8071378 |
|
|
13311511 |
|
|
|
|
60963850 |
Aug 6, 2007 |
|
|
|
Current U.S.
Class: |
514/44R ;
435/455; 435/6.11; 435/7.1; 436/501; 506/9; 530/350; 530/387.3;
530/388.2; 530/389.8; 536/23.5 |
Current CPC
Class: |
C07K 14/4702 20130101;
C12N 2799/027 20130101; A61P 35/00 20180101 |
Class at
Publication: |
514/44.R ;
536/23.5; 530/350; 530/389.8; 530/387.3; 530/388.2; 435/6.11;
436/501; 435/455; 506/9; 435/7.1 |
International
Class: |
A61K 31/713 20060101
A61K031/713; C07K 14/47 20060101 C07K014/47; C07K 16/18 20060101
C07K016/18; C40B 30/04 20060101 C40B030/04; G01N 33/566 20060101
G01N033/566; C12N 15/85 20060101 C12N015/85; A61P 35/00 20060101
A61P035/00; C07H 21/04 20060101 C07H021/04; C12Q 1/68 20060101
C12Q001/68 |
Claims
1. An isolated polynucleotide comprising a sequence that has at
least 90% nucleic acid sequence identity to a native ZNF206
polynucleotide and that hybridizes selectively to the native ZNF206
polynucleotide.
2-3. (canceled)
4. The polynucleotide of claim 1 comprising a sequence at least 100
nucleotides in length that has at least 90% nucleic acid sequence
identity to a native ZNF206 polynucleotide.
5-6. (canceled)
7. The polynucleotide of claim 1 comprising at least 15 contiguous
nucleotides of a native ZNF206 polynucleotide, wherein the isolated
polynucleotide hybridizes selectively to a native ZNF206
polynucleotide.
8-9. (canceled)
10. The isolated polynucleotide of claim 7 comprising a full-length
protein-coding sequence of a native ZNF206 mRNA or cDNA.
11. The isolated polynucleotide of claim 1 that encodes a
polypeptide that has ZNF206 activity.
12-19. (canceled)
20. An isolated polypeptide of at least 11 amino acids that
comprises at least 4 contiguous amino acids of a native ZNF206
polypeptide, and, that when introduced into a mammal, elicits
production of an antibody that binds selectively to a native ZNF206
polypeptide.
21-36. (canceled)
37. The isolated polypeptide of claim 20 that has ZNF206
activity.
38. (canceled)
39. An antibody that binds selectively to a native ZNF206
polypeptide.
40. The antibody of claim 39, wherein the antibody is selected from
the group consisting of monoclonal antibody, polyclonal antibody,
chimeric antibody, humanized antibody, and single chain
antibody.
41-48. (canceled)
49. A method of detecting the presence of a ZNF206 polynucleotide
in a sample comprising the ZNF206 polynucleotide, the method
comprising contacting the sample with (a) a probe or primer
comprising a polynucleotide sequence that binds selectively to the
ZNF206 polynucleotide and detecting binding of the probe or primer
to the ZNF206 mRNA, or (b) contacting the sample with an antibody
that binds selectively to the ZNF206 polypeptide and detecting
binding of the antibody to the ZNF206 polypeptide.
50. The method of claim 49 comprising (a) contacting the sample
with a first primer that comprises the polynucleotide sequence that
hybridizes selectively to the ZNF206 mRNA and a second primer
comprising a polynucleotide sequence that hybridizes to the ZNF206
mRNA, (b) performing an amplification reaction to produce an
amplification product that indicates the presence of the ZNF206
mRNA in the sample, and (c) detecting the amplification
product.
51-56. (canceled)
56. A method of assessing the pluripotency of a cell comprising (a)
measuring ZNF206 polypeptide or polynucleotide levels in a sample
comprising the cell, and (b) comparing the ZNF206 polypeptide or
polynucleotide levels in the sample to a reference.
57. The method of claim 56 comprising measuring the ZNF206
polypeptide level in the cell by contacting the sample with an
antibody that binds selectively to ZNF206 polypeptide, and
measuring binding of the antibody to ZNF206 polypeptide in the
sample.
58-60. (canceled)
61. The method of claim 56 comprising measuring the ZNF206 mRNA
level in the cell by contacting the sample with a probe or primer
that hybridizes selectively to ZNF206 mRNA and measuring
hybridization of the probe or primer to the ZNF206 mRNA in the
sample.
62-69. (canceled)
70. A method of promoting differentiation of a cell by reducing
ZNF206 expression of the cell, the method comprising expressing in
the cell a vector comprising (a) a promoter suitable for expression
in the cell operably linked to (b) an isolated polynucleotide
comprising a sequence that has at least 90% nucleic acid sequence
identity to a native ZNF206 polynucleotide and that hybridizes
selectively to the native ZNF206 polypeptide, wherein expression of
the polynucleotide in the cell causes a reduction in ZNF206
polypeptide levels in the cell.
71. A method of diagnosing a cancer characterized by elevated
levels of ZNF206 comprising (a) obtaining a sample comprising a
cell, (b) determining ZNF206 polypeptide or polynucleotide levels
in the sample, and (c) comparing the ZNF206 polypeptide or
polynucleotide levels in the sample with a reference.
72. A method of treating a cancer characterized by elevated levels
of ZNF206 comprising administering to a patient in need of such
treatment a composition comprising a vector comprising (a) a
promoter suitable for expression in a cell of the patient operably
linked to (b) an isolated polynucleotide comprising a sequence at
least 100 nucleotides in length that has at least 90% nucleic acid
sequence identity to a native ZNF206 polynucleotide, wherein
expression of the polynucleotide in the cell reduces ZNF206
polypeptide levels in the cell.
73-79. (canceled)
Description
TECHNICAL FIELD
[0001] The present invention relates to stem cell research,
particularly to genes involved in regulation of self-renewal and
pluripotency of stem cells, such as, for example, human embryonic
stem cells.
BACKGROUND INFORMATION
[0002] Several transcriptional factors have been implicated in
human embryonic stem cell (hESC) self-renewal supporting a view
that this process is regulated at the level of transcriptional
control (Chambers, Cloning Stem Cells 6:386-391, 2004).
[0003] The transcription factor POU5F1 (OCT4) is essential for
embryonic stem cell (ESC) pluripotency and appears to regulate a
number of ESC properties. OCT4 is specifically expressed in ESCs,
pre-implantation embryos, epiblast, and germ cells (Okamoto et al.,
Cell 60:461-472, 1990; Scholer et al., EMBO J. 9:2185-2195, 1990).
Inactivation of OCT4 in mouse embryos and ESCs results in loss of
pluripotency and spontaneous differentiation into the trophoblast
lineage (Niwa et al., Nat. Genet. 24:372-376, 2000). Mouse ESCs
(mESCs), even when constitutively expressing OCT4 from an exogenous
promoter, still require LIF for self-renewal suggesting that LIF
and OCT4 function in different pathways. However, overexpression of
OCT4 induces mESCs to differentiate into PE (Niwa et al., Nat.
Genet. 24:372-376, 2000).
[0004] The homeodomain-containing transcription factor NANOG is
another critical ESC factor recently identified (Chambers et al.,
Cell 113:643-655, 2003; Mitsui et al., Cell 113:631-642, 2003). The
NANOG-deficient ICM fails to generate an epiblast and only produces
extraembryonic primitive endoderm (PE). Similarly in culture,
NANOG-deficient ESCs lose pluripotency and differentiate to a PE
lineage. Unlike POU5F1/OCT-4, NANOG overexpression can maintain ESC
self-renewal without LIF (Chambers et al., Cell 113:643-655, 2003).
It has been proposed that NANOG maintains ESC self-renewal through
repression of genes that promote differentiation, e.g., GATA4 and
GATA6, which are upregulated in NANOG-deficient cells. That NANOG
also binds sequences in the GATA6 gene supports this hypothesis
(Mitsui et al., Cell 113:631-642, 2003).
[0005] Together, these observations suggest that NANOG is a
critical factor underlying pluripotency in both ICM and ESCs by
repressing their differentiation into PE, and that NANOG and OCT4
work together in the maintenance of the undifferentiated state by
virtue of overlapping functions. Two cell fate decisions have to be
made during pre-implantation development. The first is that cells
of the morula remain pluripotent or differentiate into
trophectoderm. The second is that cells of the ICM remain
pluripotent as epiblast or differentiate into PE. OCT4 is the key
determinant of the first decision (since OCT4-null ESCs
differentiate into trophectoderm), while NANOG is the crucial
determinant of the second decision (since ESCs lacking NANOG
differentiate into PE) (Mitsui et al., Cell 113:631-642, 2003).
FIG. 1 shows transcription factors involved in controlling
self-renewal of human embryonic stem cells by repressing early
lineage commitment.
[0006] Two other transcription factors have been identified that
interact with OCT4: the forkhead transcription factor FOXD3 and the
Sry-related factor SOX2. FOXD3 is expressed in the blastocyst and
later in the post-implantation egg cylinder epiblast. FOXD3
physically interacts with OCT4 to activate the ostopontin enhancer,
which is expressed in ESCs (Guo et al., Proc. Natl. Acad. Sci.
U.S.A. 99:3663-3667, 2002). Sox2 is expressed in ESCs as well as in
multipotent embryonic and extra-embryonic lineages. Disrupting Sox2
results in pre-implantation embryonic lethality (Avilion et al.,
Genes Dev. 17:126-140, 2003). Sox2 was identified as a co-factor of
OCT4 for activating FGF4, which is restrictively expressed in
undifferentiated ESCs, and is essential for post-implantation mouse
development and limb patterning and growth (Yuan et al., Genes Dev.
9:2635-2645, 1995). Transcriptional regulation of NANOG itself is
also regulated by OCT4 and SOX2 (Rodda et al., J. Biol. Chem. 280:
24731-24737, 2005). Another OCT4 and SOX2 co-regulated gene is the
ESC-specific transcription factor UTF1 (Nishimoto et al., Mol.
Cell. Biol. 19:5453-5465, 1999). Taken together, these studies
suggest that the SOX2-OCT4 complex is at the apex of a regulatory
hierarchy of the "pluripotency genetic regulatory network".
[0007] FIG. 1 shows transcription factors involved in controlling
self-renewal by repressing early lineage commitment.
[0008] In summary, ESC identity is determined by cell-intrinsic
transcription factors that need to be expressed at particular
levels in order to function appropriately. However, the molecular
basis of the regulation of pluripotency and early lineage
commitment of hESCs is still poorly understood. Additional
intrinsic pathway-specific transcription factors presumably exist
that maintain expression of the thousands of genes that are
expressed in ESCs and control different types of renewal and
differentiation pathways. Understanding how hESCs maintain their
pluripotency and self-renewal and execute precise differentiation
programs will require extending our understanding of the
transcriptional regulatory hierarchy of hESC function, including
identifying new intrinsic transcription factors.
SUMMARY OF THE INVENTION
[0009] We have identified zinc finger protein 206 (ZNF206), a novel
repressor of human embryonic stem cell (hESC) differentiation.
Repressing extra-embryonic endoderm development preserves the
pluripotent state of human embryonic stem cells, and, conversely
downregulating expression of ZNF206 in hESCs causes them to
upregulate the expression of genes associated with the
extra-embryonic endodermal lineage, down-regulate genes associated
with the pluripotent state, and may lead to the further emergence
of genes associated with even more differentiated lineages and
phenotypes.
[0010] According to one aspect of the invention, isolated
polynucleotides are provided that comprise a sequence that has at
least 90%, or 95%, or 100% nucleic acid sequence identity to a
native ZNF206 polynucleotide and that hybridize selectively to the
native ZNF206 polynucleotide. The isolated polynucleotide of claim
1 wherein the sequence that has at least 95% identity to a native
ZNF206 polynucleotide. According to another embodiment, such
isolated polynucleotides comprise a sequence at least 100
nucleotides in length that has at least 90%, 95%, or 99% nucleic
acid sequence identity to a native ZNF206 polynucleotide.
[0011] According to another embodiment of the invention, isolated
polynucleotides are provided that comprise at least 15, 20, or 30
contiguous nucleotides of a native ZNF206 polynucleotide, wherein
the isolated polynucleotide hybridizes selectively to a native
ZNF206 polynucleotide. According to one embodiment, the isolated
polynucleotide comprises a full-length protein-coding sequence of a
native ZNF206 mRNA or cDNA. According to another embodiment, the
isolated polynucleotide encodes a polypeptide that has ZNF206
activity.
[0012] According to another embodiment of the invention, cells are
provided that comprise any of the isolated polynucleotides
described above. According to another embodiment, cells, vectors
(including, but not limited to expression vectors), probes and
primers are provided that comprise any of the isolated
polynucleotides described above. Also provided are cells that
comprise such vectors.
[0013] According to another embodiment of the invention, kits are
provided that comprise: (a) a first primer comprising at least 15
contiguous nucleotides of a native ZNF206 polynucleotide, wherein
the first primer hybridizes selectively to a native ZNF206
polynucleotide; (b) a second primer comprising at least 15
contiguous nucleotides of the native ZNF206 polynucleotide; and (c)
suitable packaging enclosing the first primer and the second
primer, wherein an amplification reaction performed using the first
primer, the second primer, and a sample comprising a ZNF206 mRNA
produces an amplification product that indicates the presence of
the ZNF206 mRNA in the sample.
[0014] According to another embodiment of the invention, isolated
polypeptides of at least 11 amino acids are provided that comprise
at least 4, 5, 6, 7, 8, 9, or 10 contiguous amino acids of a native
ZNF206 polypeptide, and, that when introduced into a mammal,
elicits production of an antibody that binds selectively to a
native ZNF206 polypeptide. According to another embodiment of the
invention, isolated polypeptides are provided that comprise at
least 11, 12, 15, 20, or 30 contiguous amino acids of a native
ZNF206 polypeptide, and, that when introduced into a mammal,
elicits production of an antibody that binds selectively to a
native ZNF206 polypeptide, including but not limited to a
full-length native ZNG206 polypeptide.
[0015] According to another embodiment of the invention, isolated
polypeptides are provided that comprise a sequence that has at
least 90%, 91, 92, 93, 94, 95%, 96%, 97%, 98%, 99% or 100% amino
acid sequence identity to a native ZNF206 polypeptide, wherein
introduction of the isolated polypeptide into a mammal elicits
production of an antibody that selectively binds to ZNF206.
According to another embodiment of the invention, such isolated
polypeptides comprise a sequence at least 15, 16, 17, 18, 19, 20,
30, 40 or more amino acids in length that has such a degree of
amino acid sequence identity. According to another embodiment of
the invention, such isolated polypeptides have ZNF206 activity.
[0016] According to another embodiment of the invention, isolated
polynucleotides are provided that encode any of the aforementioned
polypeptides.
[0017] According to another embodiment of the invention, antibodies
are provided that bind selectively to a native ZNF206 polypeptide,
including, but not limited to, monoclonal, polyclonal, chimeric,
humanized, single-chain, and fragment antibodies, for example.
[0018] According to another embodiment of the invention, methods
are provided for making an antibody that binds selectively to a
native ZNF206 polypeptide comprising introducing into a mammal (a)
an expression vector comprising one of the aforementioned
polynucleotides, or (b) one of the aforementioned isolated
polypeptides, thereby eliciting production of the antibody.
[0019] According to another embodiment of the invention,
pharmaceutical compositions are provided that comprise (a) a vector
comprising a promoter suitable for expression in the cell operably
linked to an isolated polynucleotide comprising a sequence that has
at least 90% nucleic acid sequence identity to a native ZNF206
polynucleotide and that hybridizes selectively to the native ZNF206
polypeptide, wherein expression of the polynucleotide in the cell
causes a reduction in ZNF206 polypeptide levels in the cell, and
(b) a pharmaceutically acceptable carrier.
[0020] According to another embodiment of the invention, methods
are provided for making a medicament for treating a patient with a
cancer or at risk for developing the cancer, the method comprising
formulating the medicament with a pharmaceutically effective amount
of a vector comprising a promoter suitable for expression in the
cell operably linked to an isolated polynucleotide comprising a
sequence that has at least 90% nucleic acid sequence identity to a
native ZNF206 polynucleotide and that hybridizes selectively to the
native ZNF206 polypeptide, wherein expression of the polynucleotide
in the cell causes a reduction in ZNF206 polypeptide levels in the
cell.
[0021] According to another embodiment of the invention, methods
are provided for detecting the presence of a ZNF206 polynucleotide
in a sample comprising the ZNF206 polynucleotide, the method
comprising contacting the sample with a probe or primer comprising
a polynucleotide sequence that binds selectively to the ZNF206
polynucleotide and detecting binding of the probe or primer to the
ZNF206 mRNA. One such embodiment, comprises (a) contacting the
sample with a first primer that comprises the polynucleotide
sequence that hybridizes selectively to the ZNF206 mRNA and a
second primer comprising a polynucleotide sequence that hybridizes
to the ZNF206 mRNA, (b) performing an amplification reaction to
produce an amplification product that indicates the presence of the
ZNF206 mRNA in the sample, and (c) detecting the amplification
product, including, but not limited to, a PCR reaction.
[0022] According to another embodiment of the invention, methods
are provided for detecting the presence of a ZNF206 polypeptide in
a sample comprising the ZNF206 polypeptide, the method comprising
(a) contacting the sample with an antibody (including, but not
limited to, a monoclonal antibody) that binds selectively to the
ZNF206 polypeptide and (b) detecting binding of the antibody to the
ZNF206 polypeptide. Such methods may, for example, comprise
performing ELISA or bio-barcode assays.
[0023] According to another embodiment of the invention, methods
are provided for assessing the pluripotency of a cell by various
means. According to one such embodiment, such methods comprise (a)
measuring ZNF206 polypeptide or polynucleotide levels in a sample
comprising the cell, and (b) comparing the ZNF206 polypeptide or
polynucleotide levels in the sample to a reference. According to
another such embodiment, such methods comprise measuring the ZNF206
polypeptide level in the cell by contacting a sample comprising the
cell with an antibody that binds selectively to ZNF206 polypeptide
(including but not limited to a monoclonal antibody) and measuring
binding of the antibody to ZNF206 polypeptide in the sample, such
as, for example, by an ELISA or bio-barcode assay. According to
another embodiment, such methods comprise measuring the ZNF206 mRNA
level in the cell by contacting a sample comprising the cell with a
probe or primer that hybridizes selectively to ZNF206 mRNA and
measuring hybridization of the probe or primer to the ZNF206 mRNA
in the sample. According to another embodiment, such methods
comprise measuring the ZNF206 mRNA level in the cell by (a)
contacting the sample comprising the cell with one or more primers
that comprise a polynucleotide sequence that hybridizes selectively
to the ZNF206 mRNA, (b) performing an amplification reaction
(including but not limited to a PCR reaction or bio-barcode assay)
to produce an amplification product that indicates the presence of
ZNF206 mRNA in the sample, and (c) measuring the amplification
product. In any of the foregoing methods for assessing the
pluripotency of a cell, the sample may be, for example, a tissue
sample.
[0024] According to another embodiment of the invention, methods
are provided for maintaining or increasing the pluripotency of a
cell comprising expressing in the cell a vector comprising (a) a
promoter suitable for expression in the cell operably linked to (b)
an isolated polynucleotide comprising a sequence at least 100
nucleotides in length that has at least 90% nucleic acid sequence
identity to a native ZNF206 polynucleotide, wherein expression of
the polynucleotide in the cell produces a polypeptide that reduces
or prevents differentiation of the cell.
[0025] According to another embodiment of the invention, methods
are provided for promoting differentiation of a cell comprising
reducing ZNF206 expression of the cell. According to one
embodiment, such a method comprises expressing in the cell a vector
comprising (a) a promoter suitable for expression in the cell
operably linked to (b) an isolated polynucleotide comprising a
sequence that has at least 90% nucleic acid sequence identity to a
native ZNF206 polynucleotide and that hybridizes selectively to the
native ZNF206 polypeptide, wherein expression of the polynucleotide
in the cell causes a reduction in ZNF206 polypeptide levels in the
cell.
[0026] According to another embodiment of the invention, methods
are provided for diagnosing a cancer characterized by elevated
levels of ZNF206 comprising (a) obtaining a sample comprising a
cell, (b) determining ZNF206 polypeptide or polynucleotide levels
in the sample, and (c) comparing the ZNF206 polypeptide or
polynucleotide levels in the sample with a reference.
[0027] According to another embodiment of the invention, methods
are provided for treating a cancer characterized by elevated levels
of ZNF206 comprising administering to a patient in need of such
treatment a composition comprising a vector comprising (a) a
promoter suitable for expression in a cell of the patient operably
linked to (b) an isolated polynucleotide comprising a sequence at
least 100 nucleotides in length that has at least 90% nucleic acid
sequence identity to a native ZNF206 polynucleotide, wherein
expression of the polynucleotide in the cell reduces ZNF206
polypeptide levels in the cell.
[0028] According to another embodiment of the invention, methods
are provided for diagnosing a disease state resulting from a
mutation in a ZNF206 polynucleotide comprising (a) providing a
sample from a patient comprising a cell and (b) determining whether
the sample comprises a mutated ZNF206 polynucleotide. The presence
of a mutated ZNF 206 polynucleotide in the sample may be
determined, for example by: contacting the sample with a
polynucleotide probe or primer that hybridizes specifically to a
mutated ZNF206 polynucleotide sequence; by contacting the sample
with one or more primers that comprise a polynucleotide sequence
that hybridizes selectively to the mutated ZNF206 polynucleotide,
and performing an amplification reaction (e.g., a PCR or
bio-barcode assay) to produce an amplification product that
indicates the presence of the mutated ZNF206 polynucleotide in the
sample; by detecting a restriction fragment length polymorphism; or
by contacting the sample with an antibody probe that hybridizes
specifically to a mutated ZNF polypeptide sequence encoded by the
mutated ZNF polynucleotide.
[0029] Any of the aforementioned methods may be automated.
[0030] The foregoing and other aspects of the invention will become
more apparent from the following detailed description, accompanying
drawings, and the claims.
[0031] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, suitable methods and materials are described
below. All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
BRIEF DESCRIPTION OF THE FIGURES
[0032] FIG. 1 shows transcription factors involved in controlling
self-renewal of human embryonic stem cells by repressing early
lineage commitment.
[0033] FIG. 2 shows high and unique expression of ZNF206 in
undifferentiated hESCs. [A] ZNF206 and NANOG were highly expressed
in hESC line WA09 (H9) but not in PE-like (PEL) cells derived from
them. [B] Quantitative RT-PCR analysis of ZNF206 expression in H9
hESCs, in PEL cells derived from H9 hESCs, and in adult human
tissues.
[0034] FIG. 3 shows that ZNF206 expression is downregulated upon
hESC differentiation into extraembryonic endoderm cells. HESCs
(from lines WA09 [H9] and WA01 [H1]) were treated for various
times--0, 48, 96 hrs--with BMP2 (50 ng/ml) followed by Quantitative
RT-PCR to analyze the expression of NANOG [A], ZNF206[B], GATA6
[C], and GATA4 [D].
[0035] FIG. 4 shows the predicted protein sequence of three
isoforms of ZNF206. The ZNF206 gene contains five introns and five
exons. [A] Primers were specifically designed to amplify and to
clone the different spliced ZNF206 mRNA isoforms expressed in
undifferentiated hESCs. [B] Four different ZNF206 mRNA isoforms
were cloned from undifferentiated hESCs. Isoform 1 is 2568 bp,
isoform 2 is 2343 bp, and isoform 3 is 2075 bp. [C] Isoform 1 and 4
are predicted to encode truncated ZNF206 proteins containing a
"Novel" and "SCAN" domain. The Novel domain contains a sumoylation
site and the SCAN domain has been previously reported to mediate
protein-protein interactions. On the other hand, ZNF206 isoform 2
is predicted to contain 780 amino acids containing the Novel, SCAN
and 14 C2H2 Zinc finger domains. The C2H2 zinc finger domains often
mediate DNA binding.
[0036] FIG. 5 shows a diagram of three C-terminally tagged ZNF206
lentivirus expression vectors that we have successfully made.
[0037] FIG. 6 shows the knock-down efficiency of lentiviral ZNF206
shRNA expression constructs. Human kidney 293FT-ZNF206-V5
expressing cell lines were infected with lentiviral particles
containing ZNF206 shRNA expression constructs. After puromycin
selection and expansion of infected 293FT cells, we performed
quantitative RT-PCR [A] and Western blot analysis [B].
[0038] FIG. 7 shows the generation of a polyclonal rabbit antibody
against the human ZNF206 proteins. Underlined is the peptide (amino
acids 711-726) used to immunize rabbits against the human ZNF206
protein. The polyclonal antibody detects a protein that is
approximately 80 kD in size in undifferentiated hES cell line H9
and not in the hES-derived PEL differentiated cells.
[0039] FIG. 8 shows the effects of ZNF206 knockdown on OCT-4 and
NANOG expression in hESCs. hESCs were infected with three different
shRNA lentiviral expression particles (ZNF206 shRNA-A, ZNF206
shRNA-B, ZNF206 shRNA-C) and the control lentiviral empty vector.
Four days after infection of undifferentiated hESC lines H9 (WA09)
and H1 (WA01), the mRNA and protein expression of ZNF206, Oct-4 and
NANOG were determined by quantitative RT PCR.
[0040] FIG. 9 shows the hypothesized Role of ZN206 in hESCs. [A] In
our model, OCT4 is the key inhibitor of trophoblast differentiation
in hESCs (since specific down-regulation of OCT-4 in hESCs leads to
trophoblast differentiation), while NANOG and ZNF206 are key
inhibitiors of extra-embryonic endoderm lineage differentiation
(since specific down-regulation of NANOG or ZNF206 leads to
extra-embryonic endoderm lineage differentiation). For example,
down-regulation of ZNF206 expression in hESCs causes them to
upregulate genes associated with the extra-embryonic endoderm
lineage (e.g., GATA4, GATA6, SOX17, AFP and HNF4A). [B] We further
hypothesize that extra-embryonic endoderm differentiation may be
the earliest default pathways for hESCs, particularly when
dissociated into single cells and grown in defined, serum-free,
feeder-free conditions. This default lineage may then help instruct
the emergence of other lineages, e.g., neuroectoderm (perhaps
giving the appearance of being default).
[0041] FIG. 10 shows the DNA sequence for four isoforms of
ZNF206.
DETAILED DESCRIPTION OF THE INVENTION
Definitions and Methods
[0042] The following definitions and methods are provided to better
define the present invention and to guide those of ordinary skill
in the art in the practice of the present invention. Unless
otherwise noted, terms are to be understood according to
conventional usage by those of ordinary skill in the relevant art.
Definitions of common terms in molecular biology may also be found
in Rieger et al., Glossary of Genetics: Classical and Molecular,
5th edition, Springer-Verlag: New York, 1991; and Lewin, Genes V,
Oxford University Press: New York, 1994. The nomenclature for DNA
bases as set forth at 37 CFR 1.822 is used. The standard one- and
three-letter nomenclature for amino acid residues is used.
Polynucleotides
[0043] As used herein, the term "ZNF206 polynucleotide" refers to
the ZNF206 genomic DNA, mRNA, and cDNA corresponding to the mRNA as
present in humans (including any of the several human isoforms of
ZNF206) or non-human species, such as, for example, in the
chimpanzee, mouse or chicken (Bernot et al., Genomics 50:147-160,
1998). Also encompassed by the term "ZNF206 polynucleotides" are,
for example: fragments or portions of the ZNF206 mRNA or cDNA,
including but not limited to, a ZNF206 polynucleotide; fragments
that encode antigenic determinants of ZNF206 (e.g., those that
elicit antibodies that bind selectively to ZNF206 polypeptide);
probes and primers that hybridize selectively to ZNF206
polynucleotides; etc. Also included are mutated or variant
polynucleotides that include one or more nucleotide insertions,
deletions, or substitutions from the wild-type ZNF206 sequence, but
that, for example: retain the ability to bind selectively to
ZNF206; encode a polypeptide that includes a ZNF206 antigenic
determinant; encode a polypeptide having ZNF206 activity; etc.
[0044] As used herein, the term "hybridizes selectively" refers to
binding of a probe, primer or other polynucleotide, under stringent
hybridization conditions, to a target polynucleotide, such as a
native, or wild-type, ZNF206 mRNA or cDNA, to a substantially
higher degree than to other polynucleotides. Probes and primers
that hybridize selectively to ZNF206 include sequences that are
unique to ZNF206. In particular, a probe that "hybridizes
selectively" to ZNF206 does not hybridize substantially to ZNF206
under stringent hybridization conditions and therefore can be used
to distinguish a ZNF206 polynucleotide (e.g., a ZNF206 mRNA) from a
ZNF206 polynucleotide. Similarly, a primer that "hybridizes
selectively" to ZNF206, when used in an amplification reaction such
as PCR, results in amplification of ZNF206 without resulting in
substantial amplification of ZNF206 under suitable amplification
conditions. Thus, all or substantially all of a ZNF206-selective
probe or primer hybridizes to the target ZNF206 polynucleotide
under suitable conditions, as can be determined given the
sensitivity of a particular procedure. Similarly, as used herein,
the term "selective for" in reference to a polynucleotide,
indicates that the polynucleotide hybridizes selectively to a
target polynucleotide.
[0045] Similarly, a probe or primer that includes a sequence that
is unique to ZNF206 hybridizes selectively to ZNF206. In
particular, a probe that hybridizes selectively to ZNF206 does not
hybridize substantially to ZNF206 under stringent hybridization
conditions and therefore can be used to distinguish a ZNF206
polynucleotide (e.g., a ZNF206 mRNA) from a ZNF206 polynucleotide.
Similarly, a primer that hybridizes selectively to a ZNF206
polynucleotide, when used in an amplification reaction such as PCR,
results in amplification of the ZNF206 polynucleotide without
resulting in substantial amplification of ZNF206 polynucleotide.
Thus, all or substantially all of a ZNF206-selective probe or
primer hybridizes to the target ZNF206 polynucleotide, as can be
determined given the sensitivity of a particular procedure.
[0046] As used herein, the terms "wild-type" or "native" in
reference to a polynucleotide are used interchangeably to refer to
a polynucleotide that has 100% sequence identity with a reference
polynucleotide that can be found in a cell or organism, or a
fragment thereof.
[0047] Polynucleotide (e.g., DNA or RNA) sequences may be
determined by sequencing a polynucleotide molecule using an
automated DNA sequencer. A polynucleotide sequence determined by
this automated approach can contain some errors. The actual
sequence can be confirmed by resequencing the polynucleotide by
automated means or by manual sequencing methods well known in the
art.
[0048] Unless otherwise indicated, each "nucleotide sequence" set
forth herein is presented as a sequence of deoxyribonucleotides
(abbreviated A, G, C and T). However, the term "nucleotide
sequence" of a DNA molecule as used herein refers to a sequence of
deoxyribonucleotides, and for an RNA molecule, the corresponding
sequence of ribonucleotides (A, G, C and U) where each thymidine
deoxynucleotide (T) in the specified deoxynucleotide sequence in is
replaced by the ribonucleotide uridine (U).
[0049] By "isolated" polynucleotide is intended a polynucleotide
that has been removed from its native environment. For example,
recombinant polynucleotides contained in a vector are considered
isolated for the purposes of the present invention. Further
examples of isolated polynucleotides include recombinant
polynucleotides maintained in heterologous host cells or purified
(partially or substantially) polynucleotides in solution. Isolated
RNA molecules include in vivo or in vitro RNA transcripts of the
DNA molecules of the present invention. Isolated polynucleotides
according to the present invention further include such molecules
produced synthetically.
[0050] Polynucleotides can be in the form of RNA, such as mRNA, or
in the form of DNA, including, for instance, cDNA and genomic DNA.
The DNA can be double-stranded or single-stranded. A
single-stranded DNA or RNA can be a coding strand, also known as
the sense strand, or it can be a non-coding strand, also referred
to as the anti-sense strand. Polynucleotides can include
non-naturally occurring nucleotide or ribonucleotide analogs.
[0051] The term "fragment" (of a polynucleotide) as used herein
refers to polynucleotides that are part of a longer polynucleotide
having a length of at least about 15, 20, 25, 30, 35, or 40
nucleotides (nt) in length, which are useful, for example, as
probes and primers. Thus, for example, a fragment of ZNF206 at
least 20 nucleotides in length includes 20 or more contiguous
nucleotides from the nucleotide sequence of the ZNF206 full-length
cDNA. Such DNA fragments may be generated by the use of automated
DNA synthesizers or by restriction endonuclease cleavage or
shearing (e.g., by sonication) a full-length ZNF206 cDNA, for
example.
[0052] Also encompassed by the present invention are isolated
polynucleotides that hybridize under stringent hybridization
conditions to a ZNF206 polynucleotide such as, for example, a
ZNF206 transcript (i.e., mRNA). By "stringent hybridization
conditions" is intended overnight incubation at 42.degree. C. in a
solution comprising: 50% formamide, 5.times.SSC (750 mM NaCl, 75 mM
trisodium citrate), 50 mM sodium phosphate (pH7.6),
5.times.Denhardt's solution, 10% dextran sulfate, and 20 .mu.g/ml
denatured, sheared salmon sperm DNA, followed by washing the
filters in 0.1.times.SSC at about 65.degree. C. Alternatively,
stringent hybridizations are conditions used for performance of a
polymerase chain reaction (PCR). Such hybridizing polynucleotides
are useful diagnostically as a probe according to conventional DNA
hybridization techniques or as primers for amplification of a
target sequence by the polymerase chain reaction (PCR).
[0053] As used herein, the term "hybridizes (or binds)
specifically" is used interchangeably with the term "hybridizes (or
binds) selectively" means that most or substantially all
hybridization of a probe or primer is to a particular
polynucleotide in a sample under stringent hybridization
conditions.
[0054] The present invention also provides polynucleotides that
encode all or a portion of a polypeptide, e.g., a full-length
ZNF206 polypeptide or a portion thereof. Such protein-coding
polynucleotides may include, but are not limited to, those
sequences that encode the amino acid sequence of the particular
polypeptide or fragment thereof and may also include together with
additional, non-coding sequences, including for example, but not
limited to introns and non-coding 5' and 3' sequences, such as the
transcribed, non-translated sequences that play a role in
transcription, mRNA processing--including splicing and
polyadenylation signals, e.g., ribosome binding and stability of
mRNA; an additional coding sequence which codes for additional
amino acids, such as those which provide additional
functionalities. In addition, the sequence encoding the polypeptide
can be fused to a heterogeneous polypeptide or peptide sequence,
such as, for example a marker sequence that facilitates
purification of the fused polypeptide. One example of such a marker
sequence is a hexa-histidine peptide, such as the tag provided in a
pQE vector (Qiagen, Inc.). As described in Gentz et al., Proc.
Natl. Acad. Sci. USA 86:821-824 (1989), for instance,
hexa-histidine provides for convenient purification of the fusion
protein. The "HA" tag is another peptide useful for purification
which corresponds to an epitope derived from the influenza
hemagglutinin (HA) protein (Wilson et al., Cell 37:767, 1984).
[0055] The present invention further relates to variants of the
native, or wild-type, polynucleotides of the present invention,
which encode portions, analogs or derivatives of a ZNF206
polypeptide. Variants can occur naturally, such as a natural
allelic variant, i.e., one of several alternate forms of a gene
occupying a given locus on a chromosome of an organism.
Non-naturally occurring variants can be produced, e.g., using known
mutagenesis techniques or by DNA synthesis. Such variants include
those produced by nucleotide substitutions, deletions or additions.
The substitutions, deletions or additions can involve one or more
nucleotides. The variants can be altered in coding or non-coding
regions or both. Alterations in the coding regions can produce
conservative or non-conservative amino acid substitutions,
deletions or additions. Also included are silent substitutions,
additions and deletions, which do not alter the properties and
activities of the ZNF206 polypeptide or portions thereof.
[0056] Further embodiments of the invention include isolated
polynucleotide molecules have, or comprise a sequence having, a
high degree of sequence identity with a native, or wild type,
ZNF206 polynucleotide, for example, at least 90%, 95%, 96%, 97%,
98% or 99% identical thereto.
[0057] A polynucleotide is considered to have a nucleotide sequence
at least, for example, 95% "identical" to a reference nucleotide
sequence if it is identical to the reference sequence except that
it includes up to five mutations (additions, deletions, or
substitutions) per each 100 nucleotides of the reference nucleotide
sequence. These mutations of the reference sequence can occur at
the 5' or 3' terminal positions of the reference nucleotide
sequence or anywhere between those terminal positions, interspersed
either individually among nucleotides in the reference sequence or
in one or more contiguous groups within the reference sequence.
Nucleotide sequence identity may be determined conventionally using
known computer programs such as the BESTFIT program (Wisconsin
Sequence Analysis Package, Version 8 for Unix, Genetics Computer
Group, University Research Park, 575 Science Drive, Madison, Wis.
53711. BESTFIT uses the local homology algorithm of Smith and
Waterman, Adv. Appl. Math. 2:482-489 (1981), to find the best
segment of homology between two sequences. When using BESTFIT or
any other sequence alignment program to determine whether a
particular sequence is, for instance, 95% identical to a reference
sequence according to the present invention, the parameters are
set, of course, such that the percentage of identity is calculated
over the full length of the reference nucleotide sequence and that
gaps in homology of up to 5% of the total number of nucleotides in
the reference sequence are allowed.
Recombinant Constructs; Vectors and Host Cells
[0058] The present invention also provides recombinant
polynucleotide constructs that comprise a ZNF206 polynucleotide,
including but not limited to vectors. The present invention also
provides host cells comprising such vectors and the production of
ZNF206 polypeptides or fragments thereof by recombinant or
synthetic techniques.
[0059] "Operably Linked". A first nucleic-acid sequence is
"operably linked" with a second nucleic-acid sequence when the
first nucleic-acid sequence is placed in a functional relationship
with the second nucleic-acid sequence. For instance, a promoter is
operably linked to a coding sequence if the promoter affects the
transcription or expression of the coding sequence. Generally,
operably linked DNA sequences are contiguous and, where necessary
to join two protein coding regions, in reading frame.
[0060] "Recombinant". A "recombinant" polynucleotide is made by an
artificial combination of two otherwise separated segments of
sequence, e.g., by chemical synthesis or by the manipulation of
isolated segments of polynucleotides by genetic engineering
techniques. Techniques for nucleic-acid manipulation are well-known
(see, e.g., Sambrook et al., 1989, and Ausubel et al., 1992).
Methods for chemical synthesis of polynucleotides are discussed,
for example, in Beaucage and Carruthers, Tetra. Letts.
22:1859-1862, 1981, and Matteucci et al., J. Am. Chem. Soc.
103:3185, 1981. Chemical synthesis of polynucleotides can be
performed, for example, on commercial automated oligonucleotide
synthesizers.
[0061] Recombinant vectors are produced by standard recombinant
techniques and may be introduced into host cells using well known
techniques such as infection, transduction, transfection,
transvection, electroporation and transformation. The vector may
be, for example, a phage, plasmid, viral or retroviral vector.
Retroviral vectors may be replication competent or replication
defective. In the latter case, viral propagation generally will
occur only in complementing host cells.
[0062] Expression vectors include sequences that permit expression
of a polypeptide encoded by a polynucleotide of interest in a
suitable host cell. Such expression may be constitutive or
non-constitutive, e.g., inducible by an environmental factor or a
chemical inducer that is specific to a particular cell or tissue
type, for example. Expression vectors include chromosomal-,
episomal- and virus-derived vectors, e.g., vectors derived from
bacterial plasmids, bacteriophage, yeast episomes, yeast
chromosomal elements, viruses such as baculoviruses, papova
viruses, vaccinia viruses, adenoviruses, fowl pox viruses,
pseudorabies viruses and retroviruses, and vectors derived from
combinations thereof, such as cosmids and phagemids.
[0063] In expression vectors, a polynucleotide insert is operably
linked to an appropriate promoter. The promoter may be a homologous
promoter, i.e., a promoter or functional portion thereof, that is
associated with the polynucleotide insert in nature, for example, a
ZNF206 promoter with a ZNF206 or ZNF206 protein coding region.
Alternatively, the promoter may be a heterologous promoter, i.e., a
promoter or functional portion thereof, that is not associated with
the polynucleotide insert in nature, for example, a bacterial
promoter used for high-level protein expression in bacterial cells
(or, for that matter, any promoter other than a ZNF206 promoter)
operably linked to a ZNF206 protein coding region. The expression
constructs will further contain sites for transcription initiation,
termination and, in the transcribed region, a ribosome binding site
for translation. The coding portion of the mature transcripts
expressed by the constructs will include a translation initiating
AUG at the beginning and a termination codon appropriately
positioned at the end of the polypeptide to be translated.
[0064] Vectors may include one or more selectable marker suitable
for selection of a host cell into which such a vector has been
introduced. Such markers include dihydrofolate reductase or
neomycin resistance for eukaryotic cell culture and tetracycline or
ampicillin resistance genes for culturing in E. coli and other
bacteria. Representative examples of appropriate hosts include
bacterial cells, such as E. coli, Streptomyces and Salmonella
typhimurium cells; fungal cells, such as yeast cells; insect cells
such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such
as CHO, COS and Bowes melanoma cells; and plant cells. Appropriate
culture media and conditions for the above-described host cells are
known in the art.
[0065] Bacterial promoters suitable include the E. coli lad and
lacZ promoters, the T3 and T7 promoters, the gpt promoter, the
lambda PR and PL promoters and the trp promoter. Eukaryotic
promoters include the CMV immediate early promoter, the HSV
thymidine kinase promoter, the early and late SV40 promoters, the
promoters of retroviral LTRs, such as those of the Rous sarcoma
virus (RSV), and metallothionein promoters, such as the mouse
metallothionein-I promoter.
[0066] For secretion of the translated protein into the lumen of
the endoplasmic reticulum, into the periplasmic space or into the
extracellular environment, appropriate secretion signals may be
incorporated into the expressed polypeptide. The signals may be
endogenous to the polypeptide or they may be heterologous
signals.
[0067] A polypeptide of interest may be expressed in a modified
form, such as a fusion protein, and may include not only secretion
signals but also additional heterologous functional regions. For
instance, a region of additional amino acids, particularly charged
amino acids, may be added to the N-terminus of the polypeptide to
improve stability and persistence in the host cell, during
purification or during subsequent handling and storage. Also,
peptide moieties may be added to the polypeptide to facilitate
purification. Such regions may be removed prior to final
preparation of the polypeptide. The addition of peptide moieties to
polypeptides to engender secretion or excretion, to improve
stability and to facilitate purification, among others, are
familiar and routine techniques in the art.
[0068] An expressed polypeptide of interest can be recovered and
purified from recombinant cell cultures by well-known methods
including ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography.
[0069] Polypeptides of the present invention include naturally
purified products, products of chemical synthetic procedures, and
products produced by recombinant techniques from a prokaryotic or
eukaryotic host, including, for example, bacterial, yeast, higher
plant, insect and mammalian cells. Depending upon the host employed
in a recombinant production procedure, the polypeptides of the
present invention may be glycosylated or may be non-glycosylated.
In addition, polypeptides of the invention may also include an
initial modified methionine residue, in some cases as a result of
host-mediated processes.
[0070] Polynucleotide constructs can also be used to reduce
expression of ZNF206 in a cell. For example, antisense constructs,
ribozymes, short interfering RNA (siRNA) or small hairpin RNA
(shRNA), and other such constructs can be used for this
purpose.
[0071] A "small interfering RNA" or "short interfering RNA" (siRNA)
or "short hairpin RNA" (shRNA) is a double-stranded RNA molecule
that is complementary to a target nucleic acid sequence, for
example, VEGF-C. A double-stranded RNA molecule is formed by the
complementary pairing between a first RNA portion and a second RNA
portion. The length of each portion generally is less than 30
nucleotides in length (e.g., 29, 28, 27, 26, 25, 24, 23, 22, 21,
20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides). In some
embodiments, the length of each portion is 19 to 25 nucleotides in
length. In some siRNA molecules, the complementary first and second
portions of the RNA molecule are the "stem" of a hairpin structure.
The two portions can be joined by a linking sequence, which can
form the "loop" in the hairpin structure. The linking sequence can
vary in length. In some embodiments, the linking sequence can be 5,
6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length. The first and
second portions are complementary but may not be completely
symmetrical, as the hairpin structure may contain 3' or 5' overhang
nucleotides (e.g., a 1, 2, 3, 4, or 5 nucleotide overhang).
[0072] RNA molecules have been shown by many researchers to be
effective in suppressing mRNA accumulation. siRNA-mediated
suppression of nucleic acid expression is specific as even a single
base pair mismatch between siRNA and the targeted nucleic acid can
abolish the action of RNA interference. siRNAs generally do not
elicit anti-viral responses.
[0073] There are well-established criteria for designing siRNAs
(see, e.g., Elbashire et al., Nature, 411:494 8, 2001; Amarzguioui
et al., Biochem. Biophys. Res. Commun., 316:1050 8, 2004; Reynolds
et al., Nat. Biotech., 22:326-30, 2004). Details can be found in
the websites of several commercial vendors such as Ambion,
Dharmacon, GenScript, and OligoEngine. The sequence of any
potential siRNA candidate generally is checked for any possible
matches to other nucleic acid sequences or polymorphisms of nucleic
acid sequence using the BLAST alignment program (see
ncbi.nlm.nih.gov on the World Wide Web). Typically, a number of
siRNAs have to be generated and screened in order to compare their
effectiveness.
[0074] Once designed, the siRNAs of the present invention can be
generated by any method known in the art, for example, by in vitro
transcription, recombinantly, or by synthetic means (e.g., having
either a TT or a UU overhang at the 3' end). siRNAs can be
generated in vitro by using a recombinant enzyme, such as T7 RNA
polymerase, and DNA oligonucleotide templates, or can be prepared
in vivo, for example, in cultured cells (see, for example, Elbashir
et al., supra; Brummelkamp et al., supra; and Lee et al., Nat.
Biotech., 20:500-505, 2002).
[0075] In addition, strategies have been described for producing a
hairpin siRNA from vectors containing a RNA polymerase III
promoter. Various vectors have been constructed for generating
hairpin siRNAs in host cells using either an H1-RNA or an snU6 RNA
promoter. A RNA molecule as described above (e.g., a first portion,
a linking sequence, and a second portion) can be operably linked to
such a promoter. When transcribed by RNA polymerase III, the first
and second portions form a duplexed stem of a hairpin and the
linking sequence forms a loop. The pSuper vector (OligoEngines
Ltd., Seattle, Wash.) also can be used to generate siRNA.
[0076] A TTTTT penta-nucleotide usually is attached to the end of
the second portion (i.e., the antisense strand) in a vector to
serve as a terminator for RNA polymerase III transcription. For
that reason, siRNA candidates that contain more than three
consecutive Ts should be avoided since four or more consecutive Ts
in the template nucleic acid triggers termination of RNA polymerase
III transcription.
[0077] Several techniques can be used to test the effect of
different siRNA constructs on cellular mRNA and/or protein levels.
For example, dual-GFP transfection, CHO-cell double transfection
based on an antibody/epitope specificity, quantitative RT-PCR,
Northern blots, Western blots, immunofluorescence, and Hygro/Neo
selection. These methods are well known in the art.
Polypeptides
[0078] As used herein, the phrase "a ZNF206 polypeptide" refers to
a polypeptide at least 10, 11, 12, 12, 14, 15, 20, 30, 40, 49, 50,
100 or more amino acid residues in length and have a high degree of
sequence identity with the full-length native, or wild-type, ZNF206
polypeptide or a fragment thereof. Included are variant forms of
ZNF206 polypeptides that include deletions, insertions or
substitutions of one or more amino acid residues in a native ZNF206
polypeptide sequence, including without limitation polypeptides
that exhibit activity similar, but not necessarily identical, to an
activity of the full-length native, or wild-type, ZNF206
polypeptide or fragment thereof as measured in a relevant
biological assay.
[0079] As used herein, the terms "wild-type" or "native" in
reference to a peptide or polypeptide are used interchangeably to
refer to a polypeptide that has 100% sequence identity with a
reference polypeptide that can be found in a cell or organism, or a
fragment thereof.
[0080] As used herein, the term "ZNF206 activity" refers to a
biological activity of a native ZNF206 polypeptide including, but
not limited to, repressing PE or PE-like differentiation,
regulation of pluripotency gene expression, DNA binding, etc.
[0081] As used herein, the terms "peptide" and "oligopeptide" are
considered synonymous and, as used herein, each term refers to a
chain of at least two amino acids coupled by peptidyl linkages. As
used herein, the terms "polypeptide" and "protein" are considered
synonymous and each term refers to a chain of more than about ten
amino acid residues. All oligopeptide and polypeptide formulas or
sequences herein are written from left to right and in the
direction from amino terminus to carboxy terminus.
[0082] As used herein, the term "isolated" polypeptide or protein
refers to a polypeptide or protein removed from its native
environment. For example, recombinantly produced polypeptides and
proteins expressed in host cells are considered isolated for
purposes of the invention as are native or recombinant polypeptides
and proteins which have been substantially purified by any suitable
technique.
[0083] As used herein, the term "binds selectively" is
interchangeable with the term "binds specifically, and, when used
in reference to a ZNF206 polypeptide, refers to binding of an
antibody, ligand, receptor, substrate, or other binding agent to
the target ZNF206 polypeptide to a substantially higher degree than
to other polypeptides. According to some embodiments, all or
substantially all binding of an antibody or other binding agent is
to the target ZNF206 polynucleotide, as can be determined given the
sensitivity of a particular procedure. An antibody, ligand,
receptor, substrate or other binding agent is said to be "selective
for" or specific for" a polypeptide or other target molecule, such
as ZNF206, if it binds selectively to the target molecule.
[0084] The amino acid sequence of a ZNF206 polypeptide or peptide
can be varied without significant effect on the structure or
function of the protein. In general, it is possible to replace
residues which contribute to the tertiary structure of the
polypeptide or peptide, provided that residues performing a similar
function are used. In other instances, the type of residue may be
completely unimportant if the alteration occurs at a non-critical
region of the protein.
[0085] Thus, the invention further includes variations of ZNF206
polypeptide or peptide that show substantial ZNF206 activity. Such
mutants include deletions, insertions, inversions, repeats, and
type substitutions (for example, substituting one hydrophilic
residue for another, but not strongly hydrophilic for strongly
hydrophobic as a rule). Small changes or such "neutral" amino acid
substitutions will generally have little effect on activity.
[0086] Typically seen as conservative substitutions are the
replacements, one for another, among the aliphatic amino acids Ala,
Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,
exchange of the acidic residues Asp and Glu, substitution between
the amide residues Asn and Gln, exchange of the basic residues Lys
and Arg and replacements among the aromatic residues Phe, Tyr.
[0087] Guidance concerning which amino acid changes are likely to
be phenotypically silent (i.e., are not likely to have a
significant deleterious effect on a function) can be found, for
example, in Bowie et al., Science 247:1306-1310, 1990.
[0088] Thus, a fragment, derivative or analog of a native, or
wild-type ZNF206 polypeptide, may be (i) one in which one or more
of the amino acid residues are substituted with a conserved or
non-conserved amino acid residue and such substituted amino acid
residue may or may not be one encoded by the genetic code, or (ii)
one in which one or more of the amino acid residues includes a
substituent group, or (iii) one in which the mature polypeptide is
fused with another compound, such as a compound to increase the
half-life of the polypeptide (for example, polyethylene glycol), or
(iv) one in which the additional amino acids are fused to the
mature polypeptide, such as an IgG Fc fusion region peptide or
leader or secretory sequence or a sequence that is employed for
purification of the mature polypeptide or a proprotein
sequence.
[0089] Charged amino acids may be substituted with another charged
amino acid. Charged amino acids may also be substituted with
neutral or negatively charged amino acids, resulting in proteins
with reduced positive charge. The prevention of aggregation is
highly desirable to avoid a loss of activity and increased
immunogenicity (Pinckard et al., Clin Exp. Immunol. 2:331-340,
1967; Robbins et al., Diabetes 36:838-845, 1987; Cleland et al.,
Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377, 1993).
[0090] The replacement of amino acids can also change the
selectivity of protein binding to cell surface receptors. Ostade et
al., Nature 361:266-268 (1993) describes certain mutations
resulting in selective binding of TNF-.alpha. to only one of the
two known types of TNF receptors.
[0091] It is well known in the art that one or more amino acids in
a native sequence can be substituted with other amino acid(s), the
charge and polarity of which are similar to that of the native
amino acid, i.e., a conservative amino acid substitution, resulting
in a silent change. Conservative substitutes for an amino acid
within the native polypeptide sequence can be selected from other
members of the class to which the amino acid belongs. Amino acids
can be divided into the following four groups: (1) acidic amino
acids, (2) basic amino acids, (3) neutral polar amino acids, and
(4) neutral, nonpolar amino acids. Representative amino acids
within these various groups include, but are not limited to, (1)
acidic (negatively charged) amino acids such as aspartic acid and
glutamic acid; (2) basic (positively charged) amino acids such as
arginine, histidine, and lysine; (3) neutral polar amino acids such
as glycine, serine, threonine, cysteine, cystine, tyrosine,
asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic)
amino acids such as alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan, and methionine. Conservative amino acid
substitution within the native polypeptide sequence can be made by
replacing one amino acid from within one of these groups with
another amino acid from within the same group. In one aspect,
biologically functional equivalents of the proteins or fragments
thereof of the present invention can have ten or fewer, seven or
fewer, five or fewer, four or fewer, three or fewer, two, or one
conservative amino acid changes. The encoding nucleotide sequence
will thus have corresponding base substitutions, permitting it to
encode biologically functional equivalent forms of the proteins or
fragments of the present invention.
[0092] It is understood that certain amino acids may be substituted
for other amino acids in a protein structure without appreciable
loss of interactive binding capacity with structures such as, for
example, antigen-binding regions of antibodies or binding sites on
substrate molecules. Because it is the interactive capacity and
nature of a protein that defines that protein's biological
functional activity, certain amino acid sequence substitutions can
be made in a protein sequence and, of course, its underlying DNA
coding sequence and, nevertheless, a protein with like properties
can still be obtained. It is thus contemplated by the inventors
that various changes may be made in the peptide sequences of the
proteins or fragments of the present invention, or corresponding
DNA sequences that encode said peptides, without appreciable loss
of their biological utility or activity. It is understood that
codons capable of coding for such amino acid changes are known in
the art.
[0093] In making such changes, the hydropathic index of amino acids
may be considered. The importance of the hydropathic amino acid
index in conferring interactive biological function on a protein is
generally understood in the art (Kyte and Doolittle, J. Mol. Biol.
157:105-132, 1982). It is accepted that the relative hydropathic
character of the amino acid contributes to the secondary structure
of the resultant protein, which in turn defines the interaction of
the protein with other molecules, for example, enzymes, substrates,
receptors, DNA, antibodies, antigens, and the like. Each amino acid
has been assigned a hydropathic index on the basis of its
hydrophobicity and charge characteristics (Kyte and Doolittle, J.
Mol. Biol. 157:105-132, 1982); these are: isoleucine (+4.5), valine
(+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine/cystine
(+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4),
threonine (-0.7), serine (-0.8), tryptophan (-0.9), tyrosine
(-1.3), proline (-1.6), histidine (-3.2), glutamate (-3.5),
glutamine (-3.5), aspartate (-3.5), asparagine (-3.5), lysine
(-3.9), and arginine (4.5). In making such changes, the
substitution of amino acids whose hydropathic indices may be within
.+-.2, or .+-.1, or within .+-.0.5.
[0094] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. U.S. Pat. No. 4,554,101 states that the greatest
local average hydrophilicity of a protein, as govern by the
hydrophilicity of its adjacent amino acids, correlates with a
biological property of the protein.
[0095] As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0), lysine (+3.0), aspartate (+3.0.+-0.1), glutamate
(+3.0.+-0.1), serine (+0.3), asparagine (+0.2), glutamine (+0.2),
glycine (0), threonine (-0.4), proline (-0.5.+-0.1), alanine
(-0.5), histidine (-0.5), cysteine (-1.0), methionine (-1.3),
valine (-1.5), leucine (-1.8), isoleucine (-1.8), tyrosine (-2.3),
phenylalanine (-2.5), and tryptophan (-3.4). In making changes to a
native polypeptide or peptide sequence, the substitution of amino
acids whose hydrophilicity values may be within .+-.2, or within
.+-.1, or within .+-.0.5.
[0096] Of course, the number of amino acid substitutions a skilled
artisan would make depends on many factors, including those
described above. Generally speaking, the number of substitutions
for any given ZNF206 polypeptide will not be more than 50, 40, 30,
20, 10, 5, 3, or 2.
[0097] Amino acids in the ZNF206 protein of the present invention
that are essential for function can be identified by methods known
in the art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081-1085, 1989).
The latter procedure introduces single alanine mutations at every
residue in the molecule. The resulting mutant molecules are then
tested for biological activity such as in vitro or in vivo ligand
or receptor binding or other characteristic biological activities.
Sites that are critical for ligand-receptor binding can also be
determined by structural analysis such as crystallization, nuclear
magnetic resonance or photoaffinity labeling (Smith et al., J. Mol.
Biol. 224:899-904, 1992; de Vos et al. Science 255:306-312,
1992).
[0098] The polypeptides and peptides of the present invention
include native, or wild-type polypeptides and peptides, and
polypeptides or peptide variants that are at least 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98% or 99% identical to (or have such a degree
of identity with) the native ZNF206 polypeptide and fragments
thereof.
[0099] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a reference amino acid sequence is
intended that the amino acid sequence of the polypeptide is
identical to the reference sequence except that the polypeptide
sequence may include up to five amino acid alterations per each 100
amino acids of the reference amino acid sequence of the reference
polypeptide. In other words, to obtain a polypeptide having an
amino acid sequence at least 95% identical to a reference amino
acid sequence, up to 5% of the amino acid residues in the reference
sequence may be deleted or substituted with another amino acid, or
a number of amino acids up to 5% of the total amino acid residues
in the reference sequence may be inserted into the reference
sequence. These alterations of the reference sequence may occur at
the amino- or carboxy-terminal positions of the reference amino
acid sequence or anywhere between those terminal positions,
interspersed either individually among residues in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0100] As a practical matter, whether any particular polypeptide
has a particular degree of amino acid sequence identity when
compared to a reference polypeptide can be determined
conventionally using known computer programs such the Bestfit
program (Wisconsin Sequence Analysis Package, Version 8 for Unix,
Genetics Computer Group, University Research Park, 575 Science
Drive, Madison, Wis. 53711. When using Bestfit or any other
sequence alignment program to determine whether a particular
sequence is, for instance, 95% identical to a reference sequence
according to the present invention, the parameters are set, of
course, such that the percentage of identity is calculated over the
full length of the reference amino acid sequence and that gaps in
homology of up to 5% of the total number of amino acid residues in
the reference sequence are allowed.
[0101] Fragments of the polypeptides described herein may, for
example, comprise: the full-length amino acid sequence of ZNF206; a
less than full-length amino acid sequence that retains ZNF206
activity; a sequence that comprises one or more antigenic
determinants of ZNF206, for example, those that elicit antibodies
that bind selectively to ZNF206; etc. Also included are fragments
that include both sequences that are unique to ZNF206 and sequences
from another protein. The polypeptide fragments of the present
invention can be used for numerous purposes, for example, to elicit
antibody production in a mammal, as molecular weight markers on
SDS-PAGE gels or on molecular sieve gel filtration columns using
methods well known to those of skill in the art, etc.
[0102] Polypeptides of the present invention can be used to raise,
or elicit, polyclonal and monoclonal antibodies that bind
selectively to a native ZNF206 polypeptide, which are useful in
diagnostic assays for detecting ZNF206 expression or for other
purposes. Further, such polypeptides can be used in the yeast
two-hybrid system to "capture" binding proteins (Fields and Song,
Nature 340:245-246, 1989). For eliciting ZNF206-specific antibody
production, the fragment may comprise, for example, a polypeptide
of at least 11 amino acids, including at least 4, 5, 6, 7, 8, 9,
10, 11, or more contiguous amino acids of a native ZNF206
polypeptide. Of course, longer fragments with complete sequence
homology with the ZNF206 polypeptide, including fragments
constituting the full-length ZNF206 polypeptide, may be used for
eliciting antibody production. Alternatively, for eliciting
ZNF206-specific antibody production, a longer polypeptide may be
employed that has at least 70%, or 80%, or 85%, or 90%, or 95%, or
100% amino acid sequence identity to a native ZNF206 polypeptide.
Such a longer polypeptide may be at least 15, or 20, or 30, or 40
or more amino acids in length.
[0103] In another aspect, the invention provides a peptide or
polypeptide comprising an epitope-bearing portion of a polypeptide
of the invention. The epitope of this polypeptide portion is an
immunogenic or antigenic epitope of a polypeptide of the invention.
An "immunogenic epitope" is defined as a part of a protein that
elicits an antibody response when the whole protein is the
immunogen. These immunogenic epitopes are believed to be confined
to a few loci on the molecule. On the other hand, a region of a
protein molecule to which an antibody can bind is defined as an
"antigenic epitope." The number of immunogenic epitopes of a
protein generally is less than the number of antigenic epitopes.
See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA
81:3998-4002, 1984).
[0104] As to the selection of peptides or polypeptides bearing an
antigenic epitope (i.e., that contain a region of a protein
molecule to which an antibody can bind), it is well known in that
art that relatively short synthetic peptides that mimic part of a
protein sequence are routinely capable of eliciting an antiserum
that reacts with the partially mimicked protein. See, for instance,
Sutcliffe et al., Science 219:660-666, 1983). Peptides capable of
eliciting protein-reactive sera are frequently represented in the
primary sequence of a protein, can be characterized by a set of
simple chemical rules, and are confined neither to immunodominant
regions of intact proteins (i.e., immunogenic epitopes) nor to the
amino or carboxyl terminals. Peptides that are extremely
hydrophobic and those of six or fewer residues generally are
ineffective at inducing antibodies that bind to the mimicked
protein; longer, soluble peptides, especially those containing
proline residues, usually are effective (Sutcliffe et al., supra,
at 661).
[0105] Antigenic epitope-bearing peptides and polypeptides of the
invention are useful for eliciting the production of antibodies,
including monoclonal antibodies, which bind selectively to a
polypeptide of the invention. A high proportion of hybridomas
obtained by fusion of spleen cells from donors immunized with an
antigen epitope-bearing peptide generally secrete antibody reactive
with the native protein (Sutcliffe et al., supra, at 663). The
antibodies raised by antigenic epitope-bearing peptides or
polypeptides are useful to detect the mimicked protein, and
antibodies to different peptides may be used for tracking the fate
of various regions of a protein precursor which undergoes
post-translational processing. The peptides and anti-peptide
antibodies may be used in a variety of qualitative or quantitative
assays for the mimicked protein, for instance in competition assays
since it has been shown that even short peptides (e.g., about 9
amino acids) can bind and displace the larger peptides in
immunoprecipitation assays. See, for example, Wilson et al., Cell
37:767-778, 1984). The anti-peptide antibodies of the invention
also are useful for protein purification, e.g., by adsorption
chromatography using known methods.
[0106] Antigenic epitope-bearing peptides and polypeptides of the
invention designed according to the above guidelines may contain a
sequence of at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or 30 or
more amino acids contained within the amino acid sequence of a
polypeptide of the invention. However, peptides or polypeptides
comprising a larger portion of an amino acid sequence of a
polypeptide of the invention, containing about 30 to about 50 amino
acids, or any length up to and including the entire amino acid
sequence of a polypeptide of the invention, also are considered
epitope-bearing peptides or polypeptides of the invention and also
are useful for inducing antibodies that react with the mimicked
protein.
[0107] The amino acid sequence of the epitope-bearing peptide may
be selected to provide substantial solubility in aqueous solvents
(i.e., sequences including relatively hydrophilic residues and
highly hydrophobic sequences may be avoided).
[0108] The epitope-bearing peptides and polypeptides of the
invention may be produced by any conventional means for making
peptides or polypeptides including recombinant means using nucleic
acid molecules of the invention. For instance, a short
epitope-bearing amino acid sequence may be fused to a larger
polypeptide which acts as a carrier during recombinant production
and purification, as well as during immunization to produce
anti-peptide antibodies. Epitope-bearing peptides also may be
synthesized using known methods of chemical synthesis. For
instance, Houghten has described a simple method for synthesis of
large numbers of peptides, such as 10-20 mg of 248 different 13
residue peptides representing single amino acid variants of a
segment of the HA1 polypeptide which were prepared and
characterized (by binding studies employing an enzyme-linked
immunosorbent assay [ELISA]) in less than four weeks (Houghten,
Proc. Natl. Acad. Sci. USA 82:5131-5135, 1985; and U.S. Pat. No.
4,631,211). In this procedure the individual resins for the
solid-phase synthesis of various peptides are contained in separate
solvent-permeable packets, enabling the optimal use of the many
identical repetitive steps involved in solid-phase methods. A
completely manual procedure allows 500-1000 or more syntheses to be
conducted simultaneously.
[0109] Epitope-bearing peptides and polypeptides of the invention
are used to induce antibodies according to methods well known in
the art. See, for instance, Sutcliffe et al., supra; Wilson et al.,
supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and
Bittle et al., J. Gen. Virol. 66:2347-2354, 1985). Generally,
animals may be immunized with free peptide; however, anti-peptide
antibody titer may be boosted by coupling of the peptide to a
macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or
tetanus toxoid. For instance, peptides containing cysteine may be
coupled to carrier using a linker such as
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other
peptides may be coupled to carrier using a more general linking
agent such as glutaraldehyde. Animals such as rabbits, rats and
mice are immunized with either free or carrier-coupled peptides,
for instance, by intraperitoneal and/or intradermal injection of
emulsions containing about 100 .mu.g peptide or carrier protein and
Freund's adjuvant. Several booster injections may be needed, for
instance, at intervals of about two weeks, to provide a useful
titer of anti-peptide antibody which can be detected, for example,
by ELISA assay using free peptide adsorbed to a solid surface. The
titer of anti-peptide antibodies in serum from an immunized animal
may be increased by selection of anti-peptide antibodies, for
instance, by adsorption to the peptide on a solid support and
elution of the selected antibodies according to methods well known
in the art.
[0110] Immunogenic epitope-bearing peptides of the invention, i.e.,
those parts of a protein that elicit an antibody response when the
whole protein is the immunogen, are identified according to methods
known in the art. For instance, Geysen et al. (1984), supra,
discloses a procedure for rapid concurrent synthesis on solid
supports of hundreds of peptides of sufficient purity to react in
an enzyme-linked immunosorbent assay. Interaction of synthesized
peptides with antibodies is then easily detected without removing
them from the support. In this manner a peptide bearing an
immunogenic epitope of a desired protein may be identified
routinely by one of ordinary skill in the art. For instance, the
immunologically important epitope in the coat protein of
foot-and-mouth disease virus was located by Geysen et al. with a
resolution of seven amino acids by synthesis of an overlapping set
of all 208 possible hexapeptides covering the entire 213 amino acid
sequence of the protein. Then, a complete replacement set of
peptides in which all 20 amino acids were substituted in turn at
every position within the epitope were synthesized, and the
particular amino acids conferring specificity for the reaction with
antibody were determined. Thus, peptide analogs of the
epitope-bearing peptides of the invention can be made routinely by
this method. U.S. Pat. No. 4,708,781 to Geysen (1987) further
describes this method of identifying a peptide bearing an
immunogenic epitope of a desired protein.
[0111] U.S. Pat. No. 5,194,392 to Geysen (1990) describes a general
method of detecting or determining the sequence of monomers (amino
acids or other compounds) which is a topological equivalent of the
epitope (i.e., a "mimotope") which is complementary to a particular
paratope (antigen binding site) of an antibody of interest. More
generally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a
method of detecting or determining a sequence of monomers which is
a topographical equivalent of a ligand which is complementary to
the ligand binding site of a particular receptor of interest.
Similarly, U.S. Pat. No. 5,480,971 discloses linear C.sub.1-7-alkyl
peralkylated oligopeptides and sets and libraries of such peptides,
as well as methods for using such oligopeptide sets and libraries
for determining the sequence of a peralkylated oligopeptide that
preferentially binds to an acceptor molecule of interest. Thus,
non-peptide analogs of the epitope-bearing peptides of the
invention also can be made routinely by these methods.
[0112] Polypeptides of the present invention and the
epitope-bearing fragments thereof described above can be combined
with parts of the constant domain of immunoglobulins (IgG),
resulting in chimeric polypeptides. These fusion proteins
facilitate purification and show an increased half-life in vivo.
This has been shown, e.g., for chimeric proteins consisting of the
first two domains of the human CD4-polypeptide and various domains
of the constant regions of the heavy or light chains of mammalian
immunoglobulins (EPA 394,827; Traunecker et al., Nature 331:84-86,
1988). Fusion proteins that have a disulfide-linked dimeric
structure due to the IgG part can also be more efficient in binding
and neutralizing other molecules than the monomeric ZNF206 protein
or protein fragment alone (Fountoulakis et al., J. Biochem.
270:3958-3964, 1995).
Antibodies
[0113] ZNF206-selective antibodies for use in the present invention
can be raised against the intact ZNF206 or an antigenic polypeptide
fragment thereof, which may presented together with a carrier
protein, such as an albumin, to an animal system (such as rabbit or
mouse) or, if it is long enough (at least about 25 amino acids),
without a carrier.
[0114] As used herein, the term "antibody" (Ab) or "monoclonal
antibody" (Mab) is meant to include intact molecules as well as
antibody fragments (or "fragment antibodies") (such as, for
example, Fab and F(ab').sub.2 fragments) which are capable of
selectively binding to ZNF206. Fab and F(ab').sub.2 fragments lack
the Fc portion of intact antibody, clear more rapidly from the
circulation, and may have less non-specific tissue binding of an
intact antibody (Wahl et al., J. Nucl. Med. 24:316-325, 1983). Also
included are single-chain antibodies.
[0115] The antibodies of the present invention may be prepared by
any of a variety of methods. For example, cells expressing the
ZNF206 or an antigenic fragment thereof can be administered to an
animal in order to induce the production of sera containing
polyclonal antibodies. In one method, a preparation of ZNF206
protein is prepared and purified as described above to render it
substantially free of natural contaminants. Such a preparation is
then introduced into an animal in order to produce polyclonal
antisera of greater specific activity.
[0116] The antibodies of the present invention include monoclonal
antibodies (or ZNF206 binding fragments thereof). Such monoclonal
antibodies can be prepared using hybridoma technology (Colligan,
Current Protocols in Immunology, Wiley Interscience, New York
(1990-1996); Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1988), Chapters 6-9,
Current Protocols in Molecular Biology, Ausubel, infra, Chapter
11). In general, such procedures involve immunizing an animal (for
example, a mouse or rabbit) with a ZNF206 antigen or with a
ZNF206-expressing cell. Suitable cells can be recognized by their
capacity to bind anti-ZNF206 antibody. Such cells may be cultured
in any suitable tissue culture medium, such as Earle's modified
Eagle's medium supplemented with 10% fetal bovine serum
(inactivated at about 56.degree. C.), and supplemented with about
10 .mu.g/l of nonessential amino acids, about 1,000 U/ml of
penicillin, and about 100 .mu.g/ml of streptomycin. The splenocytes
of such mice are extracted and fused with a suitable myeloma cell
line. Any suitable myeloma cell line may be employed in accordance
with the present invention. After fusion, the resulting hybridoma
cells are selectively maintained in HAT medium, and then cloned by
limiting dilution as described by Wands et al., Gastroenterology
80:225-232, 1981); Harlow and Lane, infra, Chapter 7. The hybridoma
cells obtained through such a selection are then assayed to
identify clones which secrete antibodies capable of binding the
ZNF206 antigen.
[0117] Alternatively, additional antibodies capable of binding to
the ZNF206 antigen may be produced in a two-step procedure through
the use of anti-idiotypic antibodies. Such a method makes use of
the fact that antibodies are themselves antigens, and therefore it
is possible to obtain an antibody which binds to a second antibody.
In accordance with this method, ZNF206-selective antibodies are
used to immunize an animal, such as a mouse. The splenocytes of
such an animal are then used to produce hybridoma cells, and the
hybridoma cells are screened to identify clones which produce an
antibody whose ability to bind to the ZNF206-selective antibody can
be blocked by the ZNF206 antigen. Such antibodies comprise
anti-idiotypic antibodies to the ZNF206-selective antibody and can
be used to immunize an animal to induce formation of further
ZNF206-selective antibodies.
[0118] It will be appreciated that Fab and F(ab').sub.2 and other
fragments of the antibodies of the present invention may be used
according to the methods disclosed herein. Such fragments are
typically produced by proteolytic cleavage, using enzymes such as
papain (to produce Fab fragments) or pepsin (to produce
F(ab').sub.2 fragments). Alternatively, ZNF206-binding fragments
can be produced through recombinant DNA technology or protein
synthesis.
Diagnostic Methods
[0119] The present invention provides methods for detecting the
presence of ZNF206 polynucleotides (for example, ZNF206 mRNA) or
polypeptides in a sample, such as a biological sample from an
individual; for quantitating ZNF206 polynucleotides or polypeptides
in a sample; for determining a ZNF206/ZNF206 polynucleotide or
polypeptide ratio in a sample, etc.
[0120] In the methods of the present invention, a measurement of
ZNF206 polypeptide or polynucleotide or a ZNF206/ZNF206 ratio is
compared to a "reference." Depending on the embodiment of the
invention, such a reference can include a measurement in a control
sample; a standard value obtained by measurements of a population
of individuals; a baseline value determined for the same individual
at an earlier timepoint, e.g., before commencing a course of
treatment; or any other suitable reference used for similar
methods.
[0121] As used herein, the term "individual" or "patient" refers to
a mammal, including, but not limited to, a mouse, rat, rabbit, cat,
dog, monkey, ape, human, or other mammal.
[0122] By "biological sample" is intended any biological sample
obtained from an individual, including but not limited to, a body
fluid, cell, tissue, tissue culture, or other source that contains
ZNF206 protein or mRNA. Methods for obtaining such biological
samples from mammals are well known in the art.
[0123] Detection of mRNA. Total cellular RNA can be isolated from a
biological sample using any suitable technique such as the
single-step guanidinium-thiocyanate-phenol-chloroform method
described in Chomczynski and Sacchi, Anal. Biochem. 162:156-159
(1987). Levels of mRNA encoding ZNF206 are then assayed using any
appropriate method. These include Northern blot analysis, S1
nuclease mapping, the polymerase chain reaction (PCR), reverse
transcription in combination with the polymerase chain reaction
(RT-PCR), and reverse transcription in combination with the ligase
chain reaction (RT-LCR).
[0124] Northern blot analysis can be performed as described in
Harada et al., Cell 63:303-312, 1990). Briefly, total RNA is
prepared from a biological sample as described above. For the
Northern blot, the RNA is denatured in an appropriate buffer (such
as glyoxal/dimethyl sulfoxide/sodium phosphate buffer), subjected
to agarose gel electrophoresis, and transferred onto a
nitrocellulose filter. After the RNAs have been linked to the
filter by a UV linker, the filter is prehybridized in a solution
containing formamide, SSC, Denhardt's solution, denatured salmon
sperm, SDS, and sodium phosphate buffer. ZNF206 cDNA labeled
according to any appropriate method (such as a .sup.32P-multiprimed
DNA labeling system is used as probe. After hybridization
overnight, the filter is washed and exposed to x-ray film. cDNA for
use as probe according to the present invention is described in the
sections above.
[0125] S1 mapping can be performed as described in Fujita et al.,
Cell 49:357-367, 1987). To prepare probe DNA for use in S1 mapping,
the sense strand of above-described cDNA is used as a template to
synthesize labeled antisense DNA. The antisense DNA can then be
digested using an appropriate restriction endonuclease to generate
further DNA probes of a desired length. Such antisense probes are
useful for visualizing protected bands corresponding to the target
mRNA (i.e., mRNA encoding ZNF206). Northern blot analysis can be
performed as described above.
[0126] According to one embodiment, levels of mRNA encoding ZNF206
are assayed using a polynucleotide amplification method, including
but not limited to a polymerase chain reaction (PCR). One PCR
method that is useful in the practice of the present invention is
the RT-PCR method described in Makino et al., Technique 2:295-301,
1990), for example. By this method, the radioactivity of the DNA
products of the amplification, i.e., the "amplification products"
or "amplicons," in the polyacrylamide gel bands is linearly related
to the initial concentration of the target mRNA. Briefly, this
method involves adding total RNA isolated from a biological sample
in a reaction mixture containing a RT primer and appropriate
buffer. After incubating for primer annealing, the mixture can be
supplemented with a RT buffer, dNTPs, DTT, RNase inhibitor and
reverse transcriptase. After incubation to achieve reverse
transcription of the RNA, the RT products are then subject to PCR
using labeled primers. Alternatively, rather than labeling the
primers, a labeled dNTP can be included in the PCR reaction
mixture. PCR amplification can be performed in a DNA thermal cycler
according to conventional techniques. After a suitable number of
rounds to achieve amplification, the PCR reaction mixture is
electrophoresed on a polyacrylamide gel. After drying the gel, the
radioactivity of the appropriate bands (corresponding to the mRNA
encoding ZNF206 is quantified using an imaging analyzer. RT and PCR
reaction ingredients and conditions, reagent and gel
concentrations, and labeling methods are well known in the art.
[0127] According to one embodiment of an amplification method of
the invention, primers are employed that selectively amplify a
ZNF206 polynucleotide in a sample, for example, a primer pair
including at least one primer that selectively hybridizes to ZNF206
mRNA (e.g., that includes sequences from the region of the ZNF206
mRNA that encodes the ZNF206 polypeptide. The second primer can
include any sequence from the target ZNF206 polynucleotide, whether
such a sequence is unique to ZNF206 or is shared by ZNF206 and
another polynucleotide. This embodiment is useful for amplifying
only a ZNF206 transcript (mRNA) in a sample, for example.
[0128] According to another embodiment of the invention, primers
are employed that selectively amplify a ZNF206 polynucleotide, for
example, a primer pair that includes at least one primer that
selectively hybridizes to ZNF206 mRNA. The second primer can
include any sequence from the target ZNF206 polynucleotide, whether
such a sequence is unique to ZNF206 or is shared by ZNF206 and
another polynucleotide. This embodiment is useful for amplifying
only a ZNF206 transcript (mRNA) in a sample, for example.
[0129] According to another embodiment of the invention, primers
are employed that amplify both a ZNF206 polynucleotide and a second
reference polynucleotide. For example, two primer pairs (e.g., four
primers) can be used, one pair that selectively amplifies ZNF206
and a second pair that selectively amplifies the reference
polynucleotide, so as to produce amplification products that can be
distinguished from one another, for example by length. This
embodiment is useful, for example, for determining the ratio of
ZNF206 mRNA to a reference mRNA in a sample.
[0130] The skilled artisan will be able to produce additional
primers, primer pairs, and sets of primers for PCR and other
amplification methods based on the sequences taught herein.
[0131] One embodiment of the present invention is a kit that
includes primers useful for amplification methods according to the
present invention. Such kits also include suitable packaging,
instructions for use, or both.
[0132] Another PCR method useful for detecting the presence of
and/or quantitating ZNF206 mRNA and protein in a biological sample
is through the use of "bio-barcode" nanoparticles. For detection
and/or quantitation of proteins, for example, two types of capture
particles are employed: one is a micro-size magnetic particle
bearing an antibody selective for a target protein, and the other
is a nanoparticle with attached antibodies selective for the same
protein. The nanoparticle also carries a large number (e.g.,
.about.100) of unique, covalently attached oligonucleotides that
are bound by hybridization to complementary oligonucleotides. The
latter are the "bio-barcodes" that serve as markers for a selected
protein. Because the nanoparticle probe carries many
oligonucleotides per bound protein, there is substantial
amplification, relative to protein. There is a second amplification
of signal in a silver enhancement step. The result is 5-6 orders of
magnitude greater sensitivity for proteins than ELISA-based assays,
by detecting tens to hundreds of molecules. See, e.g., U.S. Pat.
No. 6,974,669. See also, e.g., Stoeva et al., J. Am. Chem. Soc.
128:8378-8379, 2006, for an example of detection of protein cancer
markers with bio-barcoded nanoparticle probes. The bio-barcode
method can also be used for detecting and/or quantitating mRNA and
other polynucleotides in a sample (Huber et al., Nucl. Acids Res.
32:e137, 2004; Cheng et al., Curr. Opin. Chem. Biol. 10:11-19,
2006; Thaxton et al., Clin. Chim. Acta 363:120-126, 2006; U.S. Pat.
No. 6,974,669).
[0133] Detection of polypeptide. Assaying the presence of, or
quantitating, ZNF206 polypeptide in a biological sample can occur
using any method known in the art.
[0134] Antibody-based techniques are useful for detecting the
presence of and/or quantitating ZNF206 levels in a biological
sample. For example, ZNF206 expression in tissues can be studied
with classical immunohistological methods. In these, the specific
recognition is provided by the primary antibody (polyclonal or
monoclonal) but the secondary detection system can utilize
fluorescent, enzyme, or other conjugated secondary antibodies. As a
result, an immunohistological staining of tissue section for
pathological examination is obtained. Tissues can also be
extracted, e.g., with urea and neutral detergent, for the
liberation of ZNF206 for Western-blot or dot/slot assay (Jalkanen
et al., J. Cell. Biol. 101:976-985, 1985; Jalkanen et al., J. Cell.
Biol. 105:3087-3096, 1987). In this technique, which is based on
the use of cationic solid phases, quantitation of ZNF206 can be
accomplished using isolated ZNF206 as a standard. This technique
can also be applied to body fluids. With these samples, a molar
concentration of ZNF206 will aid to set standard values of ZNF206
content for different tissues, fecal matter, body fluids (serum,
plasma, urine, synovial fluid, spinal fluid), etc. The normal
appearance of ZNF206 amounts can then be set using values from
healthy individuals, which can be compared to those obtained from a
test subject.
[0135] Other antibody-based methods useful for detecting ZNF206
levels include immunoassays, such as the enzyme linked
immunosorbent assay (ELISA), the radioimmunoassay (RIA), and the
"bio-barcode" assays described above. For example, ZNF206-selective
monoclonal antibodies can be used both as an immunoadsorbent and as
an enzyme-labeled probe to detect and quantify the ZNF206. The
amount of ZNF206 present in the sample can be calculated by
reference to the amount present in a standard preparation using a
linear regression computer algorithm. Such an ELISA for detecting a
tumor antigen is described in Iacobelli et al., Breast Cancer
Research and Treatment 11:19-30, 1988. In another ELISA assay, two
distinct selective monoclonal antibodies can be used to detect
ZNF206 in a sample. In this assay, one of the antibodies is used as
the immunoadsorbent and the other as the enzyme-labeled probe.
[0136] The above techniques may be conducted essentially as a
"one-step" or "two-step" assay. The "one-step" assay involves
contacting ZNF206 with immobilized antibody and, without washing,
contacting the mixture with the labeled antibody. The "two-step"
assay involves washing before contacting the mixture with the
labeled antibody. Other conventional methods may also be employed
as suitable. It is usually desirable to immobilize one component of
the assay system on a support, thereby allowing other components of
the system to be brought into contact with the component and
readily removed from the sample.
[0137] Suitable enzyme labels include, for example, those from the
oxidase group, which catalyze the production of hydrogen peroxide
by reacting with substrate. Glucose oxidase, for example, has good
stability and its substrate (glucose) is readily available.
Activity of an oxidase label may be assayed by measuring the
concentration of hydrogen peroxide formed by the enzyme-labeled
antibody/substrate reaction. Besides enzymes, other suitable labels
include radioisotopes, such as iodine (.sup.125I, .sup.121I) carbon
(.sup.14C), sulfur (.sup.35S), tritium (.sup.3H), indium
(.sup.112In), and technetium (.sup.99Tc), and fluorescent labels,
such as fluorescein and rhodamine, and biotin.
[0138] In addition to assaying ZNF206 levels in a biological sample
obtained from an individual, ZNF206 can also be detected in vivo by
imaging. Antibody labels or markers for in vivo imaging of ZNF206
include those detectable by X-radiography, NMR or ESR. For
X-radiography, suitable labels include radioisotopes such as barium
or cesium, which emit detectable radiation but are not overtly
harmful to the subject. Suitable markers for NMR and ESR include
those with a detectable characteristic spin, such as deuterium,
which may be incorporated into the antibody by labeling of
nutrients for the relevant hybridoma.
[0139] A ZNF206-selective antibody or antibody fragment which has
been labeled with an appropriate detectable imaging moiety, such as
a radioisotope (for example, .sup.131I, .sup.112In, .sup.99mTc), a
radio-opaque substance, or a material detectable by nuclear
magnetic resonance, is introduced (for example, parenterally,
subcutaneously or intraperitoneally) into the mammal to be examined
for a disorder. It will be understood in the art that the size of
the subject and the imaging system used will determine the quantity
of imaging moieties needed to produce diagnostic images. In the
case of a radioisotope moiety, for a human subject, the quantity of
radioactivity injected will normally range from about 5 to 20
millicuries of .sup.99 mTc. The labeled antibody or antibody
fragment will then preferentially accumulate at the location of
cells which contain ZNF206. In vivo tumor imaging is described in
Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies
and Their Fragments" (Chapter 13 in Tumor Imaging: The
Radiochemical Detection of Cancer, Burchiel and Rhodes, eds.,
Masson Publishing Inc., 1982).
[0140] Where in vivo imaging is used to detect enhanced levels of
ZNF206 for diagnosis in humans, one may use "humanized" chimeric
monoclonal antibodies. Such antibodies can be produced using
genetic constructs derived from hybridoma cells producing the
monoclonal antibodies described above. Methods for producing
chimeric antibodies, including humanized chimeric antibodies, are
known in the art. See, for review, Morrison, Science 229:1202,
1985; Oi et al., BioTeclmiques 4:214, 1986; Cabilly et al., U.S.
Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al.,
EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO
8702671; Boulianne et al., Nature 312:643, 1984; Neuberger et al.,
Nature 314:268, 1985.
[0141] Further suitable labels for the ZNF206-selective antibodies
of the present invention are provided below. Examples of suitable
enzyme labels include malate dehydrogenase, staphylococcal
nuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase,
alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase,
peroxidase, alkaline phosphatase, asparaginase, glucose oxidase,
beta-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholine
esterase.
[0142] Examples of suitable radioisotopic labels include .sup.3H,
.sup.111In, .sup.125I, .sup.131I, .sup.32P, .sup.35S, .sup.14C,
.sup.51Cr, .sup.57To, .sup.58Co, .sup.59Fe, .sup.75Se, .sup.152Eu,
.sup.90Y, .sup.67Cu, .sup.217Ci, .sup.211At, .sup.212Pb, .sup.47Sc,
.sup.09Pd, etc. .sup.111In has advantages where in vivo imaging is
used since its avoids the problem of dehalogenation of the
.sup.125I- or .sup.131I-labeled monoclonal antibody by the liver.
In addition, this radionucleotide has a more favorable gamma
emission energy for imaging (Perkins et al., Eur. J. Nucl. Med.
10:296-301, 1985); Carasquillo et al., J. Nucl. Med. 28:281-287,
1987). For example, .sup.111In coupled to monoclonal antibodies
with 1-(P-isothiocyanatobenzyl)-DPTA has shown little uptake in
non-tumorous tissues, particularly the liver, and therefore
enhances specificity of tumor localization (Esteban et al., J.
Nucl. Med. 28:861-870, 1987).
[0143] Examples of suitable non-radioactive isotopic labels include
.sup.157Gd, .sup.55Mn, .sup.162Dy, .sup.52Tr, and .sup.56Fe.
[0144] Examples of suitable fluorescent labels include .sup.152Eu
label, fluorescein, isothiocyanate, rhodamine, phycoerythrin,
phycocyanin, allophycocyanin, o-phthaldehyde, and
fluorescamine.
[0145] Examples of suitable toxin labels include diphtheria toxin,
ricin, and cholera toxin. Examples of chemiluminescent labels
include luminal, isoluminal, aromatic acridinium ester, imidazole,
acridinium salt, oxalate ester, luciferin, luciferase, and
aequorin.
[0146] Examples of nuclear magnetic resonance contrasting agents
include heavy metal nuclei such as Gd, Mn, and Fe.
[0147] Typical techniques for binding the above-described labels to
antibodies are provided by Kennedy et al. (Clin. Chim. Acta
70:1-31, 1976), and Schurs et al. (Clin. Chim. Acta 81:1-40, 1977).
Coupling techniques mentioned in the latter are the glutaraldehyde
method, the periodate method, the dimaleimide method, the
m-maleimidobenzyl-N-hydroxy-succinimide ester method.
Diagnosing Disease States Resulting from Mutations in ZNF206
[0148] Given the effect of ZNF206 on the pluripotency of a cell,
mutations in ZNF206 may result in an aberrant pluripotency state in
a cell, leading to cancerous or other disease states. According to
one embodiment of the invention, methods are provided for
diagnosing a disease state resulting from a mutation in a ZNF206
polynucleotide comprising (a) providing a sample from a patient
comprising a cell and (b) determining whether the sample comprises
a mutated ZNF206 polynucleotide. The presence of a mutated ZNF 206
polynucleotide in the sample may be determined, for example by:
contacting the sample with a polynucleotide probe or primer that
hybridizes specifically to a mutated ZNF206 polynucleotide
sequence; by contacting the sample with one or more primers that
comprise a polynucleotide sequence that hybridizes selectively to
the mutated ZNF206 polynucleotide, and performing an amplification
reaction (e.g., a PCR or bio-barcode assay) to produce an
amplification product that indicates the presence of the mutated
ZNF206 polynucleotide in the sample; by detecting a restriction
fragment length polymorphism; or by contacting the sample with an
antibody probe that hybridizes specifically to a mutated ZNF
polypeptide sequence encoded by the mutated ZNF polynucleotide.
Pharmaceutical Compositions and Methods
[0149] The Oct3/4 gene, a POU (Pit-Oct-Unc) family of transcription
factors was once thought to be expressed only in embryonic stem
cells and in tumor cells. With the availability of normal adult
human stem cells, tests for the expression of Oct3/4 gene and the
stem cell theory in human carcinogenesis became possible. Human
breast, liver, pancreas, kidney, mesenchyme, and gastric stem
cells, HeLa and MCF-7 cells, and canine tumors were tested with
antibodies and polymerase chain reaction (PCR) primers for Oct3/4.
Adult human breast stem cells, immortalized nontumorigenic and
tumor cell lines, but not normal differentiated cells, expressed
Oct3/4. Adult human differentiated cells lose their Oct-4
expression. Oct3/4 is expressed in a few cells found in the basal
layer of human skin epidermis. The data demonstrate that normal
adult stem cells and cancer stem cells maintain expression of
Oct3/4, consistent with the stem cell hypothesis of carcinogenesis.
These Oct-4-positive cells may represent "cancer stem cells."
(Carcinogenesis, 26:495-502, 2005). One therapeutic strategy is to
suppress the Oct-4 gene in order to cause such "cancer stem cells"
to differentiate.
[0150] Expression of a ZNF206-encoding construct in an ESC is a way
of maintaining the cell in a pluripotent state and preventing
differentiation of the ESC, particularly default differentiation
towards the extra-embryonic lineage. In fact, ZNF206 expression in
differentiated cells may be used to "reprogram" such cells to
become pluripotent. The ability to reduce ZNF206 expression, and
thereby promote the differentiation of pluripotent cells has
pharmaceutical applications. Reducing ZNF206 expression may be used
to treat certain cancers, or to reduce the risk of developing a
cancer, characterized by cells that that have elevated levels of
ZNF206 expression. In support of this approach, pluripotent stem
cells were induced from mouse embryonic or adult fibroblasts by
introducing stem cell transcription factors Oct 3/4, SOX2, c-Myc
and Klf4 (Takahashi and Yamanka, Cell 126:663-676, 2006; Wernig et
al., In vitro reprogramming of fibroblasts into a pluripotent
ES-cell-like state, Nature advance online publication 6 Jun. 2007
[doi:10.1038/nature05944]; Okita et al., Generation of
germline-competent induced pluripotent stem cells, Nature advance
online publication 6 Jun. 2007 [doi:10.1038/nature05934]). ZNF
could be used to induce pluripotent stem cells from human embryonic
or adult cells, such as, for example, fibroblast cells, by itself
or in combination with one or more stem cell transcription factors
such as Oct 3/4, SOX2, c-Myc or Klf4, for example, under ES cell
culture conditions.
[0151] The invention will be better understood by reference to the
following Examples, which are intended to merely illustrate the
best mode now known for practicing the invention. The scope of the
invention is not to be considered limited thereto.
Example 1
Materials and Methods
[0152] Human embryonic stem cell (hESC) culture. hESC lines WA01
(H1) and WA09 (H9) (WiCell, Madison Wis.) were initially maintained
on irradiated mouse embryonic fibroblast (MEF) feeder cells in
medium that consisted of DMEM/F-12 (80%), Knockout Serum
Replacement (20%), L-alanyl-L-glutamine (GlutaMax; 2 mM), MEM
nonessential amino acids (1.times.), b-Mercaptoethanol (100 mM)
(all from Invitrogen, Carlsbad, Calif.), and bFGF (4 ng/ml)
(PeproTech Inc., Rocky Hill, N.J.) as described previously (Thomson
et al., 1998), then transferred to human feeder layers (HS27 line,
ATCC). For feeder-free growth, cells were transferred to Matrigel
(growth factor-reduced, Becton Dickinson, Bedford, Mass.) or human
purified laminin-coated dishes, and cultured in the same medium
with a higher concentration of bFGF (20 ng/ml). HESCs were
mechanically passaged every 5 to 7 days by cutting undifferentiated
hESC colonies into small pieces using a 27 G PrecisionGlide Needle
attached to a 1 ml syringe (Becton Dickinson, Bedford, Mass.).
[0153] Isolation of hESC-derived PEL cells. WA09 hESC-derived PEL
cells were isolated from the differentiated cells surrounding the
periphery of undifferentiated hESC colonies grown in feeder-free
defined culture. A two-step mechanical/enzymatic treatment method
was employed: first, all of the morphologically distinct hESC
colonies were mechanically dissected away from the cultures, then
the remaining cells were lifted by brief treatment with 0.05%
trypsin and then transferred to new Matrigel- or laminin-coated
plates containing hESC medium. The PEL cells were further purified
by repeating the isolation procedure multiple times until no
morphologically hESC-like cells were observed. POU5F1/OCT4 staining
confirmed that no positive cells remained and GATA6 staining showed
that the PEL cells homogeneously expressed this marker. The PEL
cells maintained a normal diploid karyotype identical to the
parental hESC cells for at least 20 passages. For production of
feeder layers, PEL cells were irradiated in the same manner as
human fibroblast cell lines.
[0154] Production of lentivirus particles and infection of hESCs.
Briefly, lentiviral vectors were produced by co-transfecting the
transfer vector pFUGW, the HIV-1 packaging vector 8.9, and the VSVG
envelope glycoprotein into 293 fibroblasts and concentrated as
described previously. Undifferentiated hESCs (line WA01 [passage
49] and line WA09 [passage 45]) that had been growing in
feeder-free culture for 4 days were incubated with lentiviral
vector particles and polybrene (6 .mu.g/ml; Sigma) overnight and
the medium was changed the next day. After 7 days of continuous
culturing in the defined conditions, hESC colonies that displayed
homogenous expression of eGFP were each mechanically picked and
individually transferred to wells of 6 well plates. The
eGFP-positive undifferentiated hESC subcultures were maintained
under the defined culture conditions. For testing growth of
colonies from single cells, eGFP-positive colonies were dissociated
and sorted by FACS into 96 well plates (see below). Colonies that
were observed to be derived from single cells were expanded and
characterized.
[0155] Fluorescence Activated Cell Sorting (FACS) and single-cell
culture. Undifferentiated eGFP-hESCs were dissociated with 0.05%
trypsin/0.53 mM EDTA (Invitrogen) into a suspension of single cells
and small clusters. Dissociated cells were filtered through
85-.mu.m Nitex mesh to remove aggregates and then sorted on a
FACSVantage SE equipped with DiVa electronics and software (Becton
Dickinson Biosciences). The GFP signal was excited with an argon
laser tuned to 488 nm at 200 mW of power and the emission signal
was collected through a 530/30 bandpass filter. The eGFP-positive
cells were sorted into wells of a 96 well plate (1 eGFP cell/well)
at 15 psi using a 100-.mu.m nozzle tip. Propidium iodide was used
to exclude dead cells and only live cells were used for sorting.
PEL cell conditioned medium was generated by 48 hours incubation at
37.degree. C. in serum-free medium containing ITS supplement
(Invitrogen) and 100 ng/ml bFGF but no serum or serum replacement.
Colony-forming efficiency was measured by plating a known number of
cells (1000) into 6-well dishes containing the appropriate feeder
layer or conditioned medium.
[0156] Microarray analysis. RNA was isolated from cultured cells
using the Qiagen RNEasy kit (Qiagen, Inc, Valencia, Calif.). Two
PEL cultures, 2 undifferentiated hESC (WA09) cultures, and 2 HS27
human foreskin fibroblast (HFF) cultures were harvested separately
and served as biological replicates. To assure that only
undifferentiated hESCs were isolated, colonies were isolated by
hand using a micropipette. Sample preparation and analysis was
performed as previously described (Cai et al., Stem Cells
24:516-530, 2006; Schwartz et al., Stem Cells Dev. 14:517-534,
2005). Briefly, amplification was performed using 100 ng of total
RNA using the Illumina RNA Amplification kit (Ambion, Inc., Austin,
Tex.) following the manufacturer's instructions; labeling was done
by incorporating of biotin-16-UTP (Perkin Elmer Life and Analytical
Sciences, Boston, Mass.) present at a ratio of 1:1 with unlabeled
UTP. Labeled, amplified material (700 ng per array) was hybridized
to the Illumina Sentrix Human 6 BeadChip according to the
manufacturer's instructions (Illumina, Inc., San Diego, Calif.).
Arrays were washed, and then stained with Amersham fluorolink
streptavidin-Cy3 (GE Healthcare Bio-Sciences, Little Chalfont, UK)
according to methods provided by the manufacturer. Arrays were
scanned with an Illumina BeadArray Reader confocal scanner and
array data processing and analysis were performed using Illumina
BeadStudio software. The Illumina BeadArrays have an average of 30
beads of each type (50-mer complementary oligonucleotides) in each
array, so for each set of biological replicates we obtained
approximately 60 independent measurements of hybridization for each
transcript. Differential expression of individual genes between
groups was calculated by the t-test.
[0157] RT-PCR. Expression of several gene transcripts was probed by
semiquantitative RT-PCR. Initial denaturation was carried out at
94.degree. C. for 2 minutes, followed by 35 cycles of PCR
(94.degree. C. for 30 seconds, 55.degree. C. for 30 seconds,
72.degree. C. for 1 minute). Primers used and their expected
products are:
TABLE-US-00001 Size Product (bp) Primers Activin A 262
5'-CTTGAAGAAGAGACCCGAT-3' (Inhibin 5'-CTTCTGCACGCTCCACTAC-3' beta
A) Activin 556 5'-ACACGGGAGTGCATCTACTACAACG-3' Receptor IIB
5'-TTCATGAGCTGGGCCTTCCAGACAC-3'; (ACTRIIB-2B) AFP 676
5'-AGAACCTGTCACAAGCTGTG-3' 5'-CACAGCAAGCTGAGGATGTC-3' beta-Actin
400 5'-TGGCACCACACC TTTCTACAATGAGC-3' 5'-GCACAGCTTCTCCTTAA
TGTCACGC-3' CDX2 563 5'-GAACCTGTGCGAGTGGATGCG-3'
5'-GGTCTATGGCTGTGGGTGGGAG-3' DNMT3B 433 5'-CTCTTACCTTACCATCGACC-3'
5'-CTCCAGAGCATGGTACATGG-3' GATA4 218 5'-CATCAAGACGGAGCCTGGCC-3'
5'-TGACTGTCGGCCAAGACCAG-3' HNF4 762 5'-GCTTGGTTCTCGTTGAGTGG-3'
5'-CAGGAGCTTATAGGGGCTCAGAC-3' LIN-28 420 5'-AGTAAGCTGCACATGGAAGG-3'
5'-ATTGTGGCTCAATTCTGTGC-3' SOX2 370 5'-CCGCATG TACAACATGATGG-3'
5'-CTTCTTCATGAGCGTCT TGG-3' GATA-6 213 5' -CCATGACTCCAACTTCCACC-3'
5' -ACGGAGGACGTGACTTCGGC-3' NANOG 493 5'-GGCAAACAACCCACTTCTGC-3'
5'-TGTT CCAGGCCTGATTGTTC-3' POU5F1 247
5'-CGTGAAGCTGGAGAAGGAGAAGCTG-3' 5'-CAAGGGCCGCAGCTTACACATGTTC-3' SOX
17 181 5'-CGCACGGAATTTGAACAGTA -3' 5'-GGATCAGGGACCTGTCACAC-3'
[0158] Immunocytochemistry. Cultures were fixed with 4%
paraformaldehyde and blocked in 1.times.PBS containing 0.2% Triton
X-100 and 2% BSA. The cells were incubated with the primary
antibody in 0.1% Triton X-100 in PBS at 4.degree. C. overnight.
Then, secondary antibody (Invitrogen) was added and incubated at RT
for 45 min. After staining with DAPI, cells were visualized with a
fluorescence microscope. Primary antibody to AFP, GATA6,
POU51/OCT4, SSEA-4, and Tra-1-81 were obtained from Santa Cruz
Biotechnology.
[0159] Teratoma formation. Approximately 10.sup.4 hESCs were
injected beneath the kidney capsule of adult male Severe Combined
Immunodeficient (SCID) mice. After 21 to 90 days, mice were
sacrificed and teratomas were dissected, fixed in Bouin's fixative
overnight, processed for paraffin sections and stained with
hematoxylin and eosin. Sections were examined for evidence of
tissue differentiation using bright field light microscopy and
photographed as appropriate. All procedures involving mice were
carried out in accordance with Institutional and NIH
guidelines.
Results
[0160] Identification of ZNF206 as a potential transcriptional
repressor of PE-like differentiation. The molecular mechanisms
regulating early lineage commitment from the ICM (or its in vitro
counterpart, the human embryonic stem cell [hESC]) to primitive
endoderm (PE) are poorly understood. NANOG is the only known
transcription factor that regulates hESC self-renewal by inhibiting
PE differentiation.
[0161] To identify other transcription factors that could act as
specific repressors of the PE (with similar proprieties as NANOG)
we performed microarray analysis on an isolated population of hESC
derived PE-like (PEL) cells and on their parental undifferentiated
clonally-related hESC line. From these analyses, we found many
genes that were uniquely expressed in hESCs and not expressed in
PEL cells. Among the many genes that exhibited unique expression
was a zinc finger protein (ZNF206) and NANOG (FIG. 2A).
[0162] The identification of NANOG among the genes uniquely
expressed in undifferentiated hESCs offered us confidence that our
microarray analysis had, indeed, revealed genes that might be
involved in regulating self-renewal and early lineage commitment to
the PE. Since our goal was to find novel transcription factors that
might act as transcriptional repressors, we decided to focus on
ZNF206 since zinc finger proteins often act as transcriptional
regulators. Therefore, we hypothesized that it may be a novel
repressor of PE (or PE-like) differentiation. To determine whether
ZNF206 is uniquely expressed in hESCs, we performed quantitative
RT-PCR on many human tissues and found it to be expressed only in
hESCs and not in differentiated PEL cells or any of the
differentiated human tissues tested (FIG. 2B).
[0163] To test further whether ZNF206 expression is regulated
during early differentiation into the PE, we treated hESCs with
BMP2, a factor previously reported to induce hESCs to differentiate
into PE (Pera et al., J. Cell Sci. 117:1269-1280, 2004).
[0164] Indeed, NANOG and ZNF206 expression were both down-regulated
in BMP2-treated hESCs (FIGS. 3A and 3B) while expression of PE
markers GATA6 and GATA4 were induced (FIGS. 3C and 3D). The
similarity in the expression patterns of NANOG and ZNF206 suggested
to us that ZNF206 may have a similar function as NANOG in promoting
self-renewal by inhibiting PE differentiation.
[0165] Human ZNF206 Cloning and Expression Analysis. FIG. 4 shows
the predicted protein sequence of three isoforms of ZNF206. The
ZNF206 gene contains five introns and five exons. To begin to
understand the function of human ZNF206, primers were specifically
designed to amplify and to clone the different spliced ZNF206 mRNA
isoforms expressed in undifferentiated hESCs by RT-PCR (FIG. 4A).
Four different ZNF206 mRNA isoforms were cloned from
undifferentiated hESCs; isoform 1 is 2568 bp, isoform 2 is 2343 bp,
and isoform 3 is 2075 bp (FIG. 4B). These isoforms likely result
from alternative splicing that takes place in undifferentiated
hESCs. The ZNF206 isoform 2 is predicted encode the 780 amino-acid
full-length functional ZNF206 protein that contains the Novel and
SCAN domains and 14 C.sub.2H.sub.2 zinc fingers (FIG. 4C). The
Novel domain contains a sumoylation site, and the SCAN domain has
been previously reported to mediate protein-protein interactions.
Zinc fingers often mediate DNA binding. ZNF206 isoform 3 is
predicted to encode a protein that contains a SCAN domain and 13
C.sub.2H.sub.2 zinc fingers (FIG. 4C). ZNF206 isoforms 1 and 4 are
predicted to encode short truncated proteins containing the Novel
and SCAN domains but lacking the 14 C.sub.2H.sub.2 zinc finger
domains (FIG. 4C).
[0166] The ZNF206 mRNA transcripts for the four isoforms are
similar in size; however isoform 2 is the predominant form
expressed by undifferentiated hESCs. As a result, we focused on
ZNF206 isoform 2 and generated various ZNF206 lentivirus expression
constructs containing different C-terminal tags, one a V5 tag,
another a eGFP fluorescent protein, and third containing a TAP tag
(FIG. 5). To begin analyzing the localization of ZNF206 protein, we
transfected human 293T kidneys cells and human cervical HeLa cells
with lentiviral vectors expressing ZNF206-eGFP and ZNF206-V5
protein. Our expression experiments show that both the ZNF206-eGFP
and the ZNF206-V5 tagged protein localizes to the nucleus.
[0167] Knockdown of ZNF206 protein causes the down-regulation of
pluripotency genes in hESCs. To determine the functional role of
ZNF206 in hESCs, we decided to knockdown its expression in
undifferentiated hESCs by expressing short hairpin RNAs (shRNAs)
specifically directed against the human ZNF206 mRNA. Sense and
antisense oligos for ZNF206 shRNA were annealed to form a linker
for ligation into pEN_H1 Entry vector. We successfully generated
three gateway entry clones. Each candidate ZNF206 shRNA clone was
fully sequenced to ensure that they retained 100% homology to the
ZNF206 target gene. The H1 Pol III-ZNF206 Cassettes were then
subcloned into the lentiviral expression construct pDSL_hpUGIP (a
shRNA lentiviral expression destination vector obtained from ATTC)
via the Gateway LR recombination reaction (Invitrogen) (FIG. 6A).
We then tested their ability to specifically knockdown the
expression of ZNF206 in 293FT-ZNF206-V5 expressing cells and
performed quantitative RT-PCR and Western blot analysis using an
anti-V5 antibody (Invitrogen). The V5 antibody recognizes the
C-terminal V5 epitope of the ZNF206-V5 fusion protein and allowed
us to see the protein knock-down efficiency. Our results indicated
that two lentiviral shRNA constructs specifically down-regulated
ZNF206 mRNA and protein expression but only the lentiviral shRNA
ZNF206 C expression construct was effective at down-regulating
ZNF206 protein at >90% (FIG. 6A, B).
[0168] To evaluate endogenous ZNF206 expression in undifferentiated
hESC's we generated a custom rabbit polyclonal anti-peptide
polyclonal antibody raised against amino acids 711-726 of human
ZNF206 protein sequence (FIG. 7A) we found that this antibody
specifically detected a protein that was approximately 80 kD in
undifferentiated hESC's and not in the hESC-derived PEL
differentiated cells, corresponding to the predicted full size of
the human ZNF206 protein.
[0169] To evaluate the effects of ZNF206 down-regulation, we
infected undifferentiated H9 (NIH WA09) and H1 (NIH WA01) hESC
lines with three different ZNF206 shRNA lentivirus expression
particles (ZNF 206 shRNA-A, ZNF 206 shRNA-B, or ZNF 206 shRNA-C) or
lentivirus carrying "empty" control vectors. Four days after
infection of undifferentiated hESCs, we evaluated their effects on
ZNF206, OCT-4, and NANOG mRNA levels (FIG. 8). The protein
expression was evaluated by using the commercial antibodies for
OCT4, and NANOG. The results of the knockdown experiments indicated
that infection of undifferentiated hESCs with ZNF206 shRNA-C
lentivirus particles was the most potent down-regulator of ZNF206
mRNA and protein expression levels. In addition, we preliminarily
observed that OCT-4 and NANOG expression were also indirectly
down-regulated as a result of knocking down ZNF206 protein
expression. SSEA-4, a surface marker on undifferentiated hESCs was
also down-regulated. Since OCT-4 and NANOG expression are required
to maintain hESCs undifferentiated and pluripotent, our results
strongly suggested that ZNF206 expression is associated with (and
perhaps essential) for hESC self-renewal and pluripotency.
[0170] Down-regulation of ZNF206 protein expression induces hESCs
to differentiate along the extra-embryonic endodermal lineage.
Since ZNF206 is differentially expressed between undifferentiated
hESCs and primitive endoderm-like (PEL) cells (FIG. 2A), we decided
to also determine if knocking down ZNF206 expression in
undifferentiated hESCs causes them to differentiate along the
extra-embryonic endoderm lineage. To determine this, we infected H9
hESCs with ZNF206 shRNA-C lentiviral expression particles.
Consistent with our previous experiments, after four days, the hESC
colonies that were infected with the ZNF206 shRNA-C lentiviral
expression particles had a differentiated morphology. Analysis of
the ZNF206 shRNA-C infected hESC colonies by immunofluorescence
indicated that the knockdown of ZNF206 caused the majority of the
hES cells to expressed SSEA-1, a specific surface marker of
differentiated hESCs and, within the positive population of
SSEA-1-expressing cells, were cells that co-expressed GATA6 (an
early marker of the primitive endoderm lineage). Further analysis
using RT-PCR indicated that down-regulating the expression of
ZNF206 in hESCs causes them to up-regulate the expression of genes
associated with the extra-embryonic endodermal lineage, e.g.,
GATA4, GATA6, SOX7, CouptfI and CouptfII.
[0171] The role of ZN206 in hESCs and, by extension, human
embryonic development. As indicated in the model depicted in FIG.
9, our studies show that extra-embryonic endoderm lineage appears
to be the earliest default pathway for hESC differentiation (even
prior to neuroectoderm--indeed, perhaps helping to instruct the
formation of neuroectoderm), particularly when hESCs are
dissociated into single cells and grown in defined, serum-free,
feeder-free conditions. This default lineage may then help instruct
the emergence of other lineages, e.g., neuroectoderm (perhaps
giving the appearance of being default). In our model, OCT4 is the
key inhibitor of trophoblast differentiation in hESCs (since
specific down-regulation of OCT-4 in hESCs leads to trophoblast
differentiation), while NANOG and ZNF206 are key inhibitiors of
extra-embryonic endoderm lineage differentiation (since specific
down-regulation of NANOG or ZNF206 leads to extra-embryonic
endoderm lineage differentiation). For example, down-regulation of
ZNF206 expression in hESCs causes the upregulation of genes in the
hESCs that are associated with the extra-embryonic endoderm lineage
(e.g., GATA4, GATA6, SOX17, AFP and HNF4A). Repressing
extra-embryonic endoderm development preserves the pluripotent
state of hESCs (and perhaps, by extension, the ICM), and,
conversely downregulating expression of ZNF206 in hESCs causes them
to upregulate the expression of genes associated with the
extra-embryonic endodermal lineage, down-regulate genes associated
with the pluripotent state, and perhaps lead to the further
emergence of genes associated with even more differentiated
lineages and phenotypes.
[0172] FIG. 10 provides the nucleotide sequence of four isoforms of
ZNF206.
Example 2
[0173] As discussed in Example 1 above, the discovery of ZNF206 was
one of the byproducts of having devised an entirely defined medium
for growing human embryonic stem cells (hESCs). Briefly, we
determined the minimal essential components of a defined culture
system that could stably maintain hESCs in a self-renewing
pluripotent state and serve as a platform for directing such hESCs
towards particular differentiated cell types efficiently and
exclusively using small molecules inducers, without an intervening
multi-lineage embryoid body (EB) stage. In this culture system,
hESCs spontaneously form an autogenic supportive niche composed of
what proved to be primitive endoderm (PE) cells that could, in
turn, support efficient clonal expansion and long-term self-renewal
of hESCs, presumably providing paracrine support in vitro, much as
the PE does for epiblast in vivo. High-throughput genomic and
proteomic analysis of this clonally-related hESC-derived PE--when
compared with the undifferentiated starting hESCs--allowed us to
identify a novel Zinc finger protein (ZNF206) that was found to
maintain hESC renewal and pluripotency by repressing PE lineage
commitment.
Activin A is the Predominant Paracrine Factor Enabling hESC
Growth
[0174] Our further analysis suggests that Activin A, which is
secreted by hESC-derived primitive endoderm-like (PEL) cells (and
the signal transduction pathway it activates) is the predominant
paracrine factor enabling hESC clonal growth in a feeder-free
minimal essential chemically-defined culture system.
[0175] Table 1 below provides a selective list of potential hESC
growth-supporting proteins identified specifically in PEL-(but not
human fibroblast [Hs27]-) conditioned medium (CM) by MudPit
(Multidimensional Protein Identification Technology) proteomic
analysis followed by Western blotting analysis. To meet the
criteria, a peptide had to be detected three or more times
(sequence count) and 10 percent or more of the protein sequence had
to be detected (sequence coverage).
TABLE-US-00002 TABLE 1 Potential hESC growth supporting proteins
Hs27-CM PEL-CM Accession SeCov SeCov number Protein name Seqcount
SpecCount (%) Seqcount SpecCount (%) IPI00009720 Leukemia x x x 3 9
10 inhibitory factor IPI00008780 Stanniocalcin-2 x x x 4 16 22
IPI00028670 Inhibin .beta. A x x x 12 24 30 (Activin A) IPI00007960
Periostin x x x 57 244 46 IPI00215630 Versican x x x 6 15 10
IPI00220156 Transforming x x x 3 5 11 growth factor .beta.2
SpecCount = number of times a peptide for the corresponding protein
was identified. X = Not detected
[0176] Activin A added to our minimum essential defined culture
medium (before spontaneous PE formation) can substitute in large
measure for PE paracrine factors to maintain hESC pluripotency as
assessed by the ability to promote hESC colony formation from a
single cell. We found that the PE-mediated activation of the
Activin-A receptorIIA/B-Smad2/3 signaling pathway is required to
maintain undifferentiated hESC growth. When specific inhibitors of
Activin A (anti-Activin A, soluble ACVR2A/B-FC receptors, or
SB-431542) were added to PE culture medium, the PE culture medium
lost its ability to support clonal hESC expansion for both WA09
(H9) and WA01 (H1) cells. Hence, ZNF206 appears to regulate not
only the emergence of extra-embryonic endoderm, but also the
spontaneous secretion of members of the critical Activin
pathway.
shRNA-Mediated Knock-Down of ZNF206 Causes HESCs to Lose
Pluripotency
[0177] Using Western blots, we determined that short-hairpin
(sh)RNA-mediated knock-down of ZNF206 causes hESCs to lose
pluripotency and differentiate into extra-embryonic endoderm.
ZNF206 knock-down alone was sufficient to abrogate Oct-4 and Nanog
expression, suggesting it may work either upstream or in a critical
complex with these known canonical "pluripotency genes", and likely
establishing ZNF206 as an equally pivotal mediator of
pluripotence--perhaps even essential for the proper expression and
functioning of Oct-4 and Nanog.
[0178] RT-PCR was used to demonstrate the new expression of
extra-embryonic lineage markers (GATA4, GATA6, SOX7, AFP and HNF4A)
coincident with the loss of pluripotency marker expression (Oct-4,
Nanog, Sox2); however, expression of trophoblastic markers (i.e.,
Cdx2, HCG.alpha., HGG.beta.) was not turned on.
[0179] Immunofluorescence staining was used to illustrate the
effect of ZNF206 on the actual expression of markers within H9
(WA09) hESC colonies infected with ZNF206 shRNA-C lentiviral
expression particles. Immunofluorescence demonstrated the
expression of the differentiation hESC surface marker SSEA-1 and
the emergence of expression of the primitive endoderm (PE) early
marker GATA-6 ectopically within the formerly undifferentiated
colong (i.e., PE-like cells). These studies confirmed that
knockdown of ZNF206 induces hESCs to differentiate alone the
extra-embryonic endodermal lineage.
[0180] Indeed, ZNF206-shRNA treated hESCs and PE cells have
overlapping global gene expression profiles. Microarray gene
expression was used to compare hESCs (line WA09 [H9]) treated with
ZNF206 shRNA expression particles and human heart, brain, and liver
tissues and hESC-derived primitive endoderm cells. The gene
profiles of primitive endoderm and hESCs in which ZNF206 was
suppressed were virtually identical. However there was very little
overlap when such ZNF206-suppressed HSCs were compared with other
cell types.
Overexpression of ZNF206 in PE Cells Induces Dedifferentiation into
Pluripotent Cells
[0181] Most intriguing, however, is the role that ZNF206 may play
in a reprogramming process. As indicated above, we determined that
ZNF206 could maintain hESC renewal and pluripotence by repressing
constitutive PE lineage commitment. We also found that
overexpression of ZNF206 alone in PE cells induced them to
dedifferentiate--become "reprogrammed--back into pluripotent cells,
as demonstrated in dedifferentiated PE cells that were
immunostained for intracellular (Oct4, alkaline phosphatase) and
surface markers of pluripotence (SSEA-4, Tra-1-80, Tra-1-60). Cells
reprogrammed with the single factor ZNF206 not only looked like
hESCs but also appeared to be identical to induced pluripotent
somatic cells (IPSCs) generated from skin fibroblasts using the
classical "four-factor cocktail" of Oct4, c-myc, Sox-2 and
flf-4.
[0182] This result becomes intriguing in light of recent reports
that their most efficient reprogramming occurs in "fibroblasts"
generated from hESCs. We suspect these are not actually fibroblasts
but rather PE, suggesting that ZNF206 may be a simpler biologically
faithful method for dedifferentiation. In other words, under some
circumstances, this single factor ZNF206 may be sufficient for
generating induced pluripotent somatic cells (iPSCs), rather than
the four factors usually required. The reprogrammed cells obtained
by this method appear to be identical to those obtained using Oct4,
c-myc, sox-2, & flf4 retrovirally transduced into skin
cells.
[0183] All publications, patents and patent applications are
incorporated herein by reference. While in the foregoing
specification, this invention has been described in relation to
certain embodiments thereof, and many details have been set forth
for purposes of illustration, it will be apparent to those skilled
in the art that the invention is susceptible to additional
embodiments and that certain of the details herein may be varied
considerably without departing from the basic principles of the
invention.
Sequence CWU 1
1
37119DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1cttgaagaag agacccgat 19219DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2cttctgcacg ctccactac 19325DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 3acacgggagt gcatctacta caacg
25425DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 4ttcatgagct gggccttcca gacac 25520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
5agaacctgtc acaagctgtg 20620DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 6cacagcaagc tgaggatgtc
20726DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 7tggcaccaca cctttctaca atgagc 26825DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
8gcacagcttc tccttaatgt cacgc 25921DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 9gaacctgtgc gagtggatgc g
211022DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 10ggtctatggc tgtgggtggg ag 221120DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
11ctcttacctt accatcgacc 201220DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 12ctccagagca tggtacatgg
201320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 13catcaagacg gagcctggcc 201420DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
14tgactgtcgg ccaagaccag 201520DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 15gcttggttct cgttgagtgg
201623DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 16caggagctta taggggctca gac 231720DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
17agtaagctgc acatggaagg 201820DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 18attgtggctc aattctgtgc
201920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 19ccgcatgtac aacatgatgg 202020DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
20cttcttcatg agcgtcttgg 202120DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 21ccatgactcc aacttccacc
202220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 22acggaggacg tgacttcggc 202320DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
23ggcaaacaac ccacttctgc 202420DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 24tgttccaggc ctgattgttc
202525DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 25cgtgaagctg gagaaggaga agctg 252625DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
26caagggccgc agcttacaca tgttc 252720DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
27cgcacggaat ttgaacagta 202820DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 28ggatcaggga cctgtcacac
2029148PRTHomo sapiens 29Met Leu Gly Glu Ser Val Pro Ala Ala Leu
Glu Gln Glu Gln Leu Gly1 5 10 15Glu Val Lys Leu Glu Glu Glu Glu Ala
Val Ser Pro Glu Asp Pro Arg 20 25 30Arg Pro Glu Ser Arg Leu Arg Pro
Glu Val Ala His Gln Leu Phe Arg 35 40 45Cys Phe Gln Tyr Gln Glu Asp
Met Gly Pro Arg Ala Ser Leu Ser Arg 50 55 60Leu Arg Glu Leu Cys Gly
His Trp Leu Arg Pro Ala Leu His Thr Lys65 70 75 80Lys Gln Ile Leu
Glu Leu Leu Val Leu Glu Gln Phe Leu Ser Val Leu 85 90 95Pro Pro His
Leu Leu Gly Arg Leu Gln Gly Gln Pro Leu Arg Asp Gly 100 105 110Glu
Glu Val Val Leu Leu Leu Glu Gly Ile His Arg Glu Pro Ser His 115 120
125Ala Gly Pro Leu Val Arg Gly Trp Gly Ser Gly Leu Ser Ser Met Arg
130 135 140Met Met Gly Thr14530780PRTHomo sapiens 30Met Leu Gly Glu
Ser Val Pro Ala Ala Leu Glu Gln Glu Gln Leu Gly1 5 10 15Glu Val Lys
Leu Glu Glu Glu Glu Ala Val Ser Pro Glu Asp Pro Arg 20 25 30Arg Pro
Glu Ser Arg Leu Arg Pro Glu Val Ala His Gln Leu Phe Arg 35 40 45Cys
Phe Gln Tyr Gln Glu Asp Met Gly Pro Arg Ala Ser Leu Ser Arg 50 55
60Leu Arg Glu Leu Cys Gly His Trp Leu Arg Pro Ala Leu His Thr Lys65
70 75 80Lys Gln Ile Leu Glu Leu Leu Val Leu Glu Gln Phe Leu Ser Val
Leu 85 90 95Pro Pro His Leu Leu Gly Arg Leu Gln Gly Gln Pro Leu Arg
Asp Gly 100 105 110Glu Glu Val Val Leu Leu Leu Glu Gly Ile His Arg
Glu Pro Ser His 115 120 125Ala Gly Pro Leu Asp Phe Ser Cys Asn Ala
Gly Lys Ser Cys Pro Arg 130 135 140Ala Asp Val Thr Leu Glu Glu Lys
Gly Cys Ala Ser Gln Val Pro Ser145 150 155 160His Ser Pro Lys Lys
Glu Leu Pro Ala Glu Glu Pro Ser Val Leu Gly 165 170 175Pro Ser Asp
Glu Pro Pro Arg Pro Gln Pro Arg Ala Ala Gln Pro Ala 180 185 190Glu
Pro Gly Gln Trp Arg Leu Pro Pro Ser Ser Lys Gln Pro Leu Ser 195 200
205Pro Gly Pro Gln Lys Thr Phe Gln Ala Leu Gln Glu Ser Ser Pro Gln
210 215 220Gly Pro Ser Pro Trp Pro Glu Glu Ser Ser Arg Asp Gln Glu
Leu Ala225 230 235 240Ala Val Leu Glu Cys Leu Thr Phe Glu Asp Val
Pro Glu Asn Lys Ala 245 250 255Trp Pro Ala His Pro Leu Gly Phe Gly
Ser Arg Thr Pro Asp Lys Glu 260 265 270Glu Phe Lys Gln Glu Glu Pro
Lys Gly Ala Ala Trp Pro Thr Pro Ile 275 280 285Leu Ala Glu Ser Gln
Ala Asp Ser Pro Gly Val Pro Gly Glu Pro Cys 290 295 300Ala Gln Ser
Leu Gly Arg Gly Ala Ala Ala Ser Gly Pro Gly Glu Asp305 310 315
320Gly Ser Leu Leu Gly Ser Ser Glu Ile Leu Glu Val Lys Val Ala Glu
325 330 335Gly Val Pro Glu Pro Asn Pro Glu Leu Gln Phe Ile Cys Ala
Asp Cys 340 345 350Gly Val Ser Phe Pro Gln Leu Ser Arg Leu Lys Ala
His Gln Leu Arg 355 360 365Ser His Pro Ala Gly Arg Ser Phe Leu Cys
Leu Cys Cys Gly Lys Ser 370 375 380Phe Gly Arg Ser Ser Ile Leu Lys
Leu His Met Arg Thr His Thr Asp385 390 395 400Glu Arg Pro His Ala
Cys His Leu Cys Gly His Arg Phe Arg Gln Ser 405 410 415Ser His Leu
Ser Lys His Leu Leu Thr His Ser Ser Glu Pro Ala Phe 420 425 430Leu
Cys Ala Glu Cys Gly Arg Gly Phe Gln Arg Arg Ala Ser Leu Val 435 440
445Gln His Leu Leu Ala His Ala Gln Asp Gln Lys Pro Pro Cys Ala Pro
450 455 460Glu Ser Lys Ala Glu Ala Pro Pro Leu Thr Asp Val Leu Cys
Ser His465 470 475 480Cys Gly Gln Ser Phe Gln Arg Arg Ser Ser Leu
Lys Arg His Leu Arg 485 490 495Ile His Ala Arg Asp Lys Asp Arg Arg
Ser Ser Glu Gly Ser Gly Ser 500 505 510Arg Arg Arg Asp Ser Asp Arg
Arg Pro Phe Val Cys Ser Asp Cys Gly 515 520 525Lys Ala Phe Arg Arg
Ser Glu His Leu Val Ala His Arg Arg Val His 530 535 540Thr Gly Glu
Arg Pro Phe Ser Cys Gln Ala Cys Gly Arg Ser Phe Thr545 550 555
560Gln Ser Ser Gln Leu Val Ser His Gln Arg Val His Thr Gly Glu Lys
565 570 575Pro Tyr Ala Cys Pro Gln Cys Gly Lys Arg Phe Val Arg Arg
Ala Ser 580 585 590Leu Ala Arg His Leu Leu Thr His Gly Gly Pro Arg
Pro His His Cys 595 600 605Thr Gln Cys Gly Lys Ser Phe Gly Gln Thr
Gln Asp Leu Ala Arg His 610 615 620Gln Arg Ser His Thr Gly Glu Lys
Pro Cys Arg Cys Ser Glu Cys Gly625 630 635 640Glu Gly Phe Ser Gln
Ser Ala His Leu Ala Arg His Gln Arg Ile His 645 650 655Thr Gly Glu
Lys Pro His Ala Cys Asp Thr Cys Gly His Arg Phe Arg 660 665 670Asn
Ser Ser Asn Leu Ala Arg His Arg Arg Ser His Thr Gly Glu Arg 675 680
685Pro Tyr Ser Cys Gln Thr Cys Gly Arg Ser Phe Arg Arg Asn Ala His
690 695 700Leu Arg Arg His Leu Ala Thr His Ala Glu Pro Gly Gln Glu
Gln Ala705 710 715 720Glu Pro Pro Gln Glu Cys Val Glu Cys Gly Lys
Ser Phe Ser Arg Ser 725 730 735Cys Asn Leu Leu Arg His Leu Leu Val
His Thr Gly Ala Arg Pro Tyr 740 745 750Ser Cys Thr Gln Cys Gly Arg
Ser Phe Ser Arg Asn Ser His Leu Leu 755 760 765Arg His Leu Arg Thr
His Ala Arg Glu Thr Leu Tyr 770 775 78031724PRTHomo sapiens 31Met
Leu Gly Glu Ser Val Pro Ala Ala Leu Glu Gln Glu Gln Leu Gly1 5 10
15Glu Val Lys Leu Glu Glu Glu Glu Ala Val Ser Pro Glu Asp Pro Arg
20 25 30Arg Pro Glu Ser Arg Leu Arg Pro Glu Val Ala His Gln Leu Phe
Arg 35 40 45Cys Phe Gln Tyr Gln Glu Asp Met Gly Pro Arg Ala Ser Leu
Ser Arg 50 55 60Leu Arg Glu Leu Cys Gly His Trp Leu Arg Pro Ala Leu
His Thr Lys65 70 75 80Lys Gln Ile Leu Glu Leu Leu Val Leu Glu Gln
Phe Leu Ser Val Leu 85 90 95Pro Pro His Leu Leu Gly Arg Leu Gln Gly
Gln Pro Leu Arg Asp Gly 100 105 110Glu Glu Val Val Leu Leu Leu Glu
Gly Ile His Arg Glu Pro Ser His 115 120 125Ala Gly Pro Leu Asp Phe
Ser Cys Asn Ala Gly Lys Ser Cys Pro Arg 130 135 140Ala Asp Val Thr
Leu Glu Glu Lys Gly Cys Ala Ser Gln Val Pro Ser145 150 155 160His
Ser Pro Lys Lys Glu Leu Pro Ala Glu Glu Pro Ser Val Leu Gly 165 170
175Pro Ser Asp Glu Pro Pro Arg Pro Gln Pro Arg Ala Ala Gln Pro Ala
180 185 190Glu Pro Gly Gln Trp Arg Leu Pro Pro Ser Ser Lys Gln Pro
Leu Ser 195 200 205Pro Gly Pro Gln Lys Thr Phe Gln Ala Leu Gln Glu
Ser Ser Pro Gln 210 215 220Gly Pro Ser Pro Trp Pro Glu Glu Ser Ser
Arg Asp Gln Glu Leu Ala225 230 235 240Ala Val Leu Glu Cys Leu Thr
Phe Glu Asp Val Pro Glu Asn Lys Ala 245 250 255Trp Pro Ala His Pro
Leu Gly Phe Gly Ser Arg Thr Pro Asp Lys Glu 260 265 270Glu Phe Lys
Gln Glu Glu Pro Lys Gly Ala Ala Trp Pro Thr Pro Ile 275 280 285Leu
Ala Glu Ser Gln Ala Asp Ser Pro Gly Val Pro Gly Glu Pro Cys 290 295
300Ala Gln Ser Leu Gly Arg Gly Ala Ala Ala Ser Gly Pro Gly Glu
Asp305 310 315 320Gly Ser Leu Leu Gly Ser Ser Glu Ile Leu Glu Val
Lys Val Ala Glu 325 330 335Gly Val Pro Glu Pro Asn Pro Glu Leu Gln
Phe Ile Cys Ala Asp Cys 340 345 350Gly Val Ser Phe Pro Gln Leu Ser
Arg Leu Lys Ala His Gln Leu Arg 355 360 365Ser His Pro Ala Gly Arg
Ser Phe Leu Cys Leu Cys Cys Gly Lys Ser 370 375 380Phe Gly Arg Ser
Ser Ile Leu Lys Leu His Met Arg Thr His Thr Asp385 390 395 400Glu
Arg Pro His Ala Cys His Leu Cys Gly His Arg Phe Arg Gln Ser 405 410
415Ser His Leu Ser Lys His Leu Leu Thr His Ser Ser Glu Pro Ala Phe
420 425 430Leu Cys Ala Glu Cys Gly Arg Gly Phe Gln Arg Arg Ala Ser
Leu Val 435 440 445Gln His Leu Leu Ala His Ala Gln Asp Gln Lys Pro
Pro Cys Ala Pro 450 455 460Glu Ser Lys Ala Glu Ala Pro Pro Leu Thr
Asp Val Leu Cys Ser His465 470 475 480Cys Gly Gln Ser Phe Gln Arg
Arg Ser Ser Leu Lys Arg His Leu Arg 485 490 495Ile His Ala Arg Asp
Lys Asp Arg Arg Ser Ser Glu Gly Ser Gly Ser 500 505 510Arg Arg Arg
Asp Ser Asp Arg Arg Pro Phe Val Cys Ser Asp Cys Gly 515 520 525Lys
Ala Phe Arg Arg Ser Glu His Leu Val Ala His Arg Arg Val His 530 535
540Thr Gly Glu Arg Pro Phe Ser Cys Gln Ala Cys Gly Arg Ser Phe
Thr545 550 555 560Gln Ser Ser Gln Leu Val Ser His Gln Arg Val His
Thr Gly Glu Lys 565 570 575Pro Tyr Ala Cys Pro Gln Cys Gly Lys Arg
Phe Val Arg Arg Ala Ser 580 585 590Leu Ala Arg His Leu Leu Thr His
Gly Gly Pro Arg Pro His His Cys 595 600 605Thr Gln Cys Gly Lys Ser
Phe Gly Gln Thr Gln Asp Leu Ala Arg His 610 615 620Gln Arg Ser His
Thr Gly Glu Arg Pro Tyr Ser Cys Gln Thr Cys Gly625 630 635 640Arg
Ser Phe Arg Arg Asn Ala His Leu Arg Arg His Leu Ala Thr His 645 650
655Ala Glu Pro Gly Gln Glu Gln Ala Glu Pro Pro Gln Glu Cys Val Gly
660 665 670Cys Gly Lys Ser Phe Ser Arg Ser Cys Asn Leu Leu Arg His
Leu Leu 675 680 685Val His Thr Gly Ala Arg Pro Tyr Ser Cys Thr Gln
Cys Gly Arg Ser 690 695 700Phe Ser Arg Asn Ser His Leu Leu Arg His
Leu Arg Thr His Ala Arg705 710 715 720Glu Thr Leu Tyr32156PRTHomo
sapiens 32Met Leu Gly Glu Ser Val Pro Ala Ala Leu Glu Gln Glu Gln
Leu Gly1 5 10 15Glu Val Lys Leu Glu Glu Glu Glu Ala Val Ser Pro Glu
Asp Pro Arg 20 25 30Arg Pro Glu Ser Arg Leu Arg Pro Glu Val Ala His
Gln Leu Phe Arg 35 40 45Cys Phe Gln Tyr Gln Glu Asp Met Gly Pro Arg
Ala Ser Leu Ser Arg 50 55 60Leu Arg Glu Leu Cys Gly His Trp Leu Arg
Pro Ala Leu His Thr Lys65 70 75 80Lys Gln Ile Leu Glu Leu Leu Val
Leu Glu Gln Phe Leu Ser Val Leu 85 90 95Pro Pro His Leu Leu Gly Arg
Leu Gln Gly Gln Pro Leu Arg Asp Gly 100 105 110Glu Glu Val Val Leu
Leu Leu Glu Gly Ile His Arg Glu Pro Ser His 115 120 125Ala Gly Pro
Leu Val Pro Arg Ala Pro His His Gly Gln Arg Arg Val 130 135 140Pro
Glu Ile Arg Ser Trp Arg Leu Cys Trp Ser Ala145 150
15533780PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 33Met Leu Gly Glu Ser Val Pro Ala Ala Leu Glu
Gln Glu Gln Leu Gly1 5 10 15Glu Val Lys Leu Glu Glu Glu Glu Ala Val
Ser Pro Glu Asp Pro Arg 20 25 30Arg Pro Glu Ser Arg Leu Arg Pro Glu
Val Ala His Gln Leu Phe Arg 35
40 45Cys Phe Gln Tyr Gln Glu Asp Met Gly Pro Arg Ala Ser Leu Ser
Arg 50 55 60Leu Arg Glu Leu Cys Gly His Trp Leu Arg Pro Ala Leu His
Thr Lys65 70 75 80Lys Gln Ile Leu Glu Leu Leu Val Leu Glu Gln Phe
Leu Ser Val Leu 85 90 95Pro Pro His Leu Leu Gly Arg Leu Gln Gly Gln
Pro Leu Arg Asp Gly 100 105 110Glu Glu Val Val Leu Leu Leu Glu Gly
Ile His Arg Glu Pro Ser His 115 120 125Ala Gly Pro Leu Asp Phe Ser
Cys Asn Ala Gly Lys Ser Cys Pro Arg 130 135 140Ala Asp Val Thr Leu
Glu Glu Lys Gly Cys Ala Ser Gln Val Pro Ser145 150 155 160His Ser
Pro Lys Lys Glu Leu Pro Ala Glu Glu Pro Ser Val Leu Gly 165 170
175Pro Ser Asp Glu Pro Pro Arg Pro Gln Pro Arg Ala Ala Gln Pro Ala
180 185 190Glu Pro Gly Gln Trp Arg Leu Pro Pro Ser Ser Lys Gln Pro
Leu Ser 195 200 205Pro Gly Pro Gln Lys Thr Phe Gln Ala Leu Gln Glu
Ser Ser Pro Gln 210 215 220Gly Pro Ser Pro Trp Pro Glu Glu Ser Ser
Arg Asp Gln Glu Leu Ala225 230 235 240Ala Val Leu Glu Cys Leu Thr
Phe Glu Asp Val Pro Glu Asn Lys Ala 245 250 255Trp Pro Ala His Pro
Leu Gly Phe Gly Ser Arg Thr Pro Asp Lys Glu 260 265 270Glu Phe Lys
Gln Glu Glu Pro Lys Gly Ala Ala Trp Pro Thr Pro Ile 275 280 285Leu
Ala Glu Ser Gln Ala Asp Ser Pro Gly Val Pro Gly Glu Pro Cys 290 295
300Ala Gln Ser Leu Gly Arg Gly Ala Ala Ala Ser Gly Pro Gly Glu
Asp305 310 315 320Gly Ser Leu Leu Gly Ser Ser Glu Ile Leu Glu Val
Lys Val Ala Glu 325 330 335Gly Val Pro Glu Pro Asn Pro Glu Leu Gln
Phe Ile Cys Ala Asp Cys 340 345 350Gly Val Ser Phe Pro Gln Leu Ser
Arg Leu Lys Ala His Gln Leu Arg 355 360 365Ser His Pro Ala Gly Arg
Ser Phe Leu Cys Leu Cys Cys Gly Lys Ser 370 375 380Phe Gly Arg Ser
Ser Ile Leu Lys Leu His Met Arg Thr His Thr Asp385 390 395 400Glu
Arg Pro His Ala Cys His Leu Cys Gly His Arg Phe Arg Gln Ser 405 410
415Ser His Leu Ser Lys His Leu Leu Thr His Ser Ser Glu Pro Ala Phe
420 425 430Leu Cys Ala Glu Cys Gly Arg Gly Phe Gln Arg Arg Ala Ser
Leu Val 435 440 445Gln His Leu Leu Ala His Ala Gln Asp Gln Lys Pro
Pro Cys Ala Pro 450 455 460Glu Ser Lys Ala Glu Ala Pro Pro Leu Thr
Asp Val Leu Cys Ser His465 470 475 480Cys Gly Gln Ser Phe Gln Arg
Arg Ser Ser Leu Lys Arg His Leu Arg 485 490 495Ile His Ala Arg Asp
Lys Asp Arg Arg Ser Ser Glu Gly Ser Gly Ser 500 505 510Arg Arg Arg
Asp Ser Asp Arg Arg Pro Phe Val Cys Ser Asp Cys Gly 515 520 525Lys
Ala Phe Arg Arg Ser Glu His Leu Trp Ala His Arg Arg Val His 530 535
540Thr Gly Glu Arg Pro Phe Ser Cys Gln Ala Cys Gly Arg Ser Phe
Thr545 550 555 560Gln Ser Ser Gln Leu Val Ser His Gln Arg Val His
Thr Gly Glu Lys 565 570 575Pro Tyr Ala Cys Pro Gln Cys Gly Lys Arg
Phe Val Arg Arg Ala Ser 580 585 590Leu Ala Arg His Leu Leu Thr His
Gly Gly Pro Arg Pro His His Cys 595 600 605Thr Gln Cys Gly Lys Ser
Phe Gly Gln Thr Gln Asp Leu Ala Arg His 610 615 620Gln Arg Ser His
Thr Gly Glu Lys Pro Cys Arg Cys Ser Glu Cys Gly625 630 635 640Glu
Gly Phe Ser Gln Ser Ala His Leu Ala Arg His Gln Arg Ile His 645 650
655Thr Gly Glu Lys Pro His Ala Cys Asp Thr Cys Gly His Arg Phe Arg
660 665 670Asn Ser Ser Asn Leu Ala Arg His Arg Arg Ser His Thr Gly
Glu Arg 675 680 685Pro Tyr Ser Cys Gln Thr Cys Gly Arg Ser Phe Arg
Arg Asn Ala His 690 695 700Leu Arg Arg His Leu Ala Thr His Ala Glu
Pro Gly Gln Glu Gln Ala705 710 715 720Glu Pro Pro Gln Glu Cys Val
Glu Cys Gly Lys Ser Phe Ser Arg Ser 725 730 735Cys Asn Leu Leu Arg
His Leu Leu Val His Thr Gly Ala Arg Pro Tyr 740 745 750Ser Cys Thr
Gln Cys Gly Arg Ser Phe Ser Arg Asn Ser His Leu Leu 755 760 765Arg
His Leu Arg Thr His Ala Arg Glu Thr Leu Tyr 770 775
780342568DNAHomo sapiens 34atgcttggag aatcagtccc agctgccctg
gagcaggagc agctggggga agtcaagctg 60gaggaggagg aggctgtcag cccagaggac
cccaggcgac cagagtccag gctgaggccc 120gaggtggctc accagctgtt
cagatgcttc cagtatcagg aggacatggg gccacgggcg 180tccctgagcc
ggctccggga gctctgcggc cactggctgc ggccggctct gcacaccaag
240aaacagatcc tggagctgct ggtgctggag cagttcctga gtgtgctgcc
tccgcacctc 300ctgggccgcc tgcaggggca gccgctcagg gatggggagg
aggtggtgct gctgctcgag 360ggcatccacc gggagcccag ccacgcgggg
ccgctggtga gagggtgggg cagcgggctg 420agcagcatgc ggatgatggg
gacttgatcc ccccagtgag gaatctctgg aaactccact 480ttcccccacc
tgaccattcc ttcttcacct cctaactcct cccctggctg actctaacct
540cgtttctgtc ccatgtcccc tcggagtcag gacacaggtt gccaccccgg
gagtcactta 600acttgaatgt gttttgaaca ggattttagt tgtaatgctg
gcaagagttg tccccgtgca 660gacgtcacct tggaggaaaa ggggtgtgct
tcccaggtcc ccagccacag ccccaagaag 720gaattgcctg cggaagagcc
ttcagtgctg ggcccatcgg atgagcctcc ccgaccccag 780ccaagggctg
cccagcctgc tgagccggga cagtggaggc ttcccccaag ttcaaagcag
840ccgctgagcc cggggcccca gaagacattc caggccctgc aagaaagcag
tccccagggc 900ccctcaccat ggccagagga gagttcccga gatcaggagc
tggcggctgt gctggagtgc 960ctgacctttg aggatgtgcc agagaataag
gcgtggcctg cacaccccct gggattcgga 1020agcagaaccc cagacaagga
ggaatttaaa caagaagagc ccaaaggggc tgcctggccc 1080actcccatct
tagcagagtc ccaggcagat agtcctgggg tgccgggaga gccttgcgcc
1140cagtcgctcg gacggggcgc tgcggctagc ggccctggcg aagatgggtc
ccttcttggc 1200agcagtgaaa ttttggaggt caaagtggct gagggcgtcc
ccgagcccaa tccggagttg 1260cagttcatct gcgcggactg cggggtgagc
ttcccgcagc tgtctcgcct gaaggcgcac 1320cagctgcgct cgcacccggc
tgggcgctcc ttcctgtgcc tttgctgcgg gaagagcttc 1380ggccgcagct
ccattctcaa gctgcacatg cgcactcaca cggacgagcg gccgcacgcc
1440tgccacctgt gcggccaccg cttccgccag agctcgcacc tgagcaagca
cctgctgacc 1500cactcctccg aacccgcctt cctgtgcgca gagtgcggcc
gcggcttcca gcgccgcgcc 1560agccttgtgc agcacctgct ggcgcacgcc
caggaccaga agccgccctg cgctcctgag 1620agtaaggccg aagcgccgcc
actgaccgat gtcctgtgct cccactgcgg ccagagcttc 1680cagcgccgct
ccagcctcaa gcgccacctg cggatccacg ccagggacaa ggaccgccgg
1740tcctccgaag gctccggcag ccgccgccgg gactccgacc ggaggccctt
cgtgtgcagc 1800gactgcggca aggccttccg gcgcagcgag cacctggtgg
cccaccggag ggtgcacacg 1860ggcgagcggc ccttctcctg ccaggcttgc
ggccgcagct tcacgcagag ctcgcagctg 1920gtcagccacc aacgggtgca
cacgggcgag aagccctacg cctgtccgca gtgcgggaag 1980cgctttgtgc
gccgggccag ccttgcccgc cacctgctga cccacggtgg ccctcggccc
2040caccactgca cccagtgcgg gaagagtttc ggccagaccc aggatctggc
ccgccaccag 2100cgcagccaca cgggcgagaa gccctgccgc tgcagcgagt
gcggtgaggg cttcagccag 2160agcgcccacc tggcgcgcca ccagcgcatc
cacacagggg agaagcccca cgcctgcgac 2220acctgcggcc accgtttccg
caatagctcc aacctggccc gccatcgccg cagccacacg 2280ggcgagcggc
cctacagctg tcagacgtgc ggtcgcagct tccggcgcaa cgcgcatctg
2340cggcggcacc tggctaccca tgcggagccc gggcaggagc aggccgagcc
cccgcaggag 2400tgcgtggagt gcgggaagag cttcagccgc agctgcaatc
tgctgcgaca cctgctggtg 2460cacacgggcg ccaggcccta ctcctgcacg
cagtgtggcc gcagcttcag ccgcaactcc 2520cacctgctgc gccacctgcg
cacccacgcc cgcgagacgc tgtactag 2568352343DNAHomo sapiens
35atgcttggag aatcagtccc agctgccctg gagcaggagc agctggggga agtcaagctg
60gaggaggagg aggctgtcag cccagaggac cccaggcgac cagagtccag gctgaggccc
120gaggtggctc accagctgtt cagatgcttc cagtatcagg aggacatggg
gccacgggcg 180tccctgagcc ggctccggga gctctgcggc cactggctgc
ggccggctct gcacaccaag 240aaacagatcc tggagctgct ggtgctggag
cagttcctga gtgtgctgcc tccgcacctc 300ctgggccgcc tgcaggggca
gccgctcagg gatggggagg aggtggtgct gctgctcgag 360ggcatccacc
gggagcccag ccacgcgggg ccgctggatt ttagttgtaa tgctggcaag
420agttgtcccc gtgcagacgt caccttggag gaaaaggggt gtgcttccca
ggtccccagc 480cacagcccca agaaggaatt gcctgcggaa gagccttcag
tgctgggccc atcggatgag 540cctccccgac cccagccaag ggctgcccag
cctgctgagc cgggacagtg gaggcttccc 600ccaagttcaa agcagccgct
gagcccgggg ccccagaaga cattccaggc cctgcaagaa 660agcagtcccc
agggcccctc accatggcca gaggagagtt cccgagatca ggagctggcg
720gctgtgctgg agtgcctgac ctttgaggat gtgccagaga ataaggcgtg
gcctgcacac 780cccctgggat tcggaagcag aaccccagac aaggaggaat
ttaaacaaga agagcccaaa 840ggggctgcct ggcccactcc catcttagca
gagtcccagg cagatagtcc tggggtgccg 900ggagagcctt gcgcccagtc
gctcggacgg ggcgctgcgg ctagcggccc tggcgaagat 960gggtcccttc
ttggcagcag tgaaattttg gaggtcaaag tggctgaggg cgtccccgag
1020cccaatccgg agttgcagtt catctgcgcg gactgcgggg tgagcttccc
gcagctgtct 1080cgcctgaagg cgcaccagct gcgctcgcac ccggctgggc
gctccttcct gtgcctttgc 1140tgcgggaaga gcttcggccg cagctccatt
ctcaagctgc acatgcgcac tcacacggac 1200gagcggccgc acgcctgcca
cctgtgcggc caccgcttcc gccagagctc gcacctgagc 1260aagcacctgc
tgacccactc ctccgaaccc gccttcctgt gcgcagagtg cggccgcggc
1320ttccagcgcc gcgccagcct tgtgcagcac ctgctggcgc acgcccagga
ccagaagccg 1380ccctgcgctc ctgagagtaa ggccgaagcg ccgccactga
ccgatgtcct gtgctcccac 1440tgcggccaga gcttccagcg ccgctccagc
ctcaagcgcc acctgcggat ccacgccagg 1500gacaaggacc gccggtcctc
cgaaggctcc ggcagccgcc gccgggactc cgaccggagg 1560cccttcgtgt
gcagcgactg cggcaaggcc ttccggcgca gcgagcacct ggtggcccac
1620cggagggtgc acacgggcga gcggcccttc tcctgccagg cttgcggccg
cagcttcacg 1680cagagctcgc agctggtcag ccaccaacgg gtgcacacgg
gcgagaagcc ctacgcctgt 1740ccgcagtgcg ggaagcgctt tgtgcgccgg
gccagccttg cccgccacct gctgacccac 1800ggtggccctc ggccccacca
ctgcacccag tgcgggaaga gtttcggcca gacccaggat 1860ctggcccgcc
accagcgcag ccacacgggc gagaagccct gccgctgcag cgagtgcggt
1920gagggcttca gccagagcgc ccacctggcg cgccaccagc gcatccacac
aggggagaag 1980ccccacgcct gcgacacctg cggccaccgt ttccgcaata
gctccaacct ggcccgccat 2040cgccgcagcc acacgggcga gcggccctac
agctgtcaga cgtgcggtcg cagcttccgg 2100cgcaacgcgc atctgcggcg
gcacctggct acccatgcgg agcccgggca ggagcaggcc 2160gagcccccgc
aggagtgcgt ggagtgcggg aagagcttca gccgcagctg caatctgctg
2220cgacacctgc tggtgcacac gggcgccagg ccctactcct gcacgcagtg
tggccgcagc 2280ttcagccgca actcccacct gctgcgccac ctgcgcaccc
acgcccgcga gacgctgtac 2340tag 2343362175DNAHomo sapiens
36atgcttggag aatcagtccc agctgccctg gagcaggagc agctggggga agtcaagctg
60gaggaggagg aggctgtcag cccagaggac cccaggcgac cagagtccag gctgaggccc
120gaggtggctc accagctgtt cagatgcttc cagtatcagg aggacatggg
gccacgggcg 180tccctgagcc ggctccggga gctctgcggc cactggctgc
ggccggctct gcacaccaag 240aaacagatcc tggagctgct ggtgctggag
cagttcctga gtgtgctgcc tccgcacctc 300ctgggccgcc tgcaggggca
gccgctcagg gatggggagg aggtggtgct gctgctcgag 360ggcatccacc
gggagcccag ccacgcgggg ccgctggatt ttagttgtaa tgctggcaag
420agttgtcccc gtgcagacgt caccttggag gaaaaggggt gtgcttccca
ggtccccagc 480cacagcccca agaaggaatt gcctgcggaa gagccttcag
tgctgggccc atcggatgag 540cctccccgac cccagccaag ggctgcccag
cctgctgagc cgggacagtg gaggcttccc 600ccaagttcaa agcagccgct
gagcccgggg ccccagaaga cattccaggc cctgcaagaa 660agcagtcccc
agggcccctc accatggcca gaggagagtt cccgagatca ggagctggcg
720gctgtgctgg agtgcctgac ctttgaggat gtgccagaga ataaggcgtg
gcctgcacac 780cccctgggat tcggaagcag aaccccagac aaggaggaat
ttaaacaaga agagcccaaa 840ggggctgcct ggcccactcc catcttagca
gagtcccagg cagatagtcc tggggtgccg 900ggagagcctt gcgcccagtc
gctcggacgg ggcgctgcgg ctagcggccc tggtgaagat 960gggtcccttc
ttggcagcag tgaaattttg gaggtcaaag tggctgaggg cgtccccgag
1020cccaatccgg agttgcagtt catctgcgcg gactgcgggg tgagcttccc
gcagctgtct 1080cgcctgaagg cgcaccagct gcgctcgcac ccggctgggc
gctccttcct gtgcctttgc 1140tgcgggaaga gcttcggccg cagctccatt
ctcaagctgc acatgcgcac tcacacggac 1200gagcggccgc acgcctgcca
cctgtgcggc caccgcttcc gccagagctc gcacctgagc 1260aagcacctgc
tgacccactc ctccgaaccc gccttcctgt gcgcagagtg cggccgcggc
1320ttccagcgcc gcgccagcct tgtgcagcac ctgctggcgc acgcccagga
ccagaagccg 1380ccctgcgctc ctgagagtaa ggccgaagcg ccgccactga
ccgatgtcct gtgctcccac 1440tgcggccaga gcttccagcg ccgctccagc
ctcaagcgcc acctgcggat ccacgccagg 1500gacaaggacc gccggtcctc
cgaaggctcc ggcagccgcc gccgggactc cgaccggagg 1560cccttcgtgt
gcagcgactg cggcaaggcc ttccggcgca gcgagcacct ggtggcccac
1620cggagggtgc acacgggcga gcggcccttc tcctgccagg cttgcggccg
cagcttcacg 1680cagagctcgc agctggtcag ccaccaacgg gtgcacacgg
gcgagaagcc ctacgcctgt 1740ccgcagtgcg ggaagcgctt tgtgcgccgg
gccagccttg cccgccacct gctgacccac 1800ggtggccctc ggccccacca
ctgcacccag tgcgggaaga gtttcggcca gacccaggat 1860ctggctcgcc
accagcgcag ccacacgggc gagcggccct acagctgtca gacgtgcggt
1920cgcagcttcc ggcgcaacgc gcatctgcgg cggcacctgg ctacccatgc
ggagcccggg 1980caggagcagg ccgagccccc gcaggagtgc gtggggtgcg
ggaagagctt cagccgcagc 2040tgcaatctgc tgcgacacct gctggtgcac
acgggcgcca ggccctactc ctgcacgcag 2100tgtggccgca gcttcagccg
caactcccac ctgctgcgcc acctgcgcac ccacgcccgc 2160gagacgctgt actag
2175372075DNAHomo sapiens 37atgcttggag aatcagtccc agctgccctg
gagcaggagc agctggggga agtcaagctg 60gaggaggagg aggctgtcag cccagaggac
cccaggcgac cagagtccag gctgaggccc 120gaggtggctc accagctgtt
cagatgcttc cagtatcagg aggacatggg gccacgggcg 180tccctgagcc
ggctccggga gctctgcggc cactggctgc ggccggctct gcacaccaag
240aaacagatcc tggagctgct ggtgctggag cagttcctga gtgtgctgcc
tccgcacctc 300ctgggccgcc tgcaggggca gccgctcagg gatggggagg
aggtggtgct gctgctcgag 360ggcatccacc gggagcccag ccacgcgggg
ccgctggtcc ccagggcccc tcaccatggc 420cagaggagag ttcccgagat
caggagctgg cggctgtgct ggagtgcctg acctttgagg 480atgtgccaga
gaataaggcg tggcctgcac accccctggg attcggaagc agaaccccag
540acaaggagga atttaaacaa gaagagccca aaggggctgc ctggcccact
cccatcttag 600cagagtccca ggcagatagt cctggggtgc cgggagagcc
ttgcgcccag tcgctcggac 660ggggcgctgc ggctagcggc cctggcgaag
atgggtccct tcttggcagc agtgaaattt 720tggaggtcaa agtggctgag
ggcgtccccg agcccaatcc ggagttgcag ttcatctgcg 780cggactgcgg
ggtgagcttc ccgcagctgt ctcgcctgaa ggcgcaccag ctgcgctcgc
840acccggctgg gcgctccttc ctgtgccttt gctgcgggaa gagcttcggc
cgcagctcca 900ttctcaagct gcacatgcgc actcacacgg acgagcggcc
gcacgcctgc cacctgtgcg 960gccaccgctt ccgccagagc tcgcacctga
gcaagcacct gctgacccac tcctccgaac 1020ccgccttcct gtgcgcagag
tgcggccgcg gcttccagcg ccgcgccagc cttgtgcagc 1080acctgctggc
gcacgcccag gaccagaagc cgccctgcgc tcctgagagt aaggccgaag
1140cgccgccact gaccgatgtc ctgtgctccc actgcggcca gagcttccag
cgccgctcca 1200gcctcaagcg ccacctgcgg atccacgcca gggacaagga
ccgccggtcc tccgaaggct 1260ccggcagccg ccgccgggac tccgaccgga
ggcccttcgt gtgcagcgac tgcggcaagg 1320ccttccggcg cagcgagcac
ctggtggccc accggagggt gcacacgggc gagcggccct 1380tctcctgcca
ggcttgcggc cgcagcttca cgcagagctc gcagctggtc agccaccaac
1440gggtgcacac gggcgagaag ccctacgcct gtccgcagtg cgggaagcgc
tttgtgcgcc 1500gggccagcct tgcccgccac ctgctgaccc acggtggccc
tcggccccac cactgcaccc 1560agtgcgggaa gagtttcggc cagacccagg
atctggcccg ccaccagcgc agccacacgg 1620gcgagaagcc ctgccgctgc
agcgagtgcg gtgagggctt cagccagagc gcccacctgg 1680cgcgccacca
gcgcatccac acaggggaga agccccacgc ctgcgacacc tgcggccacc
1740gtttccgcaa tagctccaac ctggcccgcc atcgccgcag ccacacgggc
gagcggccct 1800acagctgtca gacgtgcggt cgcagcttcc ggcgcaacgc
gcatctgcgg cggcacctgg 1860ctacccatgc ggagcccggg caggagcagg
ccgagccccc gcaggagtgc gtggagtgcg 1920ggaagagctt cagccgcagc
tgcaatctgc tgcgacacct gctggtgcac acgggcgcca 1980ggccctactc
ctgcacgcag tgtggccgca gcttcagccg caactcccac ctgctgcgcc
2040acctgcgcac ccacgcccgc gagacgctgt actag 2075
* * * * *