U.S. patent application number 17/466228 was filed with the patent office on 2021-12-23 for method for reducing differentiation resistance of pluripotent stem cells.
This patent application is currently assigned to KEIO UNIVERSITY. The applicant listed for this patent is KEIO UNIVERSITY. Invention is credited to Tomohiko Akiyama, Minoru Ko.
Application Number | 20210395692 17/466228 |
Document ID | / |
Family ID | 1000005826259 |
Filed Date | 2021-12-23 |
United States Patent
Application |
20210395692 |
Kind Code |
A1 |
Ko; Minoru ; et al. |
December 23, 2021 |
Method For Reducing Differentiation Resistance Of Pluripotent Stem
Cells
Abstract
In related-art methods of differentiating pluripotent stem cells
into a desired cell type, there has not been established a
differentiation induction method using human ES/iPS cells and being
stable and highly efficient. A method of inducing differentiation
into a desired cell type within a short period of time and with
high efficiency by attenuating differentiation resistance of a
pluripotent stem cell to generate a pluripotent stem cell that
actively proceeds to a differentiated cell type has been found, and
thus the present invention has been completed.
Inventors: |
Ko; Minoru; (Tokyo, JP)
; Akiyama; Tomohiko; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KEIO UNIVERSITY |
Tokyo |
|
JP |
|
|
Assignee: |
KEIO UNIVERSITY
Tokyo
JP
|
Family ID: |
1000005826259 |
Appl. No.: |
17/466228 |
Filed: |
September 3, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15770634 |
Apr 24, 2018 |
11136552 |
|
|
PCT/JP2016/082152 |
Oct 28, 2016 |
|
|
|
17466228 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2501/60 20130101;
C12N 2506/45 20130101; C12N 2510/00 20130101; C12N 5/0696 20130101;
C12N 2503/02 20130101; C12N 5/0662 20130101; C12N 15/09 20130101;
C12N 2502/45 20130101; C12N 2506/02 20130101 |
International
Class: |
C12N 5/0775 20060101
C12N005/0775; C12N 5/074 20060101 C12N005/074; C12N 15/09 20060101
C12N015/09 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 28, 2015 |
JP |
2015-211356 |
Claims
1.-16. (canceled)
17. A differentiation induction kit for differentiating a
pluripotent stem cell into a desired cell type, comprising at least
any one of the following items (1) to (3): (1) a pluripotent stem
cell having a histone in which H3K27me3 modification has been
substantially removed or reduced; (2) a pluripotent stem cell in
which JMJD3 is forcibly expressed; and (3) a pluripotent stem cell
having a gene construct carrying JMJD3 inserted into a genome
thereof, wherein the JMJD3 has demethylase activity that induces
differentiation of the pluripotent stem cell by removing a methyl
group of H3K27me3.
18. The differentiation induction kit according to claim 17,
wherein the JMJD3 is a demethylase containing only an enzymatically
active region of JMJD3.
19. The differentiation induction kit according to claim 17,
wherein the JMJD3 has an amino acid sequence set forth in any one
of SEQ ID NOS: 1 to 3.
20. The differentiation induction kit according to claim 17,
further comprising a transcription factor required for induction of
differentiation into the desired cell type.
21. The differentiation induction kit according to claim 17,
wherein the desired cell type is a skeletal muscle cell, comprising
at least any one of the following items (1) to (3): (1) a
pluripotent stem cell having a histone in which H3K27me3
modification has been substantially removed or reduced, and a
transcription factor MYOD1; (2) a pluripotent stem cell in which
JMJD3 is forcibly expressed, and a transcription factor MYOD1; and
(3) a pluripotent stem cell having a gene construct carrying JMJD3
inserted into a genome thereof, and a transcription factor
MYOD1.
22. The differentiation induction kit according to claim 17,
wherein the desired cell type is a nerve cell, comprising at least
any one of the following items (1) to (3): (1) a pluripotent stem
cell having a histone in which H3K27me3 modification has been
substantially removed or reduced, and a transcription factor
NEUROG1, NEUROG2, NEUROG3, NEUROD1, and/or NEUROD2; (2) a
pluripotent stem cell in which JMJD3 is forcibly expressed, and a
transcription factor NEUROG1, NEUROG2, NEUROG3, NEUROD1, and/or
NEUROD2; and (3) a pluripotent stem cell having a gene construct
carrying JMJD3 inserted into a genome thereof, and a transcription
factor NEUROG1, NEUROG2, NEUROG3, NEUROD1, and/or NEUROD2.
23. The differentiation induction kit according to claim 17,
wherein the desired cell type is a liver cell, comprising at least
any one of the following items (1) to (3): (1) a pluripotent stem
cell having a histone in which H3K27me3 modification has been
substantially removed or reduced, and a transcription factor HNF1A;
(2) a pluripotent stem cell in which JMJD3 is forcibly expressed,
and a transcription factor HNF1A; and (3) a pluripotent stem cell
having a gene construct carrying JMJD3 inserted into a genome
thereof, and a transcription factor HNF1A.
Description
TECHNICAL FIELD
[0001] The present disclosure relates to a method of attenuating
differentiation resistance of a pluripotent stem cell to a desired
cell type, and more specifically, to a method of differentiating a
pluripotent stem cell into a desired cell type with high efficiency
and a differentiation inducer to be used for the differentiation
method.
[0002] The present application claims priority from Japanese Patent
Application No. 2015-211356, which is incorporated herein by
reference.
BACKGROUND ART
[0003] (On Induction of Differentiation of Pluripotent Stem
Cells)
[0004] Regenerative medicine using cells obtained by inducing
differentiation of embryonic stem cells (ES cells) or induced
pluripotent stem cells (iPS cells) is a therapeutic method for
which the people have high expectations and which is desired to be
realized soon. As regenerative medicine, a transplantation therapy
with retinal pigment epithelial cells derived from iPS cells is
fresh in memory. However, a technology for generating mature
differentiated cells suited for cell transplantation rapidly and in
a sufficient amount is still under development and has huge room
for development.
[0005] A currently mainstream method of inducing differentiation of
pluripotent stem cells into a desired cell type is a method
involving sequentially adding cytokines/growth factors suited for
respective differentiation stages to a medium to cause
differentiation via an embryoid body and progenitor cells. This
method has problems in, for example, that a culture period until
differentiated cells of interest are obtained is long, that
differentiation induction efficiency is not high, and that cells of
different cell lineages are mixed with each other.
[0006] In recent years, attempts have been actively made to direct
cell differentiation by forcibly expressing, in ES/iPS cells, one
or a combination of a plurality of tissue-specifically expressed
transcription factors. This differentiation induction method using
transcription factors can directly induce ES/iPS cells into
differentiated cells of interest, and hence is expected to be very
effective means. However, even with this technique, cell
differentiation induction efficiency is low. Accordingly, under the
circumstances, it is difficult to obtain a sufficient amount of
differentiated cells of interest required for regenerative medicine
depending on the kind of cells.
[0007] In view of the foregoing, there has been a demand for
development of a novel differentiation induction method for
producing differentiated cells of interest from pluripotent stem
cells more rapidly and more uniformly with higher efficiency.
[0008] (Current Situation of Induction of Differentiation of
Pluripotent Stem Cells in Related Prior Art)
[0009] Non Patent Literatures 1 to 4, which are related art, are
each directed to a system for facilitating induction of
differentiation of ES/iPS cells. As an example, there is a
disclosure that ES/iPS cells are induced into skeletal muscle
differentiation.
CITATION LIST
Non Patent Literature
[0010] [NPL 1] Nature medicine 13: 642-648. [0011] [NPL 2] Cell
stem cell 10: 610-619. [0012] [NPL 3] Mol Ther. November; 20(11):
2153-67. [0013] [NPL 4] PLoS One. 2013 Apr. 23; 8(4): e61540.
SUMMARY OF INVENTION
Problem to be Solved by Invention
[0014] In related-art methods of differentiating pluripotent stem
cells into a desired cell type, there has not been established a
differentiation induction method using human ES/iPS cells and being
stable and highly efficient. Many attempts have been made,
including a stepwise differentiation induction method based on the
control of culture conditions or the addition of, for example,
various cell growth factors/differentiation factors to a culture
solution, but the use of complicated culture steps is a serious
problem. In addition, there are also serious problems in, for
example, that the speed of cell differentiation is low, and hence
long-period culture is required, and that the differentiation
efficiency is low, and hence it is difficult to obtain a sufficient
number of required cells.
Means for Solving Problem
[0015] The inventors of the present invention have considered that
the above-mentioned problems are partly due to the fact that
pluripotent stem cells have a property of resisting cell
differentiation by various mechanisms (stemness-maintaining
property). In view of this, the inventors have found a method of
inducing differentiation into a desired cell type within a short
period of time and with high efficiency by attenuating
differentiation resistance of a pluripotent stem cell to generate a
pluripotent stem cell that actively proceeds to a differentiated
cell type. Thus, the inventors have completed the present
invention.
[0016] That is, the present disclosure includes the following.
[0017] 1. A differentiation induction kit for differentiating a
pluripotent stem cell into a desired cell type, including at least
any one of the following items (1) to (5): [0018] (1) a pluripotent
stem cell having a histone in which H3K27me3 modification has been
substantially removed or reduced; [0019] (2) a pluripotent stem
cell in which a demethylase is forcibly expressed; [0020] (3) a
pluripotent stem cell and a demethylase gene; [0021] (4) a gene
construct carrying a demethylase gene and a pluripotent stem cell;
and [0022] (5) a pluripotent stem cell having a gene construct
carrying a demethylase gene inserted into a genome thereof. [0023]
2. A differentiation induction kit according to the above-mentioned
item 1, wherein the differentiation induction kit includes the
above-mentioned item (1), (2), or (5). [0024] 3. A differentiation
induction kit according to the above-mentioned item 1 or 2, wherein
the demethylase is JMJD3. [0025] 4. A differentiation induction kit
according to the above-mentioned item 1 or 2, wherein the
demethylase is a demethylase containing only an enzymatically
active region of JMJD3. [0026] 5. A differentiation induction kit
according to the above-mentioned item 3, wherein the demethylase
has an amino acid sequence set forth in any one of SEQ ID NOS: 1 to
3. [0027] 6. A differentiation induction kit according to any one
of the above-mentioned items 1 to 5, further including a
transcription factor required for induction of differentiation into
the desired cell type. [0028] 7. A differentiation induction kit
for differentiating a pluripotent stem cell into a skeletal muscle
cell, including at least any one of the following items (1) to (5):
[0029] (1) a pluripotent stem cell having a histone in which
H3K27me3 modification has been substantially removed or reduced,
and a transcription factor MYOD1; [0030] (2) a pluripotent stem
cell in which a demethylase is forcibly expressed, and a
transcription factor MYOD1; [0031] (3) a pluripotent stem cell, a
demethylase gene, and a transcription factor MYOD1; [0032] (4) a
gene construct carrying a demethylase gene, a pluripotent stem
cell, and a transcription factor MYOD1; and [0033] (5) a
pluripotent stem cell having a gene construct carrying a
demethylase gene inserted into a genome thereof, and a
transcription factor MYOD1. [0034] 8. A differentiation induction
kit for differentiating a pluripotent stem cell into a nerve cell,
including at least any one of the following items (1) to (5):
[0035] (1) a pluripotent stem cell having a histone in which
H3K27me3 modification has been substantially removed or reduced,
and transcription factors NEUROG1, NEUROG2, NEUROG3, NEUROD1, and
NEUROD2; [0036] (2) a pluripotent stem cell in which a demethylase
is forcibly expressed, and transcription factors NEUROG1, NEUROG2,
NEUROG3, NEUROD1, and NEUROD2; [0037] (3) a pluripotent stem cell,
a demethylase gene, and transcription factors NEUROG1, NEUROG2,
NEUROG3, NEUROD1, and NEUROD2; [0038] (4) a gene construct carrying
a demethylase gene, a pluripotent stem cell, and transcription
factors NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2; and [0039]
(5) a pluripotent stem cell having a gene construct carrying a
demethylase gene inserted into a genome thereof, and transcription
factors NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2. [0040] 9.
A differentiation induction kit for differentiating a pluripotent
stem cell into a liver cell, including at least any one of the
following items (1) to (5): [0041] (1) a pluripotent stem cell
having a histone in which H3K27me3 modification has been
substantially removed or reduced, and a transcription factor HNF1A;
[0042] (2) a pluripotent stem cell in which a demethylase is
forcibly expressed, and a transcription factor HNF1A; [0043] (3) a
pluripotent stem cell, a demethylase gene, and a transcription
factor HNF1A; [0044] (4) a gene construct carrying a demethylase
gene, a pluripotent stem cell, and a transcription factor HNF1A;
and [0045] (5) a pluripotent stem cell having a gene construct
carrying a demethylase gene inserted into a genome thereof, and a
transcription factor HNF1A. [0046] 10. A method of differentiating
a pluripotent stem cell into a desired cell type, including any one
of the following steps (1) to (7): [0047] (1) a step of adding a
demethylase gene and a transcription factor required for induction
of differentiation into the desired cell type to a pluripotent stem
cell; [0048] (2) a step of inserting a gene construct carrying a
demethylase gene and a transcription factor gene required for
induction of differentiation into the desired cell type into a
genome of a pluripotent stem cell; [0049] (3) a step of inserting a
gene construct carrying a demethylase gene into a genome of a
pluripotent stem cell, followed by addition of a transcription
factor required for induction of differentiation into the desired
cell type to the cell; [0050] (4) a step of inserting a gene
construct carrying a demethylase gene and a gene construct carrying
a transcription factor required for induction of differentiation
into the desired cell type into a genome of a pluripotent stem
cell; [0051] (5) a step of adding a transcription factor required
for induction of differentiation into the desired cell type to a
pluripotent stem cell having a histone in which H3K27me3
modification has been substantially removed or reduced; [0052] (6)
a step of adding a transcription factor required for induction of
differentiation into the desired cell type to a pluripotent stem
cell in which a demethylase is forcibly expressed; and [0053] (7) a
step of adding a demethylase and a transcription factor required
for differentiation into the desired cell type to a pluripotent
stem cell. [0054] 11. A method of differentiating a pluripotent
stem cell according to the above-mentioned item 10, wherein the
differentiation induction kit includes the above-mentioned step
(1), (3), (6), or (7). [0055] 12. A method of differentiating a
pluripotent stem cell according to the above-mentioned item 10 or
11, wherein the demethylase is JMJD3. [0056] 13. A method of
differentiating a pluripotent stem cell according to the
above-mentioned item 10 or 11, wherein the demethylase is a
demethylase containing only an enzymatically active region of
JMJD3. [0057] 14. A method of differentiating a pluripotent stem
cell into a skeletal muscle cell, including any one of the
following steps (1) to (7): [0058] (1) a step of adding a
demethylase gene and a transcription factor MYOD1 to a pluripotent
stem cell; [0059] (2) a step of inserting a gene construct carrying
a demethylase gene and a desired transcription factor MYOD1 gene
into a genome of a pluripotent stem cell; [0060] (3) a step of
inserting a gene construct carrying a demethylase gene into a
genome of a pluripotent stem cell, followed by addition of a
transcription factor MYOD1 to the cell; [0061] (4) a step of
inserting a gene construct carrying a demethylase gene and a gene
construct carrying a transcription factor MYOD1 into a genome of a
pluripotent stem cell; [0062] (5) a step of adding a transcription
factor MYOD1 to a pluripotent stem cell having a histone in which
H3K27me3 modification has been substantially removed or reduced;
[0063] (6) a step of adding a transcription factor MYOD1 to a
pluripotent stem cell in which a demethylase is forcibly expressed;
and (7) a step of adding a demethylase and a transcription factor
MYOD1 to a pluripotent stem cell. [0064] 15. A method of
differentiating a pluripotent stem cell into a nerve cell,
including any one of the following steps (1) to (7): [0065] (1) a
step of adding a demethylase gene and a transcription factor
NEUROG1, NEUROG2, NEUROG3, NEUROD1, and/or NEUROD2 to a pluripotent
stem cell; [0066] (2) a step of inserting a gene construct carrying
a demethylase gene and a desired transcription factor NEUROG1,
NEUROG2, NEUROG3, NEUROD1, and/or NEUROD2 gene into a genome of a
pluripotent stem cell; [0067] (3) a step of inserting a gene
construct carrying a demethylase gene into a genome of a
pluripotent stem cell, followed by addition of a transcription
factor NEUROG1, NEUROG2, NEUROG3, NEUROD1, and/or NEUROD2 to the
cell; [0068] (4) a step of inserting a gene construct carrying a
demethylase gene and a gene construct carrying a transcription
factor NEUROG1, NEUROG2, NEUROG3, NEUROD1, and/or NEUROD2 into a
genome of a pluripotent stem cell; [0069] (5) a step of adding a
transcription factor NEUROG1, NEUROG2, NEUROG3, NEUROD1, and/or
NEUROD2 to a pluripotent stem cell having a histone in which
H3K27me3 modification has been substantially removed or reduced;
[0070] (6) a step of adding a transcription factor NEUROG1,
NEUROG2, NEUROG3, NEUROD1, and/or NEUROD2 to a pluripotent stem
cell in which a demethylase is forcibly expressed; and [0071] (7) a
step of adding a demethylase and a transcription factor NEUROG1,
NEUROG2, NEUROG3, NEUROD1, and/or NEUROD2 to a pluripotent stem
cell. [0072] 16. A method of differentiating a pluripotent stem
cell into a liver cell, including any one of the following steps
(1) to (7): [0073] (1) a step of adding a demethylase gene and a
transcription factor HNF1A to a pluripotent stem cell; [0074] (2) a
step of inserting a gene construct carrying a demethylase gene and
a desired transcription factor HNF1A gene into a genome of a
pluripotent stem cell; [0075] (3) a step of inserting a gene
construct carrying a demethylase gene into a genome of a
pluripotent stem cell, followed by addition of a transcription
factor HNF1A to the cell; [0076] (4) a step of inserting a gene
construct carrying a demethylase gene and a gene construct carrying
a transcription factor HNF1A into a genome of a pluripotent stem
cell; [0077] (5) a step of adding a transcription factor HNF1A to a
pluripotent stem cell having a histone in which H3K27me3
modification has been substantially removed or reduced; [0078] (6)
a step of adding a transcription factor HNF1A to a pluripotent stem
cell in which a demethylase is forcibly expressed; and [0079] (7) a
step of adding a demethylase and a transcription factor HNF1A to a
pluripotent stem cell. [0080] 17. A method of differentiating a
pluripotent stem cell according to any one of the above-mentioned
items 10 to 13, wherein the transcription factor required for
induction of differentiation into the desired cell type is TCF-1,
and the desired cell type is a hepatoblast. [0081] 18. A method of
differentiating a pluripotent stem cell according to any one of the
above-mentioned items 10 to 13, wherein the transcription factor
required for induction of differentiation into the desired cell
type is SOX9, and the desired cell type is a chondrocyte. [0082]
19. A method of differentiating a pluripotent stem cell according
to any one of the above-mentioned items 10 to 13, wherein the
transcription factor required for induction of differentiation into
the desired cell type is RUNX3, and the desired cell type is an
osteoblast.
Advantageous Effects of Invention
[0083] The method of differentiating a pluripotent stem cell into a
desired cell type with high efficiency and differentiation
induction kit for differentiating a pluripotent stem cell into a
desired cell type with high efficiency of the present disclosure
have at least any one of the following effects.
(1) The period of time required for cell differentiation starting
with the pluripotent stem cell is shortened and/or the
differentiation induction efficiency is improved. (2) As modified
synthetic mRNA for a gene is used to introduce the gene into the
pluripotent stem cell, the introduced gene is not integrated into
the genome of the pluripotent stem cell, with the result that there
is no risk of canceration or the like after cell differentiation
induction. (3) In the introduction of the gene into the pluripotent
stem cell using the modified synthetic mRNA, the timing and number
of times of the addition of the mRNA for the gene can be easily
changed, and hence optimal conditions specific to each of various
desired cell types can be selected so as to differentiate the
pluripotent stem cell into the desired cell types.
BRIEF DESCRIPTION OF DRAWINGS
[0084] FIG. 1A: A schematic diagram of a method of attenuating
differentiation resistance of a pluripotent stem cell to a desired
cell type of the present disclosure. FIG. 1B: When H3K27me3 in a
human ES or iPS cell is attenuated or removed, a transcription
factor (TF) binds to the promoter site of a downstream gene to
enhance the expression of a group of
development/differentiation-related genes, resulting in
differentiation. FIG. 1C: A method of inducing differentiation of a
human ES cell or an iPS cell by introducing modified synthetic mRNA
for a demethylase, and then introducing modified synthetic mRNA for
the transcription factor (TF). FIG. 1D: A method of inducing
differentiation of a human ES cell or an iPS cell by simultaneously
introducing the modified synthetic mRNAs for the demethylase and
the transcription factor (TF).
[0085] FIG. 2: A schematic view of a differentiation induction
method using modified synthetic mRNA for a target gene.
[0086] FIG. 3: A schematic view of a differentiation step using
modified synthetic mRNA for a target gene.
[0087] FIG. 4: A method of introducing a target gene into the
genome of a pluripotent stem cell.
[0088] FIGS. 5A-5H: The generation of H3K27me3-attenuated hESCs by
JMJD3c expression. FIG. 5A: The structures of full-length JMJD3
(JMJD3f) and JMJD3c proteins. JMJD3c was designed to contain the
JmjC domain (amino acids 1376 to 1484) having demethylase activity.
FIG. 5B: hESCs were transfected with modified synthetic mRNA for
human influenza virus hemagglutinin (HA)-tagged full-length JMJD3
(HA-JMJD3f) or HA-tagged JMJD3c (HA-JMJD3c), and were stained with
an anti-HA antibody and an anti-H3K27me3 antibody. The arrowheads
indicate the transfected cells.
[0089] FIG. 5C: The effects of transfection of the HA-JMJD3f and
HA-JMJD3c mRNAs on H3K27me3 were analyzed by an immunoblotting
method. Modified synthetic mRNA for a green fluorescent protein
Emerald (Em) was transfected as a control. An anti-H3 antibody was
used as a loading control. FIG. 5D: A plasmid vector for tet-on
induction of JMJD3c in hESCs (JMJD3c-hESCs). FIG. 5E: JMJD3c-hESCs
were stained with
5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside (X-gal) 3
days after doxycycline (Dox) treatment. FIG. 5F: HA-JMJD3c-induced
H3K27me3 demethylation was detected 1 day to 3 days after DOX
treatment.
[0090] FIG. 5G: A point mutation in a JMJD3c mutant (mut) was
introduced at amino acid 1390 for demethylase inactivation. FIG.
5H: Confirmation of the influences of HA-JMJD3c and HA-JMJD3c mut
on H3K27me3.
[0091] FIGS. 6A-6C: Development/differentiation-related genes whose
gene expression are upregulated in JMJD3-hESCs. FIG. 6A:
Morphologies of JMJD3-hESCs without Dox treatment (-JMJD3c) and
with Dox treatment (+JMJD3c). FIG. 6B: Changes in H3K27me3 and
H3K4me3 after Dox treatment (Day 0 to Day 3) were analyzed by
ChIP-qPCR. POU5F1 and NANOG are stem cell genes, and T, MX1, SOX17,
FOXA2, GATA4, GATA6, GSC, and EVX1 are mesendodermal
differentiation-related genes. n=2 or 3. *P<0.05. The error bars
indicate the standard error of the mean (SEM). FIG. 6C: qRT-PCR
analyses for showing relative expression of stem cell genes and
mesendodermal differentiation-related genes under differentiation
conditions as compared with hESCs. Basal Medium represents a medium
without cytokines and growth factors, activin A represents a medium
for endodermal differentiation, activin A+BMP4+bFGF represents a
medium for mesodermal differentiation, and JMJD3c represents a
medium with Dox (forced expression of JMJD3c). The expression
levels were normalized to the expression amount of glyceraldehyde
3-phosphate dehydrogenase (GAPDH).
[0092] FIGS. 7A-7G: JMJD3c facilitates MYOD1-mediated muscle
differentiation of hESCs. FIG. 7A: A schematic of a differentiation
protocol. JMJD3c-hESCs were treated with or without Dox on from Day
1 to Day 2 after plating and were transfected with synthetic mRNA
for MYOD1 or Emerald three times on from Day 2 to Day 3. The cells
were collected on Day 5. FIG. 7B: RT-qPCR analyses of muscle
differentiation-related genes in MYOD1-differentiated cells with
Dox treatment (+JMJD3c) or without Dox treatment (-JMJD3c). -
represents no transfection, Em represents Emerald transfection, and
MYOD1 represents MYOD1 transfection. The expression levels were
normalized to GAPDH. n=3. The error bars indicate the SEM. FIG. 7C:
ChIP-qPCR analyses of H3K27me3, H3K4me3, and H3K27ac in the
promoter regions of MYOG and MEF2C genes of MYOD1-transfected cells
with Dox treatment (+JMJD3c) or without Dox treatment (-JMJD3c).
For the promoter regions of the MYOG and MEF2C genes, two regions
(FIGS. 7A to 7C) and three regions (FIG. 7A, 7B) were tested,
respectively. GAPDH, POU5F1, and T each represent a positive
control, and SOX1 represents a negative control. n=2 or 3.
P<0.05. The error bars indicate the SEM. FIG. 7D: Immunostaining
for myosin heavy chain isoform (MHC) in the cells in which JMJD3c,
MYOD1, or JMJD3c+MYOD1 are forcibly expressed. FIG. 7E: The
percentage of nuclei contained within MHC-stained cells. n=3.
*P<0.01. The error bars indicate SEM. FIG. 7F: Immunostaining
for MHC in the MYOD1-transfected cells in which JMJD3c or the
JMJD3c mutant is forcibly expressed. FIG. 7G: The percentage of
nuclei contained within MHC-stained cells. n=3. *P<0.01. The
error bars indicate the SEM. FIGS. 8A-8G: Differentiation of hESCs
and iPSCs into skeletal muscle cells by transfection of a
demethylase and a transcription factor as synthetic mRNAs. FIG. 8A:
A schematic of a differentiation induction protocol. hESC/iPSCs
were transfected with synthetic mRNAs for JMJD3c or a red
fluorescent protein mCherry twice on Day 1 and Day 2 and MYOD1
three times on Day 2 and Day 3. The cells were fixed for
immunostaining on Day 5. FIG. 8B: Immunostaining for MHC in cells
that were transfected with MYOD1 after mCherry or JMJD3c
transfection. FIG. 8C: The percentage of nuclei contained within
MHC-stained cells. n=3. *P<0.01. The error bars indicate the
SEM. FIG. 8D: Representative staining images for showing muscular
fusion (arrowheads). FIG. 8E: Induced myogenic cells were labeled
with green fluorescence and cocultured with mouse C2C12 cells
having nuclei labeled with red fluorescence. On Day 3 and Day 5
after coculturing, cell fusions were detected (arrowheads). FIG.
8F: iPSCs were transfected with mCherry or JMJD3c, followed by
MYOD1, and were immunostained for MHC. FIG. 8G: The percentage of
nuclei contained within MHC-stained cells. n=3. *P<0.01. The
error bars indicate the SEM.
[0093] FIG. 9: Increases in expression of marker genes for
hepatoblasts (TCF-1), chondrocytes (SOX9), and osteoblasts (RUNX3)
through expression of respective transcription factors TCF-1, SOX9,
and RUNX3 in combination with JMJD3c. AFP is a marker gene for
hepatoblasts, COL2 is a marker gene for chondrocytes, and COL1A1 is
a marker gene for osteoblasts. The expression levels were
standardized to GAPDH. n=2. *P<0.05. The error bars indicate the
SEM.
DESCRIPTION OF EMBODIMENTS
[0094] A method of inducing differentiation of a pluripotent stem
cell into a desired cell type with high efficiency of the present
disclosure (hereinafter sometimes referred to as "method of the
present disclosure") is described below, though the method is not
particularly limited as long as the method can attenuate
differentiation resistance of a pluripotent stem cell to the
desired cell type.
[0095] (Pluripotent Stem Cell)
[0096] The pluripotent stem cell to be used in the method of the
present disclosure is not particularly limited, but is preferably
derived from a mammal, more preferably derived from a human. The
pluripotent stem cell is, for example, a human ES cell, a human iPS
cell, or any combination thereof, is not particularly limited, and
encompasses tissue stem cells derived from tissues and organs,
dermal fibroblasts, and all kinds of cells derived from tissues or
organs.
[0097] (Attenuating Differentiation Resistance of Pluripotent Stem
Cell to Desired Cell Type)
[0098] In pluripotent stem cells, a special chromatin structure
called a "bivalent domain" is formed in each promoter region of a
group of genes involved in differentiation, and under a
stemness-maintaining state, the group of genes involved in
development/differentiation are in a standby state so as not to be
easily expressed. In Examples of the present disclosure, it has
been confirmed that "when a methyl group modification of a histone
called H3K27me3 is removed or reduced in the "bivalent domain", the
expression of differentiation genes required for induction of
differentiation into the desired cell type is rapidly and
efficiently facilitated" (see FIGS. 1A-1D).
[0099] That is, the "attenuating differentiation resistance of a
pluripotent stem cell to a desired cell type" of the present
disclosure means that the H3K27me3 modification of the pluripotent
stem cell is substantially removed or reduced.
[0100] In addition, a state in which the H3K27me3 modification of
the pluripotent stem cell has been substantially removed or reduced
may be confirmed by a comparison to the degree of the H3K27me3
modification of a pluripotent stem cell that has not been subjected
to the removing or the reducing. For example, the state (degree) in
which the H3K27me3 modification of the pluripotent stem cell has
been substantially removed or reduced is from 95 to 1, from 90 to
2, from 85 to 3, from 80 to 4, from 75 to 5, from 70 to 6, from 65
to 7, from 60 to 8, from 50 to 10, from 40 to 20, about 30, or 50
or less, 40 or less, 30 or less, 20 or less, or 10 or less when
compared to the degree of the H3K27me3 modification of the
pluripotent stem cell that has not been removed or reduced, which
is defined as 100. The degree of the H3K27me3 modification of the
pluripotent stem cell may be easily measured by using a
commercially available anti-Histone H3K27me3 antibody, and the gene
expression amount of H3K27me3 may be measured by a method known per
se.
[0101] (Method of inducing Differentiation of Pluripotent Stem Cell
into Desired Cell Type with High Efficiency of the Present
Disclosure)
[0102] As described above, the method of the present disclosure is
not particularly limited as long as the method can attenuate
differentiation resistance of the pluripotent stem cell to the
desired cell type, and may be exemplified by the following.
[0103] (Use of Modified Synthetic mRNA for Target Gene)
[0104] The method of the present disclosure includes adding
(introducing, transfecting), to a pluripotent stem cell, a gene for
a compound having an action of substantially removing or reducing
H3K27me3 modification, and a gene for a transcription factor
required for induction of differentiation of the pluripotent stem
cell into the desired cell type.
[0105] The term "gene" as used herein encompasses not only double
strands, but also their respective constituent single strands, such
as plus strands (or sense strands) or complementary strands (or
antisense strands), linear nucleic acids, and circular nucleic
acids, and encompasses DNA, RNA, mRNA, cDNA, and the like, unless
otherwise stated.
[0106] In addition, the term "target gene" is meant to encompass
both or any one of the gene for the compound having an action of
substantially removing or reducing H3K27me3 modification and the
transcription factor required for induction of differentiation into
the desired cell type.
[0107] In a step of the method of the present disclosure, a method
known per se may be used without any particular limitation as a
method of adding (introducing, transfecting) the gene for the
compound having an action of substantially removing or reducing
H3K27me3 modification and/or the transcription factor required for
induction of differentiation into the desired cell type to the
pluripotent stem cell. There is preferably used a method of
inducing differentiation by efficiently introducing synthetic mRNA
for a transcription factor into human pluripotent stem cells
through use of a gene expression method involving using synthetic
mRNA developed by Warren, Rossi, et al. (reference: Cell Stem Cell
7: 618-630, 2010.), which is a footprint-free forced gene
expression method causing no gene integration into a host genome
(see FIG. 2).
[0108] The timing at which the gene for the compound having an
action of substantially removing or reducing H3K27me3 modification
and the transcription factor required for induction of
differentiation into the desired cell type are added to the
pluripotent stem cell is not particularly limited, but it is
preferred that the gene for the compound having an action of
substantially removing or reducing H3K27me3 modification be added
to the pluripotent stem cell before the addition of the
transcription factor required for differentiation induction.
[0109] Further, with regard to the addition timing of each gene
(mRNA), the addition may be performed, for example, one or more
times, preferably two to five times, two to four times, two or
three times, or two times every 12 hours to 64 hours, but the
addition timing is not particularly limited thereto.
[0110] A more specific method may be exemplified by the following.
(Synthesis of Modified mRNA encoding Amino Acid Sequence of
Transcription Factor)
[0111] Modified mRNA is synthesized with reference to a method
described in the literature "Warren et al., Cell Stem Cell, 2010
Nov. 5; 7 (5): 618-30." More detailed, mRNA is synthesized by in
vitro transcription using a mixture of dNTPs (dNTPs: 3-0-Me-m7G
(5')ppp(5')GARCA cap analog, 5-methylcytidine triphosphate, and
pseudouridine triphosphate) obtained by modifying template DNA
encoding the amino acid sequence of the transcription factor
required for induction of differentiation into the desired cell
type.
[0112] (Generation of Sendai Virus Vector encoding Amino Acid
Sequence of Transcription Factor)
[0113] In order to express a mammalian (in particular, human)
transcription factor, a Sendai virus vector capable of expressing a
human transcription factor is preferably used. In particular, a
mutant of a Sendai virus vector, such as an F protein-deficient
mutant, has no infectivity, and is easy to handle (see Inoue et
al., J Virol. 77: 23238-3246, 2003).
[0114] (Method of inducing Differentiation of Pluripotent Stem Cell
into Desired Cell Type with High Efficiency)
[0115] A single transcription factor or a cocktail of two or more
transcription factors required for induction of differentiation
into the desired cell type is prepared. The form of the
transcription factors is not particularly limited, and may be any
of synthetic mRNAs, a Sendai virus vector having incorporated
therein a transcription factor (or a plurality of transcription
factors), and nanoparticle capsules containing synthetic mRNAs.
[0116] A method of introducing the single transcription factor or
cocktail of two or more transcription factors described above into
cells is not particularly limited, and transfection with
Lipofectamine, viral infection, or the like is utilized. A
schematic view of the differentiation induction step of the method
of the present disclosure is illustrated in FIG. 3.
[0117] (Use of Expression Vector)
[0118] In a step of the method of the present disclosure, an
expression vector known per se having introduced therein the gene
for the compound having an action of substantially removing or
reducing H3K27me3 modification and/or the transcription factor
required for induction of differentiation into the desired cell
type may be used. Examples of the expression vector to be used in
the present disclosure may include, but not particularly limited
to, an animal cell expression plasmid vector and a Sendai virus
vector.
[0119] A method of introducing the synthetic mRNA and the
expression vector into the pluripotent stem cell is not
particularly limited, for examples thereof may include a
lipofection method, a liposome method, an electroporation method, a
calcium phosphate coprecipitation method, a diethylaminoethyl
(DEAE)-dextran method, a microinjection method, and a gene gun
method. A particularly preferred example is a lipofection
method.
[0120] Another method may involve using an expression vector for
the gene for the compound having an action of substantially
removing or reducing H3K27me3 modification, and using synthetic
mRNA for the transcription factor required for induction of
differentiation into the desired cell type, or may adopt the
opposite pattern.
[0121] (Compound having Action of substantially removing or
reducing H3K27Me3 Modification)
[0122] The compound having an action of substantially removing or
reducing H3K27me3 modification of the present disclosure is not
particularly limited, and is, for example, a demethylase (in
particular, a demethylase having an action of removing a methyl
group of H3K27me3), an antibody that specifically binds to
H3K27me3, an antibody for a Polycomb-group proteins (PcG proteins)
having an H3K27me3 modification action, small interfering RNA
(siRNA), or an inhibitor.
[0123] In addition, not only by using those compounds alone, but
also by using a plurality of kinds of compounds and/or a
low-molecular-weight compound in combination, it is possible to
efficiently "attenuate differentiation resistance of a pluripotent
stem cell to a desired cell type (substantially remove or reduce
H3K27me3 modification of a pluripotent stem cell)."
[0124] Examples of the low-molecular-weight compound may include,
but not particularly limited to, histone deaceylase (HDAC)
inhibitors, such as valproic acid.
[0125] Examples of the demethylase include AOF (LSD1), AOF1 (LSD2),
FBXL11 (JHDM1A), Fbxl10 (JHDM1B), FBXL19 (JHDM1C), KIAA1718
(JHDM1D), PHF2 (JHDM1E), PHF8 (JHDM1F), JMJD1A(JHDM2A),
JMJD1B(JHDM2B), JMJD1C (JHDM2C), JMJD2A (JHDM3A), JMJD2B (JHDM3B),
JMJD2C (JHDM3C), JMJD2D (JHDM3D), RBP2 (JARID1A), PLU1 (JARID1B),
SMCX (JARID1C), SMCY (JARID1D), Jumonji (JARID2), UTX(UTX),
UTY(UTY), JMJD3 (JMJD3), JMJD4 (JMJD4), JMJD5(JMJD5), JMJD6(JMJD6),
JMJD7(JMJD7), and JMJD8(JMJD8). Of those, JMJD3 or the like is
preferred as a demethylase having an action of removing a methyl
group of H3K27me3.
[0126] In addition, the demethylase of the present disclosure may
also include the following: [0127] (1) a protected derivative,
sugar chain-modified product, acylated derivative, or acetylated
derivative of any one of the demethylases described above; [0128]
(2) an enzyme that has 90% (or 92%, 94%, 96%, 98%, or 99%) or more
homology to any one of the demethylases described above and has a
substantially equivalent action of substantially removing or
reducing H3K27me3 modification to that of the demethylase; and
[0129] (3) an enzyme that has 100 to 10, 50 to 30, 40 to 20, 10 to
5, or 5 to 1 amino acid substituted, deleted, inserted, and/or
added in any one of the demethylases described above and has a
substantially equivalent action of substantially removing or
reducing H3K27me3 modification to that of the demethylase.
[0130] Further, the gene of the demethylase of the present
disclosure includes the following: [0131] (1) a gene encoding a
polypeptide formed of the amino acid sequence of any one or more of
the enzymes described above; [0132] (2) a gene encoding a
polypeptide that has 1 to 20 (or 1 to 15, 1 to 10, 1 to 7, 1 to 5,
or 1 to 3) amino acids substituted, deleted, inserted, and/or added
in the amino acid sequence of any one or more of the enzymes
described above and has a substantially equivalent action of
substantially removing or reducing H3K27me3 modification to that of
the demethylase; and [0133] (3) a gene encoding a polypeptide that
has 90% (or 92%, 94%, 96%, 98%, or 99%) or more homology to the
amino acid sequence of any one or more of the enzymes described
above and has a substantially equivalent action of substantially
removing or reducing H3K27me3 modification to that of the
demethylase.
[0134] An enzyme having a mutation may be a naturally occurring
one, or may be one obtained by introducing a mutation on the basis
of a gene of natural origin. Means for introducing a mutation is
known per se, and for example, a site-directed mutagenesis method,
a homologous gene recombination method, a primer extension method,
a polymerase chain reaction (hereinafter abbreviated as PCR), and
the like may be used alone or in combination thereof as
appropriate.
[0135] The method may be performed in conformity with any of
methods described in the literatures ("Molecular Cloning: A
Laboratory Manual, second edition" edited by Sambrook et al., 1989,
Cold Spring Harbor Laboratory; and "Lab Manual: Genetic
Engineering" edited by Masami Muramatsu, 1988, Maruzen), or by
modifying these methods, and Ulmer's technology (Ulmer, K. M.,
"Science", 1983, volume 219, p. 666-671) may also be utilized. In
the case of a peptide, from the viewpoint of preventing alteration
of basic properties of the peptide (e.g., physical properties,
function, physiological activity, or immunological activity) in the
introduction of a mutation, for example, mutual substitution
between homologous amino acids (e.g., polar amino acids, non-polar
amino acids, hydrophobic amino acids, hydrophilic amino acids,
positively charged amino acids, negatively charged amino acids, and
aromatic amino acids) is easily conceivable.
[0136] (JMJD3)
[0137] JMJD3 is known as a demethylase for H3K27me3 of a histone
(mouse NP 001017426, human NP 001073893), and even in its full
length (NP 001073893, SEQ ID NO: 1), has an action of substantially
removing or reducing the H3K27me3 modification of pluripotent stem
cells. However, in Example 1 of the present disclosure, it has been
confirmed that JMJD3c having the JmjC domain {SEQ ID NO: 2,
catalytic domain: SEQ ID NO: 3 (amino acids 1376-1484)} has a
stronger action of substantially removing or reducing H3K27me3
modification as compared to full-length JMJD3 (see Example 2).
[0138] In addition, the JMJD3 of the present disclosure encompasses
the following embodiments as well: [0139] (1) a protected
derivative, sugar chain-modified product, acylated derivative, or
acetylated derivative of an amino acid sequence set forth in SEQ ID
NO: 1; [0140] (2) an amino acid sequence that has 90% (or 92%, 94%,
96%, 98%, or 99%) or more homology to the amino acid sequence set
forth in SEQ ID NO: 1 and has a substantially equivalent action of
substantially removing or reducing H3K27me3 modification to that of
the JMJD3; [0141] (3) an amino acid sequence that has 100 to 10, 50
to 30, 40 to 20, 10 to 5, or 5 to 1 amino acid substituted,
deleted, inserted, and/or added in the amino acid sequence set
forth in SEQ ID NO: 1 and has a substantially equivalent action of
substantially removing or reducing H3K27me3 modification to that of
the JMJD3; [0142] (4) a protected derivative, sugar chain-modified
product, acylated derivative, or acetylated derivative of an amino
acid sequence set forth in SEQ ID NO: 2; [0143] (5) an amino acid
sequence that has 90% (or 92%, 94%, 96%, 98%, or 99%) or more
homology to the amino acid sequence set forth in SEQ ID NO: 2 and
has a substantially equivalent action of substantially removing or
reducing H3K27me3 modification to that of the JMJD3c; [0144] (6) an
amino acid sequence that has 100 to 10, 50 to 30, 40 to 20, 10 to
5, or 5 to 1 amino acid substituted, deleted, inserted, and/or
added in the amino acid sequence set forth in SEQ ID NO: 2 and has
a substantially equivalent action of substantially removing or
reducing H3K27me3 modification to that of the JMJD3c; [0145] (7) a
protected derivative, sugar chain-modified product, acylated
derivative, or acetylated derivative of an amino acid sequence set
forth in SEQ ID NO: 3; [0146] (8) an amino acid sequence that has
90% (or 92%, 94%, 96%, 98%, or 99%) or more homology to the amino
acid sequence set forth in SEQ ID NO: 3 and has a substantially
equivalent action of substantially removing or reducing H3K27me3
modification to that of the JMJD3; [0147] (9) an amino acid
sequence that has 100 to 10, 50 to 30, 40 to 20, 10 to 5, or 5 to 1
amino acid substituted, deleted, inserted, and/or added in the
amino acid sequence set forth in SEQ ID NO: 3 and has a
substantially equivalent action of substantially removing or
reducing H3K27me3 modification to the JMJD3; and [0148] (10) an
amino acid sequence that includes the amino acid sequence set forth
in SEQ ID NO: 3 and has a substantially equivalent action of
substantially removing or reducing H3K27me3 modification to the
JMJD3c.
[0149] It is appropriate that the "sequence homology" be generally
70% or more, preferably 80%, more preferably 85% or more, still
more preferably 90% or more, even more preferably 95% or more, most
preferably 98% or more of an entire amino acid sequence.
[0150] Further, the JMJD3 gene of the present disclosure
encompasses the following: [0151] (1) a gene encoding a polypeptide
formed of an amino acid sequence set forth in any one of SEQ ID
NOS: 1 to 3; [0152] (2) a gene encoding a polypeptide that has 1 to
20 (or 1 to 15, 1 to 10, 1 to 7, 1 to 5, or 1 to 3) amino acids
substituted, deleted, inserted, and/or added in the amino acid
sequence set forth in any one of SEQ ID NOS: 1 to 3 and has a
substantially equivalent action of substantially removing or
reducing H3K27me3 modification to that of the amino acid sequence
set forth in any one of SEQ ID NOS: 1 to 3; [0153] (3) a gene
encoding a polypeptide that has 90% (or 92%, 94%, 96%, 98%, or 99%)
or more homology to the amino acid sequence set forth in any one of
SEQ ID NOS: 1 to 3 and has a substantially equivalent action of
substantially removing or reducing H3K27me3 modification to that of
the amino acid sequence set forth in any one of SEQ ID NOS: 1 to 3;
[0154] (4) a gene formed of a base sequence set forth in any one of
SEQ ID NOS: 4 to 6; [0155] (5) a gene encoding a polypeptide that
hybridizes with a base sequence complementary to the base sequence
set forth in any one of SEQ ID NOS: 4 to 6 under stringent
conditions and has a substantially equivalent action of
substantially removing or reducing H3K27me3 modification to that of
the amino acid sequence set forth in any one of SEQ ID NOS: 1 to 3;
[0156] (6) a gene that has a sequence of 1 to 50 (or 1 to 40, 1 to
30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 bases substituted,
deleted, inserted, and/or added in the gene (DNA) formed of the
base sequence set forth in any one of SEQ ID NOS: 4 to 6; and
[0157] (7) a gene having 90% (or 92%, 94%, 96%, 98%, or 99%) or
more homology to the gene formed of the base sequence set forth in
any one of SEQ ID NOS: 4 to 6.
[0158] (Transcription Factor required for Highly Efficient
Induction of Differentiation into Desired Cell Type)
[0159] The form of the "transcription factor required for highly
efficient induction of differentiation into the desired cell type"
to be used in the method of the present disclosure is not
particularly limited, for examples thereof may include, but not
particularly limited to, nucleic acids, such as RNA and DNA,
synthetic nucleic acids, and proteins. The following examples may
be given.
[0160] In addition, in the method of the present disclosure,
examples of the desired cell type may include a skeletal muscle
(skeletal muscle cells), the liver (liver cells), and nerve (nerve
cells).
[0161] {Transcription Factor required for Induction of
Differentiation into Skeletal Muscle (in particular, Cells present
in Skeletal Muscle)}
[0162] A method of inducing differentiation into a skeletal muscle
is as described below.
[0163] A single transcription factor, or two or more transcription
factors selected from the group consisting of MYOD1, NRF1, SALL4,
ZIC1, KLF9, ZNF281, CTCF, HES1, HOXA2, TBX5, TP73, ERG, MAB21L3,
PRDM1, NFIC, CTCFL, FOXP1, HEY1, PITX2, JUNB, KLF4, ESX1, TFAP2C,
FOS, TFE3, FOSL1, GRHL2, TBX2, NFIB, and IRF4 are introduced into a
pluripotent stem cell having a histone in which H3K27me3
modification has been substantially removed or reduced.
[0164] In particular, the JMJD3c gene (SEQ ID NO: 80) and MYOD1
(myogenic differentiation 1: SEQ ID NO: 86, SEQ ID NO: 88) are
added to pluripotent stem cells known per se.
[0165] {Transcription Factor required for Induction of
Differentiation into Liver (in particular, Cells present in Liver,
i.e., Liver Cells or Hepatoblasts)}
[0166] A method of inducing differentiation into the liver (in
particular, the liver, liver cells, or the fetal liver) is as
described below.
[0167] Liver: A single transcription factor, or two or more
transcription factors selected from TCF-1, SALL4, TGIF1, MAB21L3,
ZIC1, EGFLAM, PITX2, HNF4A, NRF1, ZNF281, CTCFL, TP73, TFE3, DLX6,
and TCF4 are introduced into human pluripotent stem cells.
[0168] Fetal liver: A single transcription factor, or two or more
transcription factors selected from TCF-1, SIX5, HNF4A, SIN3A, ID1,
and HNF1A are introduced into human pluripotent stem cells.
[0169] In particular, the JMJD3c gene (SEQ ID NO: 80) and HNF1A
(hepatocyte nuclear factor 1, alpha: SEQ ID NO: 87, SEQ ID NO: 94)
are added to pluripotent stem cells known per se.
[0170] {Transcription Factor required for Induction of
Differentiation into Neural Cells (in particular, Motoneurons or
Peripheral Motoneuron Cells)}
[0171] A method of inducing differentiation into neural cells (in
particular, motoneurons or peripheral motoneuron cells) is as
described below.
[0172] A single transcription factor, or two or more, three or
more, or four or more transcription factors selected from NEUROG1
(neurogenin 1: SEQ ID NO: 81), NEUROG2 (neurogenin 2: SEQ ID NO:
82), NEUROG3 (neurogenin 3: SEQ ID NO: 83), NEUROD1 (neurogenic
differentiation 1: SEQ ID NO: 84), and NEUROD2 (neurogenic
differentiation 2: SEQ ID NO: 85) or all of these transcription
factors are introduced into human pluripotent stem cells.
[0173] In particular, the JMJD3c gene (SEQ ID NO: 80), and NEUROG1
(SEQ ID NO: 81, SEQ ID NO: 89), NEUROG2 (SEQ ID NO: 82, SEQ ID NO:
90), NEUROG3 (SEQ ID NO: 83, SEQ ID NO: 91), NEUROD1 (SEQ ID NO:
84, SEQ ID NO: 92), and NEUROD2 (SEQ ID NO: 85, SEQ ID NO: 93) are
added to pluripotent stem cells known per se.
[0174] (Method of introducing Target Gene into Genome of
Pluripotent Stem Cell)
[0175] In a step of the method of the present disclosure, a method
known per se may be used without any particular limitation as a
method of introducing the gene for the compound having an action of
substantially removing or reducing H3K27me3 modification and/or the
transcription factor required for highly efficient induction of
differentiation into the desired cell type into the genome of the
pluripotent stem cell. There may be preferably used an expression
cassette inserted between PiggyBac transposase recognition
sequences (PB sequences) developed by Woltjen et al. (reference:
Nature 458: 766-770, 2009.), which is a mechanism by which a gene
to be introduced is actively incorporated into pluripotent stem
cells (in particular, the genome of human ES cells). The expression
cassette is a system capable of efficiently establishing a
genetically modified pluripotent stem cell line by introducing a
drug selection cassette (see FIG. 4).
[0176] (Method of introducing Target Protein into Pluripotent Stem
Cell)
[0177] In a step of the method of the present disclosure, a method
known per se may be used as a method of introducing the compound
(in particular, protein) having an action of substantially removing
or reducing H3K27me3 modification and/or the transcription factor
(protein) required for highly efficient induction of
differentiation into the desired cell type into the genome of the
pluripotent stem cell, and examples thereof may include: a method
involving using a protein transfection reagent; a method involving
using a fusion protein having added thereto a cell-penetrating
peptide; and a microinjection method.
[0178] The "cell membrane permeable peptide" of the present
disclosure is a peptide having a property of migrating into a cell,
more specifically a property of permeating a cell membrane, still
more specifically a property of permeating a cell membrane or a
nuclear membrane to permeate into cytoplasm or a nucleus. The amino
acid sequence of the peptide is not particularly limited, but
examples thereof may include TAT (GRKKRRQRRRPQ: SEQ ID NO: 7), r8
{rrrrrrrr (D-form-R): SEQ ID NO: 8}, and MPG-8
((3AFLGWLGAWGTMGWSPKKKRK: SEQ ID NO: 9).
[0179] The target protein encompasses both of the compound (in
particular, protein) having an action of substantially removing or
reducing H3K27me3 modification and/or the transcription factor
(protein) required for highly efficient induction of
differentiation into the desired cell type.
[0180] (Differentiation Induction Kit for inducing Differentiation
of Pluripotent Stem Cell into Desired Cell Type with High
Efficiency)
[0181] A differentiation induction kit for inducing differentiation
of a pluripotent stem cell into a desired cell type with high
efficiency of the present disclosure (hereinafter sometimes
referred to as "kit of the present disclosure") includes any one or
more of the following embodiments.
[0182] (1) Pluripotent Stem Cell in which H3K27me3 Modification has
been substantially removed or reduced
[0183] A pluripotent stem cell in which H3K27me3 modification has
been substantially removed or reduced can be easily generated by
the method of the present disclosure described above.
[0184] A practitioner of the present disclosure can easily induce
differentiation into the desired cell type by introducing the
transcription factor required for induction of differentiation into
the desired cell type as described above into the pluripotent stem
cell in which H3K27me3 modification has been substantially removed
or reduced.
[0185] In addition, the pluripotent stem cell in which H3K27me3
modification has been substantially removed or reduced encompasses
a pluripotent stem cell having a gene construct inducible with
doxycycline or the like inserted into the genome thereof so that a
demethylase can be transiently forcibly expressed therein.
[0186] (2) Demethylase Gene for Kit of the Present Disclosure
[0187] A practitioner of the present disclosure can easily generate
the pluripotent stem cell in which H3K27me3 modification has been
substantially removed or reduced by adding a demethylase gene for a
kit to a pluripotent stem cell known per se.
[0188] Examples of the demethylase gene for a kit may include, but
not particularly limited to, mRNAs, DNAs, and proteins of
demethylase genes (e.g., JMJD3c).
[0189] (3) Demethylase Gene for Kit and Gene containing
Transcription Factor required for Induction of Differentiation into
Desired Cell Type of the Present Disclosure.
[0190] A practitioner of the present disclosure can easily generate
the pluripotent stem cell in which H3K27me3 modification has been
substantially removed or reduced, and induce differentiation into
the desired cell type with high efficiency by adding the
demethylase gene for a kit and a gene containing the transcription
factor required for induction of differentiation into the desired
cell type to a pluripotent stem cell known per se.
[0191] The two genes may be present on one gene, or on separate
genes. When the two genes are present on separate genes, the
demethylase gene and the transcription factor required for
induction of differentiation into the desired cell type may be
added to the pluripotent stem cell simultaneously or at separate
times.
[0192] (4) Demethylase for Kit of the Present Disclosure
[0193] A practitioner of the present disclosure can easily generate
the pluripotent stem cell in which H3K27me3 modification has been
substantially removed or reduced by adding a demethylase for a kit
to a pluripotent stem cell known per se.
[0194] (5) Gene Construct carrying Demethylase Gene of the Present
Disclosure
[0195] A practitioner of the present disclosure can easily generate
the pluripotent stem cell in which H3K27me3 modification has been
substantially removed or reduced by introducing a gene construct
carrying a demethylase gene into the genome of a pluripotent stem
cell known per se.
[0196] The gene construct may contain a promoter sequence, a gene
expression-enhancing sequence, a marker gene, a reporter sequence,
a drug resistance gene, and the like as required in addition to the
demethylase gene.
[0197] (6) Gene Construct carrying Demethylase Gene and
Transcription Factor required for Induction of Differentiation into
Desired Cell Type of the Present Disclosure
[0198] A practitioner of the present disclosure can easily generate
the pluripotent stem cell in which H3K27me3 modification has been
substantially removed or reduced, and induce differentiation into
the desired cell type by introducing a gene construct carrying a
demethylase gene and a transcription factor required for induction
of differentiation into the desired cell type into the genome of a
pluripotent stem cell known per se.
[0199] The two genes may be present on one gene, or on separate
genes. When the two genes are present on separate genes, the
demethylase gene and the transcription factor required for
induction of differentiation into the desired cell type may be
introduced into the genome of the pluripotent stem cell
simultaneously or at separate times.
[0200] The gene construct may contain a promoter sequence, a gene
expression-enhancing sequence, a marker gene, a reporter sequence,
a drug resistance gene, and the like as required in addition to the
demethylase gene and the transcription factor required for
induction of differentiation into the desired cell type.
[0201] A method of differentiating a pluripotent stem cell into a
desired cell type of the present disclosure may be exemplified by,
but not particularly limited to, a method including any one of the
following steps (1) to (7): [0202] (1) a step of adding a
demethylase gene and a transcription factor required for induction
of differentiation into the desired cell type to a pluripotent stem
cell; [0203] (2) a step of inserting a gene construct carrying a
demethylase gene and a transcription factor gene required for
induction of differentiation into the desired cell type into a
genome of a pluripotent stem cell; [0204] (3) a step of inserting a
gene construct carrying a demethylase gene into a genome of a
pluripotent stem cell, followed by addition of a transcription
factor required for induction of differentiation into the desired
cell type to the cell; [0205] (4) a step of inserting a gene
construct carrying a demethylase gene and a gene construct carrying
a transcription factor required for induction of differentiation
into the desired cell type into a genome of a pluripotent stem
cell; [0206] (5) a step of adding a transcription factor required
for induction of differentiation into the desired cell type to a
pluripotent stem cell having a histone in which H3K27me3
modification has been substantially removed or reduced; [0207] (6)
a step of adding a transcription factor required for induction of
differentiation into the desired cell type to a pluripotent stem
cell in which a demethylase is forcibly expressed; and [0208] (7) a
step of adding a demethylase and a transcription factor required
for differentiation into the desired cell type to a pluripotent
stem cell.
[0209] The present disclosure also encompasses any one of the
following pluripotent stem cells for differentiation into a desired
cell type: [0210] (1) a pluripotent stem cell for differentiation
into a desired cell type, which has a histone in which H3K27me3
modification has been substantially removed or reduced; [0211] (2)
a pluripotent stem cell for differentiation into a desired cell
type, in which a demethylase is forcibly expressed; and [0212] (3)
a pluripotent stem cell for differentiation into a desired cell
type, which has a gene construct carrying a demethylase gene
inserted into the genome thereof.
[0213] The present disclosure also encompasses a use of any one of
the following pluripotent stem cells for differentiation into a
desired cell type: [0214] (1) a pluripotent stem cell for
differentiation into a desired cell type, which has a histone in
which H3K27me3 modification has been substantially removed or
reduced; [0215] (2) a pluripotent stem cell for differentiation
into a desired cell type, in which a demethylase is forcibly
expressed; and [0216] (3) a pluripotent stem cell for
differentiation into a desired cell type, which has a gene
construct carrying a demethylase gene inserted into the genome
thereof.
[0217] The present disclosure also encompasses a use of any one of
the following pluripotent stem cells for differentiation into a
desired cell type, in production of a differentiation induction kit
for differentiating a pluripotent stem cell into a desired cell
type: [0218] (1) a pluripotent stem cell for differentiation into a
desired cell type, which has a histone in which H3K27me3
modification has been substantially removed or reduced; [0219] (2)
a pluripotent stem cell for differentiation into a desired cell
type, in which a demethylase is forcibly expressed; and [0220] (3)
a pluripotent stem cell for differentiation into a desired cell
type, which has a gene construct carrying a demethylase gene
inserted into the genome thereof.
[0221] The present disclosure is specifically described below by
way of Examples. However, the present disclosure is not limited
thereto. All of these Examples were carried out after being
approved by the Ethics Committee of Keio University School of
Medicine.
Example 1
[0222] (Materials and Methods)
[0223] Examples 2 to 7 were carried out using materials and methods
described below. The details are as described below.
[0224] (Human Pluripotent Stem Cell Culture and Differentiation
Induction Methods)
[0225] A human ES cell (hESC) lineage SEES-3 was obtained from the
National Center for Child Health and Development, Japan (National
Research Institute for Child Health and Development). Human induced
pluripotent stem cells (hiPSCs) were generated from adult human
fibroblasts by introducing mRNAs for POU5F1, SOX2, KLF4, and c-MYC.
hESC/iPSCs were maintained under feeder cell-free conditions using
StemFitAK-03medium (Ajinomoto) on iMatrix-511 (Nippi)-coated
plates. A ROCK inhibitor Y-27632 was added to the medium during
cell subculture in order to prevent detachment-induced
apoptosis.
[0226] For early differentiation, the hESCs were cultured in a
differentiation medium of RPMI 1640 (Gibco) supplemented with
growth factors (100 ng/ml activin A for endodermal differentiation
and 100 ng/ml activin A on Day 1, which was replaced with 10 ng/ml
BMP4 and ng/ml bFGF for mesodermal differentiation). For myogenic
differentiation, the hPSCs were cultured in a medium of a MEM
(Gibco) supplemented with 5% KSR, 1 mM sodium pyruvate, 0.1 mM
non-essential amino acids, 2 mM glutamine, 0.1 mM
.beta.-mercaptoethanol, and penicillin/streptomycin (50 U/50
.mu.g/ml) on iMatrix-511 or Matrigel (BD)-coated plates.
[0227] (Generation of JMJD3c-hESCs)
[0228] A full-length human JMJD3 clone was obtained from Addgene
(plasmid ID #24167). A point mutation in the catalytic domain was
introduced using PrimeSTAR Mutagenesis Basal Kit (Takara).
HA-tagged JMJD3c and a mutant thereof were subcloned into a
PiggyBac construct containing a tetracycline-responsive element
IRES-.beta.geo, and a puromycin resistance gene controlled by a PGK
promoter. Vectors were simultaneously introduced with PiggyBac
transposase vectors into hESCs that consistently expressed a
reverse tetracycline transactivator (SEE3-1v) using a GeneJuice
transfection reagent (Novagen). Stable clones were established by
puromycin selection. Inducible expression with doxycycline
treatment was confirmed by X-Gal staining.
[0229] (Modified mRNA Synthesis and Transfection)
[0230] The protein-coding regions (Open Reading Frames, ORFs) of a
red fluorescent protein mCherry, a green fluorescent protein
Emerald and human influenza virus hemagglutinin (Hemagglutinin,
HA)-tagged full-length or catalytic domains of JMJD3, and UTX were
subcloned into a pCRII construct containing the 5' UTR and 3' UTR
of mouse a-globin, which increased mRNA stability and translation
efficiency, to prepare templates used to synthesize mRNAs.
[0231] Modified mRNAs were synthesized on the basis of the
description of the literature "Cell stem cell 7, 618-630 (2010)".
Briefly speaking, a T7 promoter and a poly (A) tail were added
through PCR reaction using a KAPA taq kit (Kapa Biosystems). RNAs
were synthesized from PCR products using a MEGAscript T7 kit
(Ambion) together with ARCA cap analog (New England Biolabs), ATP,
GTP, 5-Methyl-CTP (TriLink), and pseudo-UTP (TriLink). The
synthetic mRNAs were purified using a MEGAclear kit (Ambion). RNA
transfections were performed with Lipofectamine 2000 (Invitrogen)
or Lipofectamine Messenger Max (Invitrogen), according to the
instructions of the accompanying manual. The B18R interferon
inhibitor (eBioscience) was added to the culture medium to increase
the viability of the transfected cells. The medium was replaced 2
hours to 3 hours after each transfection.
[0232] (Antibody)
[0233] The following antibodies were used:
[0234] HA (Abcam #ab9110 for immunoblotting method and #ab18181 for
immunostaining);
[0235] H3K4me3 (Millipore #07-473);
[0236] H3K27me3 (Millipore #07-449);
[0237] H3K27ac (Active Motif #39-133);
[0238] panH3 (Abcam #ab1791); and
[0239] MHC (R&D #MAB4470).
[0240] (Immunostaining)
[0241] The cells were fixed in 4% PFA for 10 minutes at room
temperature and permeabilized in 0.5% Triton-X-containing PBS for
10 minutes. The cells were blocked in 2% BSA-containing PBS for 10
minutes, and cultured with primary antibodies in a blocking
solution (1:500) for from 2 hours to 3 hours at room temperature or
overnight at 4.degree. C. The cells were washed twice in PBS, and
then cultured with Alexa dye-conjugated secondary antibodies
(Invitrogen) in a blocking solution (1:500) for 1 hour at room
temperature. Nuclei were counterstained with DAPI (Dako) for 5
minutes at room temperature. Immunofluorescence was visualized with
an inverted fluorescence microscope IX73 (Olympus). Images were
obtained using Olympus cellSens imaging software.
[0242] (Immunoblotting Method)
[0243] The cells were lysed with a sample buffer (50 mM Tris-HCl,
pH 6.8, 2% SDS, 6% 2-mercaptoethanol, and 500 mg/ml urea). The
proteins were separated by SDS-PAGE using a 4-15% polyacrylamide
gel (Biorad) and were electrically transferred to polyvinylidene
fluoride membranes (Biorad). The membranes were blocked for 1 hour
in 0.1% Tween-20-containing Tris-buffered saline (TBST) and 5%
skimmed milk. The membranes were washed in TBST and then incubated
with primary antibodies in 2% BSA-containing TBS (1:1,000 dilution)
overnight at 4.degree. C. The membranes were washed and then
incubated with horseradish peroxidase-conjugated secondary
antibodies (GE) for 1 hour at room temperature. The membranes were
washed in TEST, and immunoreactivity was visualized using ECL Prime
Detection Kit (GE) and detected using Luminescent Image Analyzer
(LAS-4000; Fujifilm).
[0244] (qRT-PCR)
[0245] Total RNA was isolated with TRIzol reagent (Invitrogen), and
cDNAs were generated with random hexamers using a Superscript III
First-strand Synthesis kit (Invitrogen). Real-time PCR was
performed using a SYBR Green PCR system (Takara). The primer
sequences used for RT-PCR are listed in Tables 1 and 2 below.
TABLE-US-00001 TABLE 1 qRT-PCR Forward GAPDH
GGTGGTCTCCTCTGACTTCAACA (SEQ ID NO: 10) POU5F1
CTTGAATCCCGAATGGAAAGGG (SEQ ID NO: 12) NANOG AGAAGGCCTCAGCACCTAC
(SEQ ID NO: 14) T GCCCTCTCCCTCCCCTCCACGCACAG (SEQ ID NO: 16) MSX1
CGAGAGGACCCCGTGGATGCAGAG (SEQ ID NO: 18) SOX17
CGCTTTCATGGTGTGGGCTAAGGACG (SEQ ID NO: 20) FOXA2
TGGGAGCGGTGAAGATGGAAGGGCAC (SEQ ID NO: 22) GATA4
GCTCCTTCAGGCAGTGAGAG (SEQ ID NO: 24) GATA6 GTGCCCAGACCACTTGCTAT
(SEQ ID NO: 26) GSC CGGTCCTCATCAGAGGAGTC (SEQ ID NO: 28) EVX1
CGGCTGGAGAAGGAATTCTA (SEQ ID NO: 30) MYOG GCCAGACTATCCCCTTCCTC (SEQ
ID NO: 32) MEF2C AGGTCACCTGACATCCCAAG (SEQ ID NO: 34) CKM
GAAGAGCATGACGGAGAAGG (SEQ ID NO: 36) SIX1 TGTTTGCGCATAAAGGAATG (SEQ
ID NO: 38) AFP TGGGACCCGAACTTTCCA (SEQ ID NO: 40) COL2
TTTCCCAGGTCAAGATGGTC (SEQ ID NO: 42) COL1A1 CCTGGATGCCATCAAAGTCT
(SEQ ID NO: 44) Reverse GAPDH GTGGTCGTTGAGGGCAATG (SEQ ID NO: 11)
POU5F1 GTGTATATCCCAGGGTGATCCTC (SEQ ID NO: 13) NANOG
GGCCTGATTGTTCCAGGATT (SEQ ID NO: 15) T CGGCGCCGTTGCTCACAGACCACAGG
(SEQ ID NO: 17) MSX1 GGCGGCCATCTTCAGCTTCTCCAG (SEQ ID NO: 19) SOX17
TAGTTGGGGTGGTCCTGCATGTGCTG (SEQ ID NO: 21) FOXA2
TCATGCCAGCGCCCACGTACGACGAC (SEQ ID NO: 23) GATA4
CTGTGCCCGTAGTGAGATGA (SEQ ID NO: 25) GATA6 TGGAGTCATGGGAATGGAAT
(SEQ ID NO: 27) GSC CCGAGTCCAAATCGCTTTTA (SEQ ID NO: 29) EVX1
ACACCTTGATGGTGGTTTCC (SEQ ID NO: 31) MYOG GAGGCCGCGTTATGATAAAA (SEQ
ID NO: 33) MEF2C GTTAGCCCTCCAACTCCACA (SEQ ID NO: 35) CKM
GTTGTCATTGTGCCAGATGC (SEQ ID NO: 37) SIX1 TGGGAAGGAAAATGCAAAAG (SEQ
ID NO: 39) AFP GGCCACATCCAGGACTAGTTTC (SEQ ID NO: 41) COL2
CTTCAGCACCTGTCTCACCA (SEQ ID NO: 43) COL1A1 TCTTGTCCTTGGGGTTCTTG
(SEQ ID NO: 45)
TABLE-US-00002 TABLE 2 ChIP-PCR Forward POU5F1 GGAGGTAAACCCAGCTCACA
(SEQ ID NO: 46) NANOG GCTCAGGGATGAGCATGATT (SEQ ID NO: 48) T
GGCACGGCCAAATAAGAATA (SEQ ID NO: 50) MSX1 TCCCTCATCTGATCCCAAAC (SEQ
ID NO: 52) SOX17 AGCAAGATGCTGGGTGAGTC (SEQ ID NO: 54) FOXA2
TTCTTCGCTCTCAGTGCTCA (SEQ ID NO: 56) GATA4 GATCTTCGCGACAGTTCCTC
(SEQ ID NO: 58) GATA6 TGCAGCCTACGCTCTTGTTA (SEQ ID NO: 60) GSC
GACATGACGGAGATGGGTCT (SEQ ID NO: 62) EVX1 TCACACTCTCCTCCCCAATC (SEQ
ID NO: 64) GAPDH CGGTGACTAACCCTGCGCTCCTG (SEQ ID NO: 66) MYOG_a
CCTCCGGAAAGAATGGGACT (SEQ ID NO: 68) MYOG_b TTGGAGCCAAGGTTACCAGT
(SEQ ID NO: 70) MYOG_c GGCCTCATTCACCTTCTTGA (SEQ ID NO: 72) MEF2C_a
CATGCATTTTCAGGTCACCA (SEQ ID NO: 74) MEF2C_b GCACGTTTAAGACCCCAAAG
(SEQ ID NO: 76) SOX1 CCGTCTCACTCCGTCTGAAT (SEQ ID NO: 78) Reverse
POU5F1 TTTGGCCTTAGGGTTAAGCA (SEQ ID NO: 47) NANOG
TGCCCAGTAACATCCACAAA (SEQ ID NO: 49) T GGTTCAATTCCTGGGTCGTA (SEQ ID
NO: 51) MSX1 ACCAGCTCCTACTGCGAGAA (SEQ ID NO: 53) SOX17
CTACACACCCCTGGTTTTGG (SEQ ID NO: 55) FOXA2 GGCGAGTTAAAGGTGTGTACG
(SEQ ID NO: 57) GATA4 CATGGCCAAGCTCTGATACA (SEQ ID NO: 59) GATA6
GTCAGTCAAGGCCATCCAC (SEQ ID NO: 61) GSC TGGAAGGTGCCTCACTTCTT (SEQ
ID NO: 63) EVX1 TTACAGTACCGCTGGTGACG (SEQ ID NO: 65) GAPDH
AGCTAGCCTCGCTCCACCTGACTT (SEQ ID NO: 67) MYOG_a
TCTGTTAGCTGCTCTGAGTCT (SEQ ID NO: 69) MYOG_b CTCTCACAGCGCCTCCTG
(SEQ ID NO: 71) MYOG_c TGGGCGTGTAAGGTGTGTAA (SEQ ID NO: 73) MEF2C_a
CCCCTCCACTTTGATTCGTA (SEQ ID NO: 75) MEF2C_b CGGCCTCAGCTAAATGAAAG
(SEQ ID NO: 77) SOX1 AGTGCAGGTCGGTCTCCAT (SEQ ID NO: 79)
[0246] {Chromatin Immunoprecipitation (ChIP) Analysis}
[0247] The cells were crosslinked with formaldehyde in PBS (final
concentration: 1%) at room temperature for 10 minutes. The reaction
was quenched with glycine (final concentration: 125 M). The cells
were washed with PBS, and stored at -80.degree. C. until use. The
cells were lysed in protease inhibitor cocktail-containing Lysis
buffer 3 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM
EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine).
Ultrasonication was performed using Handy Sonic UR-20P (Tomy Seiko
Co., Ltd.) so as to generate DNA fragments of from about 150 bp to
about 450 bp. The ultrasonicated lysate was diluted with protease
inhibitor cocktail-containing ChIP dilution buffer (20 mM Tris-HCl,
pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100), and then cultured
overnight at 4.degree. C. together with 30 .mu.l of protein G
magnetic beads (Invitrogen) precultured with 3 .mu.g of an
antibody. The precipitate was washed three times with RIPA buffer
(10 mM Tris-HCl, pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1%
Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate), and then washed
once with 10 mM Tris-HCl, pH 8.0, 5 mM EDTA, 10 mM NaCl. Bound
chromatin was eluted from the beads in elution buffer (20 mM
Tris-HCl, pH 7.5, 5 mM EDTA, 50 mM NaCl, 1% SDS) at 68.degree. C.,
and decrosslinked at 68.degree. C. for 6 hours. DNA was treated
with RNase A and protease K, and then purified by
phenol-chloroform-isoamyl alcohol and isopropanol precipitation.
Real-time PCR was performed using a SYBR Green PCR system (Takara).
The primer sequences are listed in Tables 1 and 2 above.
[0248] (Coculture of Myogenic Cells and C2C12 Cells)
[0249] Induced myogenic cells were labeled with green fluorescence
by introducing Emerald mRNA. The cells were cocultured with C2C12
cells expressing H2B-mCherry in a medium of DMEM (Gibco)
supplemented with 2% horse serum.
[0250] (Statistical Analysis)
[0251] The statistical significance of differences between samples
was assessed using Student's t-test for independent samples.
Example 2
[0252] (Generation of H3K27me3-deficient Pluripotent Stem Cells
(Pluripotent Stem Cells having Histone in which H3K27me3
Modification has been substantially removed or reduced))
[0253] In this Example, pluripotent stem cells in which H3K27me3
had been demethylated (H3K27me3-deficient hESCs) were generated.
Specifically, in order to demethylate the H3K27me3 of pluripotent
stem cells, two methods of manipulating the expression of the
demethylase JMJD3 were used. The details are as described
below.
[0254] (1) Use of Modified Synthetic mRNA
[0255] A forced expression system for JMJD3 was generated through
use of modified synthetic mRNAs. mRNAs encoding full-length JMJD3
(JMJD3f) and the catalytic domain-containing C-terminus (JMJD3c)
were synthesized in vitro (FIG. 5A).
[0256] The N-terminus of each of those mRNAs was tagged with a
hemagglutinin (HA) sequence for detecting a translated protein. At
8 hours after the introduction of the synthetic mRNAs into hESCs,
the demethylation of H3K27me3 was detected by immunostaining and
immunoblotting methods (FIG. 5B and FIG. 5C). The results showed
that "the introduction of the JMJD3c mRNA induced a more
significant decrease in H3K27me3 as compared to the JMJD3f mRNA,"
and the results showed that the catalytic domain of JMJD3 was able
to sufficiently demethylate a nucleosome histone.
[0257] (2) Use of Plasmid Vector having inserted therein
Demethylase Gene
[0258] A forced expression system for JMJD3c was generated through
use of a plasmid vector having introduced therein JMJD3c. More
specifically, a hESC line in which the expression of HA-JMJD3c was
regulated by doxycycline (Dox) treatment was generated
(JMJD3c-hESC) (FIG. 5D). Dox treatment (1 .mu.g/ml) induced
HA-JMJD3c expression and a significant decrease in H3K27me3 in all
hESCs (FIG. 5F). Forced expression of the JMJD3c mutant, which
lacked catalytic function (FIG. 5G), did not induce any change in
H3K27me3 (FIG. 5H). Thus, it was confirmed that JMJD3c removed or
attenuated H3K27me3 through its demethylase activity.
[0259] That is, it was confirmed that pluripotent stem cells in
which H3K27me3 modification had been substantially removed or
reduced were generated.
[0260] It was confirmed that the expression level of H3K27me3 of
pluripotent stem cells could be manipulated by each of the
above-mentioned two methods. In addition, in the use of the
modified synthetic mRNA, the timing and duration time of JMJD3c
expression can be regulated, and hence the decrease in the
expression level of H3K27me3 (or substantial removal of H3K27me3)
can be performed at specific timing at which differentiation of
pluripotent stem cells into a desired cell type is induced.
Example 3
[0261] (Confirmation of Changes in Developmental Genes in
H3K27me3-deficient Pluripotent Stem Cells)
[0262] It was revealed that forced expression of JMJD3c
(H3K27me3-deficient pluripotent stem cells) resulted in
morphological changes in hESCs toward differentiation (FIG. 6A). It
was confirmed that the morphological changes occurred even under
culture conditions for maintaining an undifferentiated state.
[0263] Chromatin immunoprecipitation (ChIP) analysis revealed that
decreases in H3K27me3 occurred in the promoters of genes whose gene
expression had been upregulated in Dox-treated JMJD3c-hESCs, but
those regions were still rich in H3K4me3 (FIG. 6B). This result
means that the chromatin structure is brought into an active
state.
[0264] In this Example, it was shown that, by demethylating
H3K27me3, JMJD3c expression was able to cause enhancement of the
expression of development/differentiation-related genes over cell
differentiation resistance (stem cell-maintaining property).
[0265] As described above, forced expression of JMJD3c upregulates
the expression of development/differentiation-related genes. In
particular, genes associated with endodermal and mesodermal
differentiation, such as SOX17, FOXA2, GATA4/6, EOMES, T, and
MIXL1, were highly expressed 3 days after the Dox treatment (FIG.
6C). Further, enhancement of the expression of those genes was
found also under undifferentiated state-maintaining culture
conditions. Typically, the differentiation of hESC/iPSCs into
mesoderm/endoderm requires changes into a differentiation medium
including various cytokines and growth factors (e.g., activin A,
BMP, and FGF). In order to evaluate the influence of JMJD3c on gene
expression enhancement for early differentiation, the expression
levels of development/differentiation-related genes under
JMJD3c-expressed conditions and conventional differentiation
conditions were compared to each other. It was confirmed by
real-time PCR analysis that JMJD3 upregulated the expression of
developmental genes in a non-differentiation medium to a degree
similar to that under differentiation conditions using cytokines
and growth factors (FIG. 6C).
[0266] Those results suggest that ectopic expression (forced
expression) of the demethylase allows a transition from a
pluripotency-maintaining state to an early differentiation state by
directly enhancing the expression of
development/differentiation-related genes, and this does not
require various cytokines and growth factors. That is, pluripotent
stem cells in which H3K27me3 modification has been substantially
removed or reduced easily undergo a transition from a pluripotent
state to an early differentiation state.
Example 4
[0267] (Confirmation of Differentiation of Pluripotent Stem Cells
in which Demethylase is forcibly expressed into Desired Cell
Type)
[0268] In the above-mentioned Examples, it was confirmed that H3K27
demethylation by JMJD3c changes the chromatin structure of hESCs to
an active form for highly efficient induction of differentiation
into a desired cell type. In view of this, it was considered that,
when a transcription factor required for induction of
differentiation into a desired cell type was introduced,
differentiation into the desired cell type was able to be induced
with high efficiency. Accordingly, in this Example, as an example
of induction of differentiation into a desired cell type, a
myogenic differentiation model using a myogenesis-regulating master
transcription factor MYOD1 was adopted. It is known that forced
expression of MYOD1 alone cannot cause sufficient epigenetic
changes and transcriptional changes in hESCs, resulting in poor
myogenic conversion (see Cell Reports 3, 661-670 (2013)).
[0269] In order to confirm whether JMJD3c was able to facilitate
MYOD1-induced muscle cell differentiation, JMJD3c was transiently
forcibly expressed in hESCs before forced expression of MYOD1 (FIG.
7A).
[0270] In this process, the JMJD3c-hESC line was used, and induced
by introducing JMJD3c and MYOD1 by means of Dox treatment and
synthetic mRNA, respectively.
[0271] Alterations in the expression of four genes (MYOG, MEF2C,
CKM, and SIX1) serving as markers for skeletal muscle
differentiation were examined. Real-time PCR analysis revealed that
forced expression of MYOD1 alone did not induce upregulation of the
expression of the muscle cell differentiation-related genes except
SIX1.
[0272] However, when JMJD3c was forcibly expressed before forced
expression of MYOD1, all of those genes showed significant
expression upregulation. However, forced expression of JMJD3c alone
did not alter the expression pattern of MYOD1 downstream genes.
Those results confirmed that JMJD3c facilitated muscle
differentiation mediated by MYOD1 gene expression.
[0273] Further, chromatin changes in the promoter regions of MYOG
and MEF2C during differentiation mediated by forced expression of
MYOD1 with or without forced expression of JMJD3c were examined
using ChIP assay. It was revealed that the levels of H3K4me3 and
H3K27me3 in those regions were lower than those of a positive
control, such as GAPDH, POU5F1, or Brachyury (T), in both the hESCs
and the differentiated cells (FIG. 7C), and there was no large
difference between a JMJD3c-positive condition and a negative
condition. Meanwhile, it was revealed that those regions were
significantly enriched for H3K27 acetylation (H3K27ac) in the
differentiated cells only under the JMJD3c-positive condition, but
not under the negative condition (FIG. 7C). H3K27ac has been known
to be directly involved in active transcription. Thus, it was
suggested that the combination of JMJD3c and MYOD1 formed an active
state of chromatin in myogenic genes.
[0274] Further, it was confirmed that JMJD3c/MYOD1-forcibly
expressing hESCs were myosin heavy chain (MHC)-positive, and
changed to myotube-like morphology at 4 days post differentiation
(FIG. 7D). The percentage of MHC-positive cells was much higher
than the percentage observed under the condition of overexpressing
MYOD1 alone (FIG. 7E). Those results show that JMJD3c facilitates
MYOD1-mediated differentiation of hESCs into skeletal muscle cells.
However, forced expression of the JMJD3c mutant did not induce
MYOD1-mediated myogenic differentiation (FIG. 7F and FIG. 7G).
Thus, it was confirmed that the demethylation of H3K27me3 was
essential to MYOD1-mediated differentiation of hESCs into muscle
cells.
[0275] As apparent from the foregoing, differentiation into a
desired cell type can be efficiently induced by introducing a
transcription factor required for induction of differentiation into
the desired cell type into H3K27me3-deficient cells (pluripotent
stem cells having a histone in which H3K27me3 modification has been
substantially removed or reduced).
Example 5
[0276] (Confirmation of Differentiation of Pluripotent Stem Cells
into Desired Cell Type using Synthetic mRNA)
[0277] In Example 4 described above, it was confirmed that forced
expression of the demethylase was able to facilitate MYOD1-mediated
differentiation of hESCs into skeletal muscle cells.
[0278] In this Example, it was confirmed whether differentiation of
hESCs into skeletal muscle cells was able to be induced by using
only synthetic mRNAs for the demethylase JMJD3c and the
transcription factor MYOD1 required for induction of
differentiation into a desired cell type without altering the DNA
of the pluripotent stem cells.
[0279] The mRNA for JMJD3c was transfected into hESCs twice,
followed by three transfections with the MYOD1 mRNA (FIG. 8A). Two
days after the last transfection of the MYOD1 mRNA, the majority of
hESCs were differentiated into MHC-positive cells (FIG. 8B and FIG.
8C). As a control, hESCs were transfected with mRNAs for mCherry
and MYOD1, but myogenic differentiation was not induced.
[0280] Some MHC-positive cells appeared to be fused cells (FIG.
8D), which was able to be further confirmed by a fusion assay with
mouse C2C12 cells (FIG. 8E). Those results were able to confirm
that the induced myotube-like cells became mature skeletal muscles
in vitro.
[0281] Further, it was confirmed that the mRNA for JMJD3c
facilitated MYOD1-mediated myogenic differentiation of
fibroblast-derived hiPSCs (FIG. 8F and FIG. 8G). This suggests that
JMJD3c facilitates direct conversion from a pluripotent state to a
terminal differentiation state.
[0282] As apparent from the foregoing, differentiation into a
desired cell type can be induced with high efficiency by
introducing (adding) a transcription factor required for induction
of differentiation into the desired cell type into pluripotent stem
cells in which H3K27me3 modification has been substantially removed
or reduced.
[0283] In related art, it is shown that skeletal muscle cells can
be induced even when MYOD1 is used alone. However, in Non Patent
Literature 4, drug selection needs to be performed in order to
stably express the MYOD1 gene, and preculture is required for about
10 days prior to the initiation of differentiation induction. In
addition, in Non Patent Literature 3, a PAX7 gene is introduced
instead of the MYOD1 gene, but differentiation induction requires
culture for about 1 month.
[0284] In addition, there is a report that skeletal muscle
differentiation is induced by introducing a gene called BAF60C, and
then introducing the MYOD1 gene (see Cell Rep. 2013 Mar. 28; 3 (3):
661-70.). However, differentiation induction takes 20 days, and
requires the use of a lentiviral vector.
Example 6
[0285] (Transcription factors differentiate Pluripotent Stem Cells
into Desired Cell Types)
[0286] In Examples 4 and 5 described above, it was confirmed that
MYOD1-mediated induction of differentiation of hESCs into skeletal
muscle cells was able to be facilitated by forcibly expressing the
demethylase or adding the synthetic mRNA for the demethylase.
[0287] In this Example, it was confirmed whether differentiation of
pluripotent stem cells into a plurality of desired cell types was
able to be induced using respective transcription factors.
[0288] With reference to the method described in Example 4,
JMJD3c-hESCs were treated with Dox (+JMJD3c) or without Dox
(-JMJD3c) on from Day 1 to Day 2 after plating, and then, during
Day 2, synthetic mRNA for TCF1, SOX9, RUNX3, or mCherry was
introduced twice. The cells were collected on Day 4, and the
expression of each differentiation marker gene was examined by
RT-qPCR analysis.
[0289] The analysis results are shown in FIG. 9. In the cells
transfected with the TCF1 transcription factor, AFP serving as a
marker gene for hepatoblasts was significantly increased. In the
cells transfected with the SOX9 transcription factor, COL2 serving
as a marker gene for chondrocytes was significantly increased. In
the cells transfected with the RUNX3 transcription factor, COL1A1
serving as a marker gene for osteoblasts was significantly
increased.
[0290] Thus, it was confirmed that differentiation into desired
cell types were able to be efficiently induced by introducing the
transcription factors required for induction of differentiation
into the desired cell types into H3K27me3-deficient cells
(pluripotent stem cells having a histone in which H3K27me3
modification has been substantially removed or reduced).
Example 7
[0291] (Examples of Differentiation into Desired Cell Types using
Pluripotent Stem Cells of the Present Disclosure)
[0292] In this Example, differentiation into various desired cell
types was confirmed using pluripotent stem cells having a histone
in which H3K27me3 modification had been substantially removed or
reduced.
[0293] (Differentiation into Skeletal Muscle Cells)
[0294] With reference to the description of Example 5, during 4-day
culture, human pluripotent stem cells were transfected with the
JMJD3c gene (SEQ ID NO: 80) twice, and then transfected with the
MYOD1 gene (SEQ ID NO: 86, SEQ ID NO: 88) three times. It was
confirmed that the cells were differentiated into skeletal muscle
cells through the 4-day culture.
[0295] (Differentiation into Liver Cells)
[0296] With reference to the description of Example 5, during 4-day
culture, human pluripotent stem cells were transfected with the
JMJD3c gene (SEQ ID NO: 80) twice, and then transfected with the
HNF1A gene (SEQ ID NO: 87, SEQ ID NO: 94) three times. It was
confirmed that the cells were differentiated into liver cells
through the 4-day culture.
[0297] (Differentiation into Nerve Cells)
[0298] With reference to the description of Example 5, during 4-day
culture, human pluripotent stem cells were transfected with the
JMJD3c gene (SEQ ID NO: 80) twice, and then transfected with the
NEUROG1 gene (SEQ ID NO: 81, SEQ ID NO: 89), the NEUROG2 gene (SEQ
ID NO: 82, SEQ ID NO: 90), the NEUROG3 gene (SEQ ID NO: 83, SEQ ID
NO: 91), the NEUROD1 gene (SEQ ID NO: 84, SEQ ID NO: 92), and the
NEUROD2 gene (SEQ ID NO: 85, SEQ ID NO: 93) three times. It was
confirmed that the cells were differentiated into nerve cells
through the 4-day culture.
[0299] (Subject Matter of the Present Invention)
[0300] It has been confirmed that, in the method of the present
disclosure, a differentiation efficiency of from 60% to 70% is
achieved in 4 days from the initiation of differentiation induction
without the addition of various cytokines and growth factors
required for causing a transition from a pluripotent state to an
early differentiation state and with only the addition of synthetic
mRNA to pluripotent stem cells. That is, in the method of the
present disclosure, differentiation induction can be achieved
within a shorter period and with higher efficiency without
requiring the various cytokines and growth factors that are
required in related-art methods.
[0301] In Examples of the present disclosure, even when there was
no environmental change, a histone demethylase {in particular, the
catalytic domain of JMJD3 (JMJD3c)} enhanced the expression of
development/differentiation-related genes in pluripotent stem
cells, and facilitated the conversion of a gene expression pattern
from a pluripotent stem cell pattern to a gene expression pattern
of a differentiated cell. This suggests that, without being limited
to JMJD3, any demethylase having an effect of removing or
attenuating methylation suppressing the expression of
development/differentiation-related genes can facilitate cell
differentiation of pluripotent stem cells into differentiated
cells.
[0302] In Examples of the present disclosure, it has been shown
that the histone demethylase JMJD3 cancels the suppression of the
expression of the differentiation-related genes by rapidly
attenuating the methylation of H3K27. Particularly when modified
synthetic mRNA for JMJD3c was used, significant attenuation of
H3K27me3 was confirmed in several hours. Those results show that
the histone demethylase antagonistically regulates H3K27
methylation by a PcG complex in human pluripotent stem cells.
[0303] In the pluripotent stem cells, forced expression of the
histone demethylase caused demethylation of H3K27me3, and
upregulated gene expression of many
development/differentiation-related genes. Those changes were also
found under human pluripotent stem cell culture conditions for
maintaining pluripotency. The mutant of the histone demethylase
(function-deficient mutant of JMJD3c) did not induce those
phenomena, revealing that specific demethylation of H3K27 by the
demethylase was directly involved in the increase of the
transcription activity of development/differentiation-related
genes.
[0304] In Examples of the present disclosure, it has also been
revealed that, in the group of development/differentiation-related
genes whose gene expression is upregulated by the demethylase
activity of JMJD3, more mesodermal/endodermal differentiation
related genes are included than genes involved in ectodermal
differentiation. This shows that the demethylase activity of the
JMJD3 gene effectively facilitates differentiation into
mesendodermal cells, specifically bone, muscle, liver, circulatory,
digestive, and reproductive cells. However, the demethylase
activity of JMJD3 gene also upregulated the expression of a group
of genes involved in ectodermal differentiation as compared to that
in pluripotent stem cells, and hence is likely involved also in
facilitating differentiation into, for example, nerve and epidermal
cells.
[0305] H3K27me3 is not present in large amounts in the promoter
regions of muscle cell differentiation-related genes in hESCs, and
hence the demethylase activity of JMJD3c is considered to be
indirectly involved in enhancement of the expression of the muscle
cell differentiation-related genes via enhancement of the
expression of a gene involved in early development/cell
differentiation.
[0306] Thus, it has been shown that the demethylase activity allows
the state of cells to undergo a transition from a
pluripotency-maintaining state to a differentiated state by
attenuating differentiation resistance of pluripotent stem cells.
The attenuation of the differentiation resistance is not limited to
only the activation of muscle differentiation-related genes, but
also facilitates the activation of other differentiated cell
genes.
INDUSTRIAL APPLICABILITY
[0307] According to the present disclosure, the novel method of
differentiating a pluripotent stem cell into a desired cell type
with high efficiency can be provided.
Sequence CWU 1
1
9411682PRTHomo sapiens 1Met His Arg Ala Val Asp Pro Pro Gly Ala Arg
Ala Ala Arg Glu Ala1 5 10 15Phe Ala Leu Gly Gly Leu Ser Cys Ala Gly
Ala Trp Ser Ser Cys Pro 20 25 30Pro His Pro Pro Pro Arg Ser Ala Trp
Leu Pro Gly Gly Arg Cys Ser 35 40 45Ala Ser Ile Gly Gln Pro Pro Leu
Pro Ala Pro Leu Pro Pro Ser His 50 55 60Gly Ser Ser Ser Gly His Pro
Ser Lys Pro Tyr Tyr Ala Pro Gly Ala65 70 75 80Pro Thr Pro Arg Pro
Leu His Gly Lys Leu Glu Ser Leu His Gly Cys 85 90 95Val Gln Ala Leu
Leu Arg Glu Pro Ala Gln Pro Gly Leu Trp Glu Gln 100 105 110Leu Gly
Gln Leu Tyr Glu Ser Glu His Asp Ser Glu Glu Ala Thr Arg 115 120
125Cys Tyr His Ser Ala Leu Arg Tyr Gly Gly Ser Phe Ala Glu Leu Gly
130 135 140Pro Arg Ile Gly Arg Leu Gln Gln Ala Gln Leu Trp Asn Phe
His Thr145 150 155 160Gly Ser Cys Gln His Arg Ala Lys Val Leu Pro
Pro Leu Glu Gln Val 165 170 175Trp Asn Leu Leu His Leu Glu His Lys
Arg Asn Tyr Gly Ala Lys Arg 180 185 190Gly Gly Pro Pro Val Lys Arg
Ala Ala Glu Pro Pro Val Val Gln Pro 195 200 205Val Pro Pro Ala Ala
Leu Ser Gly Pro Ser Gly Glu Glu Gly Leu Ser 210 215 220Pro Gly Gly
Lys Arg Arg Arg Gly Cys Asn Ser Glu Gln Thr Gly Leu225 230 235
240Pro Pro Gly Leu Pro Leu Pro Pro Pro Pro Leu Pro Pro Pro Pro Pro
245 250 255Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro Gly Leu Ala Thr
Ser Pro 260 265 270Pro Phe Gln Leu Thr Lys Pro Gly Leu Trp Ser Thr
Leu His Gly Asp 275 280 285Ala Trp Gly Pro Glu Arg Lys Gly Ser Ala
Pro Pro Glu Arg Gln Glu 290 295 300Gln Arg His Ser Leu Pro His Pro
Tyr Pro Tyr Pro Ala Pro Ala Tyr305 310 315 320Thr Ala His Pro Pro
Gly His Arg Leu Val Pro Ala Ala Pro Pro Gly 325 330 335Pro Gly Pro
Arg Pro Pro Gly Ala Glu Ser His Gly Cys Leu Pro Ala 340 345 350Thr
Arg Pro Pro Gly Ser Asp Leu Arg Glu Ser Arg Val Gln Arg Ser 355 360
365Arg Met Asp Ser Ser Val Ser Pro Ala Ala Thr Thr Ala Cys Val Pro
370 375 380Tyr Ala Pro Ser Arg Pro Pro Gly Leu Pro Gly Thr Thr Thr
Ser Ser385 390 395 400Ser Ser Ser Ser Ser Ser Asn Thr Gly Leu Arg
Gly Val Glu Pro Asn 405 410 415Pro Gly Ile Pro Gly Ala Asp His Tyr
Gln Thr Pro Ala Leu Glu Val 420 425 430Ser His His Gly Arg Leu Gly
Pro Ser Ala His Ser Ser Arg Lys Pro 435 440 445Phe Leu Gly Ala Pro
Ala Ala Thr Pro His Leu Ser Leu Pro Pro Gly 450 455 460Pro Ser Ser
Pro Pro Pro Pro Pro Cys Pro Arg Leu Leu Arg Pro Pro465 470 475
480Pro Pro Pro Ala Trp Leu Lys Gly Pro Ala Cys Arg Ala Ala Arg Glu
485 490 495Asp Gly Glu Ile Leu Glu Glu Leu Phe Phe Gly Thr Glu Gly
Pro Pro 500 505 510Arg Pro Ala Pro Pro Pro Leu Pro His Arg Glu Gly
Phe Leu Gly Pro 515 520 525Pro Ala Ser Arg Phe Ser Val Gly Thr Gln
Asp Ser His Thr Pro Pro 530 535 540Thr Pro Pro Thr Pro Thr Thr Ser
Ser Ser Asn Ser Asn Ser Gly Ser545 550 555 560His Ser Ser Ser Pro
Ala Gly Pro Val Ser Phe Pro Pro Pro Pro Tyr 565 570 575Leu Ala Arg
Ser Ile Asp Pro Leu Pro Arg Pro Pro Ser Pro Ala Gln 580 585 590Asn
Pro Gln Asp Pro Pro Leu Val Pro Leu Thr Leu Ala Leu Pro Pro 595 600
605Ala Pro Pro Ser Ser Cys His Gln Asn Thr Ser Gly Ser Phe Arg Arg
610 615 620Pro Glu Ser Pro Arg Pro Arg Val Ser Phe Pro Lys Thr Pro
Glu Val625 630 635 640Gly Pro Gly Pro Pro Pro Gly Pro Leu Ser Lys
Ala Pro Gln Pro Val 645 650 655Pro Pro Gly Val Gly Glu Leu Pro Ala
Arg Gly Pro Arg Leu Phe Asp 660 665 670Phe Pro Pro Thr Pro Leu Glu
Asp Gln Phe Glu Glu Pro Ala Glu Phe 675 680 685Lys Ile Leu Pro Asp
Gly Leu Ala Asn Ile Met Lys Met Leu Asp Glu 690 695 700Ser Ile Arg
Lys Glu Glu Glu Gln Gln Gln His Glu Ala Gly Val Ala705 710 715
720Pro Gln Pro Pro Leu Lys Glu Pro Phe Ala Ser Leu Gln Ser Pro Phe
725 730 735Pro Thr Asp Thr Ala Pro Thr Thr Thr Ala Pro Ala Val Ala
Val Thr 740 745 750Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Ala Thr
Gln Glu Glu Glu 755 760 765Lys Lys Pro Pro Pro Ala Leu Pro Pro Pro
Pro Pro Leu Ala Lys Phe 770 775 780Pro Pro Pro Ser Gln Pro Gln Pro
Pro Pro Pro Pro Pro Pro Ser Pro785 790 795 800Ala Ser Leu Leu Lys
Ser Leu Ala Ser Val Leu Glu Gly Gln Lys Tyr 805 810 815Cys Tyr Arg
Gly Thr Gly Ala Ala Val Ser Thr Arg Pro Gly Pro Leu 820 825 830Pro
Thr Thr Gln Tyr Ser Pro Gly Pro Pro Ser Gly Ala Thr Ala Leu 835 840
845Pro Pro Thr Ser Ala Ala Pro Ser Ala Gln Gly Ser Pro Gln Pro Ser
850 855 860Ala Ser Ser Ser Ser Gln Phe Ser Thr Ser Gly Gly Pro Trp
Ala Arg865 870 875 880Glu Arg Arg Ala Gly Glu Glu Pro Val Pro Gly
Pro Met Thr Pro Thr 885 890 895Gln Pro Pro Pro Pro Leu Ser Leu Pro
Pro Ala Arg Ser Glu Ser Glu 900 905 910Val Leu Glu Glu Ile Ser Arg
Ala Cys Glu Thr Leu Val Glu Arg Val 915 920 925Gly Arg Ser Ala Thr
Asp Pro Ala Asp Pro Val Asp Thr Ala Glu Pro 930 935 940Ala Asp Ser
Gly Thr Glu Arg Leu Leu Pro Pro Ala Gln Ala Lys Glu945 950 955
960Glu Ala Gly Gly Val Ala Ala Val Ser Gly Ser Cys Lys Arg Arg Gln
965 970 975Lys Glu His Gln Lys Glu His Arg Arg His Arg Arg Ala Cys
Lys Asp 980 985 990Ser Val Gly Arg Arg Pro Arg Glu Gly Arg Ala Lys
Ala Lys Ala Lys 995 1000 1005Val Pro Lys Glu Lys Ser Arg Arg Val
Leu Gly Asn Leu Asp Leu 1010 1015 1020Gln Ser Glu Glu Ile Gln Gly
Arg Glu Lys Ser Arg Pro Asp Leu 1025 1030 1035Gly Gly Ala Ser Lys
Ala Lys Pro Pro Thr Ala Pro Ala Pro Pro 1040 1045 1050Ser Ala Pro
Ala Pro Ser Ala Gln Pro Thr Pro Pro Ser Ala Ser 1055 1060 1065Val
Pro Gly Lys Lys Ala Arg Glu Glu Ala Pro Gly Pro Pro Gly 1070 1075
1080Val Ser Arg Ala Asp Met Leu Lys Leu Arg Ser Leu Ser Glu Gly
1085 1090 1095Pro Pro Lys Glu Leu Lys Ile Arg Leu Ile Lys Val Glu
Ser Gly 1100 1105 1110Asp Lys Glu Thr Phe Ile Ala Ser Glu Val Glu
Glu Arg Arg Leu 1115 1120 1125Arg Met Ala Asp Leu Thr Ile Ser His
Cys Ala Ala Asp Val Val 1130 1135 1140Arg Ala Ser Arg Asn Ala Lys
Val Lys Gly Lys Phe Arg Glu Ser 1145 1150 1155Tyr Leu Ser Pro Ala
Gln Ser Val Lys Pro Lys Ile Asn Thr Glu 1160 1165 1170Glu Lys Leu
Pro Arg Glu Lys Leu Asn Pro Pro Thr Pro Ser Ile 1175 1180 1185Tyr
Leu Glu Ser Lys Arg Asp Ala Phe Ser Pro Val Leu Leu Gln 1190 1195
1200Phe Cys Thr Asp Pro Arg Asn Pro Ile Thr Val Ile Arg Gly Leu
1205 1210 1215Ala Gly Ser Leu Arg Leu Asn Leu Gly Leu Phe Ser Thr
Lys Thr 1220 1225 1230Leu Val Glu Ala Ser Gly Glu His Thr Val Glu
Val Arg Thr Gln 1235 1240 1245Val Gln Gln Pro Ser Asp Glu Asn Trp
Asp Leu Thr Gly Thr Arg 1250 1255 1260Gln Ile Trp Pro Cys Glu Ser
Ser Arg Ser His Thr Thr Ile Ala 1265 1270 1275Lys Tyr Ala Gln Tyr
Gln Ala Ser Ser Phe Gln Glu Ser Leu Gln 1280 1285 1290Glu Glu Lys
Glu Ser Glu Asp Glu Glu Ser Glu Glu Pro Asp Ser 1295 1300 1305Thr
Thr Gly Thr Pro Pro Ser Ser Ala Pro Asp Pro Lys Asn His 1310 1315
1320His Ile Ile Lys Phe Gly Thr Asn Ile Asp Leu Ser Asp Ala Lys
1325 1330 1335Arg Trp Lys Pro Gln Leu Gln Glu Leu Leu Lys Leu Pro
Ala Phe 1340 1345 1350Met Arg Val Thr Ser Thr Gly Asn Met Leu Ser
His Val Gly His 1355 1360 1365Thr Ile Leu Gly Met Asn Thr Val Gln
Leu Tyr Met Lys Val Pro 1370 1375 1380Gly Ser Arg Thr Pro Gly His
Gln Glu Asn Asn Asn Phe Cys Ser 1385 1390 1395Val Asn Ile Asn Ile
Gly Pro Gly Asp Cys Glu Trp Phe Ala Val 1400 1405 1410His Glu His
Tyr Trp Glu Thr Ile Ser Ala Phe Cys Asp Arg His 1415 1420 1425Gly
Val Asp Tyr Leu Thr Gly Ser Trp Trp Pro Ile Leu Asp Asp 1430 1435
1440Leu Tyr Ala Ser Asn Ile Pro Val Tyr Arg Phe Val Gln Arg Pro
1445 1450 1455Gly Asp Leu Val Trp Ile Asn Ala Gly Thr Val His Trp
Val Gln 1460 1465 1470Ala Thr Gly Trp Cys Asn Asn Ile Ala Trp Asn
Val Gly Pro Leu 1475 1480 1485Thr Ala Tyr Gln Tyr Gln Leu Ala Leu
Glu Arg Tyr Glu Trp Asn 1490 1495 1500Glu Val Lys Asn Val Lys Ser
Ile Val Pro Met Ile His Val Ser 1505 1510 1515Trp Asn Val Ala Arg
Thr Val Lys Ile Ser Asp Pro Asp Leu Phe 1520 1525 1530Lys Met Ile
Lys Phe Cys Leu Leu Gln Ser Met Lys His Cys Gln 1535 1540 1545Val
Gln Arg Glu Ser Leu Val Arg Ala Gly Lys Lys Ile Ala Tyr 1550 1555
1560Gln Gly Arg Val Lys Asp Glu Pro Ala Tyr Tyr Cys Asn Glu Cys
1565 1570 1575Asp Val Glu Val Phe Asn Ile Leu Phe Val Thr Ser Glu
Asn Gly 1580 1585 1590Ser Arg Asn Thr Tyr Leu Val His Cys Glu Gly
Cys Ala Arg Arg 1595 1600 1605Arg Ser Ala Gly Leu Gln Gly Val Val
Val Leu Glu Gln Tyr Arg 1610 1615 1620Thr Glu Glu Leu Ala Gln Ala
Tyr Asp Ala Phe Thr Leu Val Arg 1625 1630 1635Ala Arg Arg Ala Arg
Gly Gln Arg Arg Arg Ala Leu Gly Gln Ala 1640 1645 1650Ala Gly Thr
Gly Phe Gly Ser Pro Ala Ala Pro Phe Pro Glu Pro 1655 1660 1665Pro
Pro Ala Phe Ser Pro Gln Ala Pro Ala Ser Thr Ser Arg 1670 1675
16802659PRTHomo sapiens 2Gln Ser Glu Glu Ile Gln Gly Arg Glu Lys
Ser Arg Pro Asp Leu Gly1 5 10 15Gly Ala Ser Lys Ala Lys Pro Pro Thr
Ala Pro Ala Pro Pro Ser Ala 20 25 30Pro Ala Pro Ser Ala Gln Pro Thr
Pro Pro Ser Ala Ser Val Pro Gly 35 40 45Lys Lys Ala Arg Glu Glu Ala
Pro Gly Pro Pro Gly Val Ser Arg Ala 50 55 60Asp Met Leu Lys Leu Arg
Ser Leu Ser Glu Gly Pro Pro Lys Glu Leu65 70 75 80Lys Ile Arg Leu
Ile Lys Val Glu Ser Gly Asp Lys Glu Thr Phe Ile 85 90 95Ala Ser Glu
Val Glu Glu Arg Arg Leu Arg Met Ala Asp Leu Thr Ile 100 105 110Ser
His Cys Ala Ala Asp Val Val Arg Ala Ser Arg Asn Ala Lys Val 115 120
125Lys Gly Lys Phe Arg Glu Ser Tyr Leu Ser Pro Ala Gln Ser Val Lys
130 135 140Pro Lys Ile Asn Thr Glu Glu Lys Leu Pro Arg Glu Lys Leu
Asn Pro145 150 155 160Pro Thr Pro Ser Ile Tyr Leu Glu Ser Lys Arg
Asp Ala Phe Ser Pro 165 170 175Val Leu Leu Gln Phe Cys Thr Asp Pro
Arg Asn Pro Ile Thr Val Ile 180 185 190Arg Gly Leu Ala Gly Ser Leu
Arg Leu Asn Leu Gly Leu Phe Ser Thr 195 200 205Lys Thr Leu Val Glu
Ala Ser Gly Glu His Thr Val Glu Val Arg Thr 210 215 220Gln Val Gln
Gln Pro Ser Asp Glu Asn Trp Asp Leu Thr Gly Thr Arg225 230 235
240Gln Ile Trp Pro Cys Glu Ser Ser Arg Ser His Thr Thr Ile Ala Lys
245 250 255Tyr Ala Gln Tyr Gln Ala Ser Ser Phe Gln Glu Ser Leu Gln
Glu Glu 260 265 270Lys Glu Ser Glu Asp Glu Glu Ser Glu Glu Pro Asp
Ser Thr Thr Gly 275 280 285Thr Pro Pro Ser Ser Ala Pro Asp Pro Lys
Asn His His Ile Ile Lys 290 295 300Phe Gly Thr Asn Ile Asp Leu Ser
Asp Ala Lys Arg Trp Lys Pro Gln305 310 315 320Leu Gln Glu Leu Leu
Lys Leu Pro Ala Phe Met Arg Val Thr Ser Thr 325 330 335Gly Asn Met
Leu Ser His Val Gly His Thr Ile Leu Gly Met Asn Thr 340 345 350Val
Gln Leu Tyr Met Lys Val Pro Gly Ser Arg Thr Pro Gly His Gln 355 360
365Glu Asn Asn Asn Phe Cys Ser Val Asn Ile Asn Ile Gly Pro Gly Asp
370 375 380Cys Glu Trp Phe Ala Val His Glu His Tyr Trp Glu Thr Ile
Ser Ala385 390 395 400Phe Cys Asp Arg His Gly Val Asp Tyr Leu Thr
Gly Ser Trp Trp Pro 405 410 415Ile Leu Asp Asp Leu Tyr Ala Ser Asn
Ile Pro Val Tyr Arg Phe Val 420 425 430Gln Arg Pro Gly Asp Leu Val
Trp Ile Asn Ala Gly Thr Val His Trp 435 440 445Val Gln Ala Thr Gly
Trp Cys Asn Asn Ile Ala Trp Asn Val Gly Pro 450 455 460Leu Thr Ala
Tyr Gln Tyr Gln Leu Ala Leu Glu Arg Tyr Glu Trp Asn465 470 475
480Glu Val Lys Asn Val Lys Ser Ile Val Pro Met Ile His Val Ser Trp
485 490 495Asn Val Ala Arg Thr Val Lys Ile Ser Asp Pro Asp Leu Phe
Lys Met 500 505 510Ile Lys Phe Cys Leu Leu Gln Ser Met Lys His Cys
Gln Val Gln Arg 515 520 525Glu Ser Leu Val Arg Ala Gly Lys Lys Ile
Ala Tyr Gln Gly Arg Val 530 535 540Lys Asp Glu Pro Ala Tyr Tyr Cys
Asn Glu Cys Asp Val Glu Val Phe545 550 555 560Asn Ile Leu Phe Val
Thr Ser Glu Asn Gly Ser Arg Asn Thr Tyr Leu 565 570 575Val His Cys
Glu Gly Cys Ala Arg Arg Arg Ser Ala Gly Leu Gln Gly 580 585 590Val
Val Val Leu Glu Gln Tyr Arg Thr Glu Glu Leu Ala Gln Ala Tyr 595 600
605Asp Ala Phe Thr Leu Val Arg Ala Arg Arg Ala Arg Gly Gln Arg Arg
610 615 620Arg Ala Leu Gly Gln Ala Ala Gly Thr Gly Phe Gly Ser Pro
Ala Ala625 630 635 640Pro Phe Pro Glu Pro Pro Pro Ala Phe Ser Pro
Gln Ala Pro Ala Ser 645 650 655Thr Ser Arg3109PRTHomo sapiens 3Gln
Leu Tyr Met Lys Val Pro Gly Ser Arg Thr Pro Gly His Gln Glu1 5 10
15Asn Asn Asn Phe Cys Ser Val Asn Ile Asn Ile Gly Pro Gly Asp Cys
20 25 30Glu Trp Phe Ala Val His Glu His Tyr Trp Glu Thr Ile Ser Ala
Phe 35 40 45Cys Asp Arg His Gly Val Asp Tyr Leu Thr Gly Ser Trp Trp
Pro Ile 50 55 60Leu Asp Asp Leu Tyr Ala Ser Asn Ile Pro Val Tyr Arg
Phe Val Gln65 70 75 80Arg Pro Gly Asp Leu Val Trp Ile Asn Ala Gly
Thr Val His Trp Val 85 90 95Gln Ala Thr Gly Trp Cys Asn Asn Ile Ala
Trp Asn Val 100 10545049DNAHomo sapiens 4atgcatcggg cagtggatcc
tccaggggcc
cgcgctgcac gggaagcctt tgcccttggg 60ggcctgagct gtgctggggc ctggagctcc
tgcccgcctc atccccctcc tcgtagcgca 120tggctgcctg gaggcagatg
ctcagccagc attgggcagc ccccgcttcc tgctccccta 180cccccttcac
atggcagtag ttctgggcac cccagcaaac catattatgc tccaggggcg
240cccactccaa gacccctcca tgggaagctg gaatccctgc atggctgtgt
gcaggcattg 300ctccgggagc cagcccagcc agggctttgg gaacagcttg
ggcaactgta cgagtcagag 360cacgatagtg aggaggccac acgctgctac
cacagcgccc ttcgatacgg aggaagcttc 420gctgagctgg ggccccgcat
tggccgactg cagcaggccc agctctggaa ctttcatact 480ggctcctgcc
agcaccgagc caaggtcctg cccccactgg agcaagtgtg gaacttgcta
540caccttgagc acaaacggaa ctatggagcc aagcggggag gtcccccggt
gaagcgagct 600gctgaacccc cagtggtgca gcctgtgcct cctgcagcac
tctcaggccc ctcaggggag 660gagggcctca gccctggagg caagcgaagg
agaggctgca actctgaaca gactggcctt 720cccccagggc tgccactgcc
tccaccacca ttaccaccac caccaccacc accaccacca 780ccaccaccac
ccctgcctgg cctggctacc agccccccat ttcagctaac caagccaggg
840ctgtggagta ccctgcatgg agatgcctgg ggcccagagc gcaagggttc
agcaccccca 900gagcgccagg agcagcggca ctcgctgcct cacccatatc
catacccagc tccagcgtac 960accgcgcacc cccctggcca ccggctggtc
ccggctgctc ccccaggccc aggcccccgc 1020cccccaggag cagagagcca
tggctgcctg cctgccaccc gtccccccgg aagtgacctt 1080agagagagca
gagttcagag gtcgcggatg gactccagcg tttcaccagc agcaaccacc
1140gcctgcgtgc cttacgcccc ttcccggccc cctggcctcc ccggcaccac
caccagcagc 1200agcagtagca gcagcagcaa cactggtctc cggggcgtgg
agccgaaccc aggcattccc 1260ggcgctgacc attaccaaac tcccgcgctg
gaggtctctc accatggccg cctggggccc 1320tcggcacaca gcagtcggaa
accgttcttg ggggctcccg ctgccactcc ccacctatcc 1380ctgccacctg
gaccttcctc accccctcca cccccctgtc cccgcctctt acgcccccca
1440ccaccccctg cctggttgaa gggtccggcc tgccgggcag cccgagagga
tggagagatc 1500ttagaagagc tcttctttgg gactgaggga cccccccgcc
ctgccccacc acccctcccc 1560catcgcgagg gcttcttggg gcctccggcc
tcccgctttt ctgtgggcac tcaggattct 1620cacacccctc ccactccccc
aaccccaacc accagcagta gcaacagcaa cagtggcagc 1680cacagcagca
gccctgctgg gcctgtgtcc tttcccccac caccctatct ggccagaagt
1740atagaccccc ttccccggcc tcccagccca gcacagaacc cccaggaccc
acctcttgta 1800cccctgactc ttgccctgcc tccagcccct ccttcctcct
gccaccaaaa tacctcagga 1860agcttcaggc gcccggagag cccccggccc
agggtctcct tcccaaagac ccccgaggtg 1920gggccggggc cacccccagg
ccccctgagt aaagcccccc agcctgtgcc gcccggggtt 1980ggggagctgc
ctgcccgagg ccctcgactc tttgattttc cccccactcc gctggaggac
2040cagtttgagg agccagccga attcaagatc ctacctgatg ggctggccaa
catcatgaag 2100atgctggacg aatccattcg caaggaagag gaacagcaac
aacacgaagc aggcgtggcc 2160ccccaacccc cgctgaagga gccctttgca
tctctgcagt ctcctttccc caccgacaca 2220gcccccacca ctactgctcc
tgctgtcgcc gtcaccacca ccaccaccac caccaccacc 2280accacggcca
cccaggaaga ggagaagaag ccaccaccag ccctaccacc accaccgcct
2340ctagccaagt tccctccacc ctctcagcca cagccaccac cacccccacc
ccccagcccg 2400gccagcctgc tcaaatcctt ggcctccgtg ctggagggac
aaaagtactg ttatcggggg 2460actggagcag ctgtttccac ccggcctggg
cccttgccca ccactcagta ttcccctggc 2520cccccatcag gtgctaccgc
cctgccgccc acctcagcgg cccctagcgc ccagggctcc 2580ccacagccct
ctgcttcctc gtcatctcag ttctctacct caggcgggcc ctgggcccgg
2640gagcgcaggg cgggcgaaga gccagtcccg ggccccatga cccccaccca
accgccccca 2700cccctatctc tgccccctgc tcgctctgag tctgaggtgc
tagaagagat cagccgggct 2760tgcgagaccc ttgtggagcg ggtgggccgg
agtgccactg acccagccga cccagtggac 2820acagcagagc cagcggacag
tgggactgag cgactgctgc cccccgcaca ggccaaggag 2880gaggctggcg
gggtggcggc agtgtcaggc agctgtaagc ggcgacagaa ggagcatcag
2940aaggagcatc ggcggcacag gcgggcctgt aaggacagtg tgggtcgtcg
gccccgtgag 3000ggcagggcaa aggccaaggc caaggtcccc aaagaaaaga
gccgccgggt gctggggaac 3060ctggacctgc agagcgagga gatccagggt
cgtgagaagt cccggcccga tcttggcggg 3120gcctccaagg ccaagccacc
cacagctcca gcccctccat cagctcctgc accttctgcc 3180cagcccacac
ccccgtcagc ctctgtccct ggaaagaagg ctcgggagga agccccaggg
3240ccaccgggtg tcagccgggc cgacatgctg aagctgcgct cacttagtga
ggggcccccc 3300aaggagctga agatccggct catcaaggta gagagtggtg
acaaggagac ctttatcgcc 3360tctgaggtgg aagagcggcg gctgcgcatg
gcagacctca ccatcagcca ctgtgctgct 3420gacgtcgtgc gcgccagcag
gaatgccaag gtgaaaggga agtttcgaga gtcctacctt 3480tcccctgccc
agtctgtgaa accgaagatc aacactgagg agaagctgcc ccgggaaaaa
3540ctcaaccccc ctacacccag catctatctg gagagcaaac gggatgcctt
ctcacctgtc 3600ctgctgcagt tctgtacaga ccctcgaaat cccatcacag
tgatccgggg cctggcgggc 3660tccctgcggc tcaacttggg cctcttctcc
accaagaccc tggtggaagc gagtggcgaa 3720cacaccgtgg aagttcgcac
ccaggtgcag cagccctcag atgagaactg ggatctgaca 3780ggcactcggc
agatctggcc ttgtgagagc tcccgttccc acaccaccat tgccaagtac
3840gcacagtacc aggcctcatc cttccaggag tctctgcagg aggagaagga
gagtgaggat 3900gaggagtcag aggagccaga cagcaccact ggaacccctc
ctagcagcgc accagacccg 3960aagaaccatc acatcatcaa gtttggcacc
aacatcgact tgtctgatgc taagcggtgg 4020aagccccagc tgcaggagct
gctgaagctg cccgccttca tgcgggtaac atccacgggc 4080aacatgctga
gccacgtggg ccacaccatc ctgggcatga acacggtgca gctgtacatg
4140aaggtgcccg gcagccgaac gccaggccac caggagaata acaacttctg
ctccgtcaac 4200atcaacattg gcccaggcga ctgcgagtgg ttcgcggtgc
acgagcacta ctgggagacc 4260atcagcgctt tctgtgatcg gcacggcgtg
gactacttga cgggttcctg gtggccaatc 4320ctggatgatc tctatgcatc
caatattcct gtgtaccgct tcgtgcagcg acccggagac 4380ctcgtgtgga
ttaatgcggg gactgtgcac tgggtgcagg ccaccggctg gtgcaacaac
4440attgcctgga acgtggggcc cctcaccgcc tatcagtacc agctggccct
ggaacgatac 4500gagtggaatg aggtgaagaa cgtcaaatcc atcgtgccca
tgattcacgt gtcatggaac 4560gtggctcgca cggtcaaaat cagcgacccc
gacttgttca agatgatcaa gttctgcctg 4620ctgcagtcca tgaagcactg
ccaggtgcaa cgcgagagcc tggtgcgggc agggaagaaa 4680atcgcttacc
agggccgtgt caaggacgag ccagcctact actgcaacga gtgcgatgtg
4740gaggtgttta acatcctgtt cgtgacaagt gagaatggca gccgcaacac
gtacctggta 4800cactgcgagg gctgtgcccg gcgccgcagc gcaggcctgc
agggcgtggt ggtgctggag 4860cagtaccgca ctgaggagct ggctcaggcc
tacgacgcct tcacgctggt gagggcccgg 4920cgggcgcgcg ggcagcggag
gagggcactg gggcaggctg cagggacggg cttcgggagc 4980ccggccgcgc
ctttccctga gcccccgccg gctttctccc cccaggcccc agccagcacg
5040tcgcgatga 504951980DNAHomo sapiens 5cagagcgagg agatccaggg
tcgtgagaag tcccggcccg atcttggcgg ggcctccaag 60gccaagccac ccacagctcc
agcccctcca tcagctcctg caccttctgc ccagcccaca 120cccccgtcag
cctctgtccc tggaaagaag gctcgggagg aagccccagg gccaccgggt
180gtcagccggg ccgacatgct gaagctgcgc tcacttagtg aggggccccc
caaggagctg 240aagatccggc tcatcaaggt agagagtggt gacaaggaga
cctttatcgc ctctgaggtg 300gaagagcggc ggctgcgcat ggcagacctc
accatcagcc actgtgctgc tgacgtcgtg 360cgcgccagca ggaatgccaa
ggtgaaaggg aagtttcgag agtcctacct ttcccctgcc 420cagtctgtga
aaccgaagat caacactgag gagaagctgc cccgggaaaa actcaacccc
480cctacaccca gcatctatct ggagagcaaa cgggatgcct tctcacctgt
cctgctgcag 540ttctgtacag accctcgaaa tcccatcaca gtgatccggg
gcctggcggg ctccctgcgg 600ctcaacttgg gcctcttctc caccaagacc
ctggtggaag cgagtggcga acacaccgtg 660gaagttcgca cccaggtgca
gcagccctca gatgagaact gggatctgac aggcactcgg 720cagatctggc
cttgtgagag ctcccgttcc cacaccacca ttgccaagta cgcacagtac
780caggcctcat ccttccagga gtctctgcag gaggagaagg agagtgagga
tgaggagtca 840gaggagccag acagcaccac tggaacccct cctagcagcg
caccagaccc gaagaaccat 900cacatcatca agtttggcac caacatcgac
ttgtctgatg ctaagcggtg gaagccccag 960ctgcaggagc tgctgaagct
gcccgccttc atgcgggtaa catccacggg caacatgctg 1020agccacgtgg
gccacaccat cctgggcatg aacacggtgc agctgtacat gaaggtgccc
1080ggcagccgaa cgccaggcca ccaggagaat aacaacttct gctccgtcaa
catcaacatt 1140ggcccaggcg actgcgagtg gttcgcggtg cacgagcact
actgggagac catcagcgct 1200ttctgtgatc ggcacggcgt ggactacttg
acgggttcct ggtggccaat cctggatgat 1260ctctatgcat ccaatattcc
tgtgtaccgc ttcgtgcagc gacccggaga cctcgtgtgg 1320attaatgcgg
ggactgtgca ctgggtgcag gccaccggct ggtgcaacaa cattgcctgg
1380aacgtggggc ccctcaccgc ctatcagtac cagctggccc tggaacgata
cgagtggaat 1440gaggtgaaga acgtcaaatc catcgtgccc atgattcacg
tgtcatggaa cgtggctcgc 1500acggtcaaaa tcagcgaccc cgacttgttc
aagatgatca agttctgcct gctgcagtcc 1560atgaagcact gccaggtgca
acgcgagagc ctggtgcggg cagggaagaa aatcgcttac 1620cagggccgtg
tcaaggacga gccagcctac tactgcaacg agtgcgatgt ggaggtgttt
1680aacatcctgt tcgtgacaag tgagaatggc agccgcaaca cgtacctggt
acactgcgag 1740ggctgtgccc ggcgccgcag cgcaggcctg cagggcgtgg
tggtgctgga gcagtaccgc 1800actgaggagc tggctcaggc ctacgacgcc
ttcacgctgg tgagggcccg gcgggcgcgc 1860gggcagcgga ggagggcact
ggggcaggct gcagggacgg gcttcgggag cccggccgcg 1920cctttccctg
agcccccgcc ggctttctcc ccccaggccc cagccagcac gtcgcgatga
19806327DNAHomo sapiens 6cagctgtaca tgaaggtgcc cggcagccga
acgccaggcc accaggagaa taacaacttc 60tgctccgtca acatcaacat tggcccaggc
gactgcgagt ggttcgcggt gcacgagcac 120tactgggaga ccatcagcgc
tttctgtgat cggcacggcg tggactactt gacgggttcc 180tggtggccaa
tcctggatga tctctatgca tccaatattc ctgtgtaccg cttcgtgcag
240cgacccggag acctcgtgtg gattaatgcg gggactgtgc actgggtgca
ggccaccggc 300tggtgcaaca acattgcctg gaacgtg 327712PRTHuman
immunodeficiency virus 7Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro
Gln1 5 1088PRTArtificial Sequencer8 8Arg Arg Arg Arg Arg Arg Arg
Arg1 5921PRTArtificial SequenceMPG-8 (The first amino acid "Ala" is
modified into "bAla.") 9Ala Phe Leu Gly Trp Leu Gly Ala Trp Gly Thr
Met Gly Trp Ser Pro1 5 10 15Lys Lys Lys Arg Lys 201023DNAArtificial
SequenceqRT-PCR forward primer for GAPDH 10ggtggtctcc tctgacttca
aca 231119DNAArtificial SequenceqRT-PCR reverse primer for GAPDH
11gtggtcgttg agggcaatg 191222DNAArtificial SequenceqRT-PCR forward
primer for POU5F1 12cttgaatccc gaatggaaag gg 221323DNAArtificial
SequenceqRT-PCR reverse primer for POU5F1 13gtgtatatcc cagggtgatc
ctc 231419DNAArtificial SequenceqRT-PCR forward primer for NANOG
14agaaggcctc agcacctac 191520DNAArtificial SequenceqRT-PCR reverse
primer for NANOG 15ggcctgattg ttccaggatt 201626DNAArtificial
SequenceqRT-PCR forward primer for T 16gccctctccc tcccctccac gcacag
261726DNAArtificial SequenceqRT-PCR reverse primer for T
17cggcgccgtt gctcacagac cacagg 261824DNAArtificial SequenceqRT-PCR
forward primer for MSX1 18cgagaggacc ccgtggatgc agag
241924DNAArtificial SequenceqRT-PCR reverse primer for MSX1
19ggcggccatc ttcagcttct ccag 242026DNAArtificial SequenceqRT-PCR
forward primer for SOX17 20cgctttcatg gtgtgggcta aggacg
262126DNAArtificial SequenceqRT-PCR reverse primer for SOX17
21tagttggggt ggtcctgcat gtgctg 262226DNAArtificial SequenceqRT-PCR
forward primer for FOXA2 22tgggagcggt gaagatggaa gggcac
262326DNAArtificial SequenceqRT-PCR reverse primer for FOXA2
23tcatgccagc gcccacgtac gacgac 262420DNAArtificial SequenceqRT-PCR
forward primer for GATA4 24gctccttcag gcagtgagag
202520DNAArtificial SequenceqRT-PCR reverse primer for GATA4
25ctgtgcccgt agtgagatga 202620DNAArtificial SequenceqRT-PCR forward
primer for GATA6 26gtgcccagac cacttgctat 202720DNAArtificial
SequenceqRT-PCR reverse primer for GATA6 27tggagtcatg ggaatggaat
202820DNAArtificial SequenceqRT-PCR forward primer for GSC
28cggtcctcat cagaggagtc 202920DNAArtificial SequenceqRT-PCR reverse
primer for GSC 29ccgagtccaa atcgctttta 203020DNAArtificial
SequenceqRT-PCR forward primer for EVX1 30cggctggaga aggaattcta
203120DNAArtificial SequenceqRT-PCR reverse primer for EVX1
31acaccttgat ggtggtttcc 203220DNAArtificial SequenceqRT-PCR forward
primer for MYOG 32gccagactat ccccttcctc 203320DNAArtificial
SequenceqRT-PCR reverse primer for MYOG 33gaggccgcgt tatgataaaa
203420DNAArtificial SequenceqRT-PCR forward primer for MEF2C
34aggtcacctg acatcccaag 203520DNAArtificial SequenceqRT-PCR reverse
primer for MEF2C 35gttagccctc caactccaca 203620DNAArtificial
SequenceqRT-PCR forward primer for CKM 36gaagagcatg acggagaagg
203720DNAArtificial SequenceqRT-PCR reverse primer for CKM
37gttgtcattg tgccagatgc 203820DNAArtificial SequenceqRT-PCR forward
primer for SIX1 38tgtttgcgca taaaggaatg 203920DNAArtificial
SequenceqRT-PCR reverse primer for SIX1 39tgggaaggaa aatgcaaaag
204018DNAArtificial SequenceqRT-PCR forward primer for AFP
40tgggacccga actttcca 184122DNAArtificial SequenceqRT-PCR reverse
primer for AFP 41ggccacatcc aggactagtt tc 224220DNAArtificial
SequenceqRT-PCR forward primer for COL2 42tttcccaggt caagatggtc
204320DNAArtificial SequenceqRT-PCR reverse primer for COL2
43cttcagcacc tgtctcacca 204420DNAArtificial SequenceqRT-PCR forward
primer for COL1A1 44cctggatgcc atcaaagtct 204520DNAArtificial
SequenceqRT-PCR reverse primer for COL1A1 45tcttgtcctt ggggttcttg
204620DNAArtificial SequenceChIP-PCR forward primer for POU5F1
46ggaggtaaac ccagctcaca 204720DNAArtificial SequenceChIP-PCR
reverse primer for POU5F1 47tttggcctta gggttaagca
204820DNAArtificial SequenceChIP-PCR forward primer for NANOG
48gctcagggat gagcatgatt 204920DNAArtificial SequenceChIP-PCR
reverse primer for NANOG 49tgcccagtaa catccacaaa
205020DNAArtificial SequenceChIP-PCR forward primer for T
50ggcacggcca aataagaata 205120DNAArtificial SequenceChIP-PCR
reverse primer for T 51ggttcaattc ctgggtcgta 205220DNAArtificial
SequenceChIP-PCR forward primer for MSX1 52tccctcatct gatcccaaac
205320DNAArtificial SequenceChIP-PCR reverse primer for MSX1
53accagctcct actgcgagaa 205420DNAArtificial SequenceChIP-PCR
forward primer for SOX17 54agcaagatgc tgggtgagtc
205520DNAArtificial SequenceChIP-PCR reverse primer for SOX17
55ctacacaccc ctggttttgg 205620DNAArtificial SequenceChIP-PCR
forward primer for FOXA2 56ttcttcgctc tcagtgctca
205721DNAArtificial SequenceChIP-PCR reverse primer for FOXA2
57ggcgagttaa aggtgtgtac g 215820DNAArtificial SequenceChIP-PCR
forward primer for GATA4 58gatcttcgcg acagttcctc
205920DNAArtificial SequenceChIP-PCR reverse primer for GATA4
59catggccaag ctctgataca 206020DNAArtificial SequenceChIP-PCR
forward primer for GATA6 60tgcagcctac gctcttgtta
206119DNAArtificial SequenceChIP-PCR reverse primer for GATA6
61gtcagtcaag gccatccac 196220DNAArtificial SequenceChIP-PCR forward
primer for GSC 62gacatgacgg agatgggtct 206320DNAArtificial
SequenceChIP-PCR reverse primer for GSC 63tggaaggtgc ctcacttctt
206420DNAArtificial SequenceChIP-PCR forward primer for EVX1
64tcacactctc ctccccaatc 206520DNAArtificial SequenceChIP-PCR
reverse primer for EVX1 65ttacagtacc gctggtgacg 206623DNAArtificial
SequenceChIP-PCR forward primer for GAPDH 66cggtgactaa ccctgcgctc
ctg 236724DNAArtificial SequenceChIP-PCR reverse primer for GAPDH
67agctagcctc gctccacctg actt 246820DNAArtificial SequenceChIP-PCR
forward primer for MYOG_a 68cctccggaaa gaatgggact
206921DNAArtificial SequenceChIP-PCR reverse primer for MYOG_a
69tctgttagct gctctgagtc t 217020DNAArtificial SequenceChIP-PCR
forward primer for MYOG_b
70ttggagccaa ggttaccagt 207118DNAArtificial SequenceChIP-PCR
reverse primer for MYOGb 71ctctcacagc gcctcctg 187220DNAArtificial
SequenceChIP-PCR forward primer for MYOG_c 72ggcctcattc accttcttga
207320DNAArtificial SequenceChIP-PCR reverse primer for MYOG_c
73tgggcgtgta aggtgtgtaa 207420DNAArtificial SequenceChIP-PCR
forward primer for MEF2C_a 74catgcatttt caggtcacca
207520DNAArtificial SequenceChIP-PCR reverse primer for MEF2C_a
75cccctccact ttgattcgta 207620DNAArtificial SequenceChIP-PCR
forward primer for MEF2C_b 76gcacgtttaa gaccccaaag
207720DNAArtificial SequenceChIP-PCR reverse primer for MEF2C_b
77cggcctcagc taaatgaaag 207820DNAArtificial SequenceChIP-PCR
forward primer for SOX1 78ccgtctcact ccgtctgaat 207919DNAArtificial
SequenceChIP-PCR reverse primer for SOX1 79agtgcaggtc ggtctccat
19802541DNAHomo sapiens 80gggttggacc ctcgtacaga agctaatacg
actcactata gggaaataag agagaaaaga 60agagtaagaa gaaatataag agccaccccg
cggtggcggc cgctctagaa ctagtggatc 120ccccgggctg caggaattcg
ataaaagcga tcgcccatca caagtttgta caaaaaagca 180ggcttagcca
ccatgtaccc atacgatgtt ccagattacg ctcctaagaa aaagaggaag
240gtgcagagcg aggagatcca gggtcgtgag aagtcccggc ccgatcttgg
cggggcctcc 300aaggccaagc cacccacagc tccagcccct ccatcagctc
ctgcaccttc tgcccagccc 360acacccccgt cagcctctgt ccctggaaag
aaggctcggg aggaagcccc agggccaccg 420ggtgtcagcc gggccgacat
gctgaagctg cgctcactta gtgaggggcc ccccaaggag 480ctgaagatcc
ggctcatcaa ggtagagagt ggtgacaagg agacctttat cgcctctgag
540gtggaagagc ggcggctgcg catggcagac ctcaccatca gccactgtgc
tgctgacgtc 600gtgcgcgcca gcaggaatgc caaggtgaaa gggaagtttc
gagagtccta cctttcccct 660gcccagtctg tgaaaccgaa gatcaacact
gaggagaagc tgccccggga aaaactcaac 720ccccctacac ccagcatcta
tctggagagc aaacgggatg ccttctcacc tgtcctgctg 780cagttctgta
cagaccctcg aaatcccatc acagtgatcc ggggcctggc gggctccctg
840cggctcaact tgggcctctt ctccaccaag accctggtgg aagcgagtgg
cgaacacacc 900gtggaagttc gcacccaggt gcagcagccc tcagatgaga
actgggatct gacaggcact 960cggcagatct ggccttgtga gagctcccgt
tcccacacca ccattgccaa gtacgcacag 1020taccaggcct catccttcca
ggagtctctg caggaggaga aggagagtga ggatgaggag 1080tcagaggagc
cagacagcac cactggaacc cctcctagca gcgcaccaga cccgaagaac
1140catcacatca tcaagtttgg caccaacatc gacttgtctg atgctaagcg
gtggaagccc 1200cagctgcagg agctgctgaa gctgcccgcc ttcatgcggg
taacatccac gggcaacatg 1260ctgagccacg tgggccacac catcctgggc
atgaacacgg tgcagctgta catgaaggtg 1320cccggcagcc gaacgccagg
ccaccaggag aataacaact tctgctccgt caacatcaac 1380attggcccag
gcgactgcga gtggttcgcg gtgcacgagc actactggga gaccatcagc
1440gctttctgtg atcggcacgg cgtggactac ttgacgggtt cctggtggcc
aatcctggat 1500gatctctatg catccaatat tcctgtgtac cgcttcgtgc
agcgacccgg agacctcgtg 1560tggattaatg cggggactgt gcactgggtg
caggccaccg gctggtgcaa caacattgcc 1620tggaacgtgg ggcccctcac
cgcctatcag taccagctgg ccctggaacg atacgagtgg 1680aatgaggtga
agaacgtcaa atccatcgtg cccatgattc acgtgtcatg gaacgtggct
1740cgcacggtca aaatcagcga ccccgacttg ttcaagatga tcaagttctg
cctgctgcag 1800tccatgaagc actgccaggt gcaacgcgag agcctggtgc
gggcagggaa gaaaatcgct 1860taccagggcc gtgtcaagga cgagccagcc
tactactgca acgagtgcga tgtggaggtg 1920tttaacatcc tgttcgtgac
aagtgagaat ggcagccgca acacgtacct ggtacactgc 1980gagggctgtg
cccggcgccg cagcgcaggc ctgcagggcg tggtggtgct ggagcagtac
2040cgcactgagg agctggctca ggcctacgac gccttcacgc tggtgagggc
ccggcgggcg 2100cgcgggcagc ggaggagggc actggggcag gctgcaggga
cgggcttcgg gagcccggcc 2160gcgcctttcc ctgagccccc gccggctttc
tccccccagg ccccagccag cacgtcgcga 2220tgaacccagc tttcttgtac
aaagtggtga tggccgctgt ttaaaacttt tatcaagctt 2280atcgataccg
tcgacctcga atgctgcctt ctgcggggct tgccttctgg ccatgccctt
2340cttctctccc ttgcacctgt acctcttggt ctttgaataa agcctgagta
ggaagtgagg 2400gtctagaact agtgtcgacg caaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 2460aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2520aaaaaaaaaa aaaaaaaaaa a
2541811222DNAHomo sapiens 81gggttggacc ctcgtacaga agctaatacg
actcactata gggaaataag agagaaaaga 60agagtaagaa gaaatataag agccaccccg
cggtggcggc cgctctagaa ctagtggatc 120ccccgggctg caggaattcg
ataaaagcga tcgcccatca caagtttgta caaaaaagca 180ggctccacca
tgccagcccg ccttgagacc tgcatctccg acctcgactg cgccagcagc
240agcggcagtg acctatccgg cttcctcacc gacgaggaag actgtgccag
actccaacag 300gcagcctccg cttcggggcc gcccgcgccg gcccgcaggg
gcgcgcccaa tatctcccgg 360gcgtctgagg ttccaggggc acaggacgac
gagcaggaga ggcggcggcg ccgcggccgg 420acgcgggtcc gctccgaggc
gctgctgcac tcgctgcgca ggagccggcg cgtcaaggcc 480aacgatcgcg
agcgcaaccg catgcacaac ttgaacgcgg ccctggacgc actgcgcagc
540gtgctgccct cgttccccga cgacaccaag ctcaccaaaa tcgagacgct
gcgcttcgcc 600tacaactaca tctgggctct ggccgagaca ctgcgcctgg
cggatcaagg gctgcccgga 660ggcggtgccc gggagcgcct cctgccgccg
cagtgcgtcc cctgcctgcc cggtccccca 720agccccgcca gcgacgcgga
gtcctggggc tcaggtgccg ccgccgcctc cccgctctct 780gaccccagta
gcccagccgc ctccgaagac ttcacctacc gccccggcga ccctgttttc
840tccttcccaa gcctgcccaa agacttgctc cacacaacgc cctgtttcat
tccttaccac 900taggacccag ctttcttgta caaagtggtg atggccgctg
tttaaaactt ttatcaagct 960tatcgatacc gtcgacctcg aatgctgcct
tctgcggggc ttgccttctg gccatgccct 1020tcttctctcc cttgcacctg
tacctcttgg tctttgaata aagcctgagt aggaagtgag 1080ggtctagaac
tagtgtcgac gcaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1140aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 1200aaaaaaaaaa aaaaaaaaaa aa 1222821357DNAHomo sapiens
82gggttggacc ctcgtacaga agctaatacg actcactata gggaaataag agagaaaaga
60agagtaagaa gaaatataag agccaccccg cggtggcggc cgctctagaa ctagtggatc
120ccccgggctg caggaattcg ataaaagcga tcgcccatca caagtttgta
caaaaaagca 180ggctccgcgg ccgccccctt caccatgttc gtcaaatccg
agaccttgga gttgaaggag 240gaagaggacg tgttagtgct gctcggatcg
gcctcccccg ccttggcggc cctgaccccg 300ctgtcatcca gcgccgacga
agaagaggag gaggagccgg gcgcgtcagg tggggcgcgt 360cggcagcgcg
gggctgaggc cgggcagggg gcgcggggcg gcgtggctgc gggtgcggag
420ggctgccggc ccgcacggct gctgggtctg gtacacgatt gcaaacggcg
cccttcccgg 480gcgcgggccg tctcccgagg cgccaagacg gccgagacgg
tgcagcgcat caagaagacc 540cgtagactga aggccaacaa ccgcgagcga
aaccgcatgc acaacctcaa cgcggcactg 600gacgcgctgc gcgaggtgct
ccccacgttc cccgaggacg ccaagctcac caagatcgag 660accctgcgct
tcgcccacaa ctacatctgg gcactcaccg agaccctgcg cctggcggat
720cactgcgggg gcggcggcgg gggcctgccg ggggcgctct tctccgaggc
agtgttgctg 780agcccgggag gcgccagcgc cgccctgagc agcagcggag
acagcccctc gcccgcctcc 840acgtggagtt gcaccaacag ccccgcgccg
tcctcctccg tgtcctccaa ttccacctcc 900ccctacagct gcactttatc
gcccgccagc ccggccgggt cagacatgga ctattggcag 960cccccacctc
ccgacaagca ccgctatgca cctcacctcc ccatagccag ggattgtatc
1020tagaagggtg ggcgcgccga cccagctttc ttgtacaaag tggtgatggc
cgctgtttaa 1080aacttttatc aagcttatcg ataccgtcga cctcgaatgc
tgccttctgc ggggcttgcc 1140ttctggccat gcccttcttc tctcccttgc
acctgtacct cttggtcttt gaataaagcc 1200tgagtaggaa gtgagggtct
agaactagtg tcgacgcaaa aaaaaaaaaa aaaaaaaaaa 1260aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1320aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 1357831153DNAHomo
sapiens 83gggttggacc ctcgtacaga agctaatacg actcactata gggaaataag
agagaaaaga 60agagtaagaa gaaatataag agccaccccg cggtggcggc cgctctagaa
ctagtggatc 120ccccgggctg caggaattcg ataaaagcga tcgcccatca
caagtttgta caaaaaagca 180ggcttcacca tgacgcctca accctcgggt
gcgcccactg tccaagtgac ccgtgagacg 240gagcggtcct tccccagagc
ctcggaagac gaagtgacct gccccacgtc cgccccgccc 300agccccactc
gcacacgggg gaactgcgca gaggcggaag agggaggctg ccgaggggcc
360ccgaggaagc tccgggcacg gcgcggggga cgcagccggc ctaagagcga
gttggcactg 420agcaagcagc gacggagtcg gcgaaagaag gccaacgacc
gcgagcgcaa tcgaatgcac 480aacctcaact cggcactgga cgccctgcgc
ggtgtcctgc ccaccttccc agacgacgcg 540aagctcacca agatcgagac
gctgcgcttc gcccacaact acatctgggc gctgactcaa 600acgctgcgca
tagcggacca cagcttgtac gcgctggagc cgccggcgcc gcactgcggg
660gagctgggca gcccaggcgg ttcccccggg gactgggggt ccctctactc
cccagtctcc 720caggctggca gcctgagtcc cgccgcgtcg ctggaggagc
gacccgggct gctgggggcc 780accttttccg cctgcttgag cccaggcagt
ctggctttct cagattttct gtgagaccca 840gctttcttgt acaaagtggt
gatggccgct gtttaaaact tttatcaagc ttatcgatac 900cgtcgacctc
gaatgctgcc ttctgcgggg cttgccttct ggccatgccc ttcttctctc
960ccttgcacct gtacctcttg gtctttgaat aaagcctgag taggaagtga
gggtctagaa 1020ctagtgtcga cgcaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 1080aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1140aaaaaaaaaa aaa
1153841609DNAHomo sapiens 84gggttggacc ctcgtacaga agctaatacg
actcactata gggaaataag agagaaaaga 60agagtaagaa gaaatataag agccaccccg
cggtggcggc cgctctagaa ctagtggatc 120ccccgggctg caggaattcg
ataaaagcga tcgcccatca caagtttgta caaaaaagca 180ggctccgcgg
ccgccccctt caccatgacc aaatcgtaca gcgagagtgg gctgatgggc
240gagcctcagc cccaaggtcc tccaagctgg acagacgagt gtctcagttc
tcaggacgag 300gagcacgagg cagacaagaa ggaggacgac ctcgaagcca
tgaacgcaga ggaggactca 360ctgaggaacg ggggagagga ggaggacgaa
gatgaggacc tggaagagga ggaagaagag 420gaagaggagg atgacgatca
aaagcccaag agacgcggcc ccaaaaagaa gaagatgact 480aaggctcgcc
tggagcgttt taaattgaga cgcatgaagg ctaacgcccg ggagcggaac
540cgcatgcacg gactgaacgc ggcgctagac aacctgcgca aggtggtgcc
ttgctattct 600aagacgcaga agctgtccaa aatcgagact ctgcgcttgg
ccaagaacta catctgggct 660ctgtcggaga tcctgcgctc aggcaaaagc
ccagacctgg tctccttcgt tcagacgctt 720tgcaagggct tatcccaacc
caccaccaac ctggttgcgg gctgcctgca actcaatcct 780cggacttttc
tgcctgagca gaaccaggac atgccccccc acctgccgac ggccagcgct
840tccttccctg tacaccccta ctcctaccag tcgcctgggc tgcccagtcc
gccttacggt 900accatggaca gctcccatgt cttccacgtt aagcctccgc
cgcacgccta cagcgcagcg 960ctggagccct tctttgaaag ccctctgact
gattgcacca gcccttcctt tgatggaccc 1020ctcagcccgc cgctcagcat
caatggcaac ttctctttca aacacgaacc gtccgccgag 1080tttgagaaaa
attatgcctt taccatgcac tatcctgcag cgacactggc aggggcccaa
1140agccacggat caatcttctc aggcaccgct gcccctcgct gcgagatccc
catagacaat 1200attatgtcct tcgatagcca ttcacatcat gagcgagtca
tgagtgccca gctcaatgcc 1260atatttcatg attagaaggg tgggcgcgcc
gacccagctt tcttgtacaa agtggtgatg 1320gccgctgttt aaaactttta
tcaagcttat cgataccgtc gacctcgaat gctgccttct 1380gcggggcttg
ccttctggcc atgcccttct tctctccctt gcacctgtac ctcttggtct
1440ttgaataaag cctgagtagg aagtgagggt ctagaactag tgtcgacgca
aaaaaaaaaa 1500aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 1560aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaa 1609851687DNAHomo sapiens 85gggttggacc
ctcgtacaga agctaatacg actcactata gggaaataag agagaaaaga 60agagtaagaa
gaaatataag agccaccccg cggtggcggc cgctctagaa ctagtggatc
120ccccgggctg caggaattcg ataaaagcga tcgcccatca caagtttgta
caaaaaagca 180ggctccgcgg ccgccccctt caccatgctg acccgcctgt
tcagcgagcc cggccttctc 240tcggacgtgc ccaagttcgc cagctggggc
gacggcgaag acgacgagcc gaggagcgac 300aagggcgacg cgccgccacc
gccaccgcct gcgcccgggc caggggctcc ggggccagcc 360cgggcggcca
agccagtccc tctccgtgga gaagagggga cggaggccac gttggccgag
420gtcaaggagg aaggcgagct ggggggagag gaggaggagg aagaggagga
ggaagaagga 480ctggacgagg cggagggcga gcggcccaag aagggcgggc
ccaagaagcg caagatgacc 540aaggcgcgct tggagcgctc caagcttcgg
cggcagaagg cgaacgcgcg ggagcgcaac 600cgcatgcacg acctgaacgc
agccctggac aacctgcgca aggtggtgcc ctgctactcc 660aagacgcaga
agctgtccaa gatcgagacg ctgcgcctag ccaagaacta tatctgggcg
720ctctcggaga tcctgcgctc cggcaagcgg ccagacctag tgtcctacgt
gcagactctg 780tgcaagggtc tgtcgcagcc caccaccaat ctggtggccg
gctgtctgca gctcaactct 840cgcaacttcc tcacggagca aggcgccgac
ggtgccggcc gcttccacgg ctcgggcggc 900ccgttcgcca tgcaccccta
cccgtacccg tgctcgcgcc tggcgggcgc acagtgccag 960gcggccggcg
gcctgggcgg cggcgcggcg cacgccctgc ggacccacgg ctactgcgca
1020gcctacgaga cgctgtatgc ggcggcaggc ggtggcggcg cgagcccgga
ctacaacagc 1080tccgagtacg agggcccgct cagccccccg ctctgtctca
atggcaactt ctcactcaag 1140caggactcct cgcccgacca cgagaaaagc
taccactact ctatgcacta ctcggcgctg 1200cccggttcgc ggcccacggg
ccacgggcta gtcttcggct cgtcggctgt gcgcgggggc 1260gtccactcgg
agaatctctt gtcttacgat atgcaccttc accacgaccg gggccccatg
1320tacgaggagc tcaatgcgtt ttttcataac tgaaagggtg ggcgcgccga
cccagctttc 1380ttgtacaaag tggtgatggc cgctgtttaa aacttttatc
aagcttatcg ataccgtcga 1440cctcgaatgc tgccttctgc ggggcttgcc
ttctggccat gcccttcttc tctcccttgc 1500acctgtacct cttggtcttt
gaataaagcc tgagtaggaa gtgagggtct agaactagtg 1560tcgacgcaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1620aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 1680aaaaaaa 1687861574DNAHomo sapiens 86gggttggacc
ctcgtacaga agctaatacg actcactata gggaaataag agagaaaaga 60agagtaagaa
gaaatataag agccaccccg cggtggcggc cgctctagaa ctagtggatc
120ccccgggctg caggaattcg ataaaagcga tcgcccatca caagtttgta
caaaaaagca 180ggctccgcgg ccgccccctt caccgctagg gataacaggg
taatagaagg agccgccacc 240atggagctac tgtcgccacc gctccgcgac
gtagacctga cggcccccga cggctctctc 300tgctcctttg ccacaacgga
cgacttctat gacgacccgt gtttcgactc cccggacctg 360cgcttcttcg
aagacctgga cccgcgcctg atgcacgtgg gcgcgctcct gaaacccgaa
420gagcactcgc acttccccgc ggcggtgcac ccggccccgg gcgcacgtga
ggacgagcat 480gtgcgcgcgc ccagcgggca ccaccaggcg ggccgctgcc
tactgtgggc ctgcaaggcg 540tgcaagcgca agaccaccaa cgccgaccgc
cgcaaggccg ccaccatgcg cgagcggcgc 600cgcctgagca aagtaaatga
ggcctttgag acactcaagc gctgcacgtc gagcaatcca 660aaccagcggt
tgcccaaggt ggagatcctg cgcaacgcca tccgctatat cgagggcctg
720caggctctgc tgcgcgacca ggacgccgcg ccccctggcg ccgcagccgc
cttctatgcg 780ccgggcccgc tgcccccggg ccgcggcggc gagcactaca
gcggcgactc cgacgcgtcc 840agcccgcgct ccaactgctc cgacggcatg
atggactaca gcggcccccc gagcggcgcc 900cggcggcgga actgctacga
aggcgcctac tacaacgagg cgcccagcga acccaggccc 960gggaagagtg
cggcggtgtc gagcctagac tgcctgtcca gcatcgtgga gcgcatctcc
1020accgagagcc ctgcggcgcc cgccctcctg ctggcggacg tgccttctga
gtcgcctccg 1080cgcaggcaag aggctgccgc ccccagcgag ggagagagca
gcggcgaccc cacccagtca 1140ccggacgccg ccccgcagtg ccctgcgggt
gcgaacccca acccgatata ccaggtgctc 1200tgagtttcct gtgaacaatt
gctcctctct taaggtagca aagggtgggc gcgccgaccc 1260agctttcttg
tacaaagtgg tgatggccgc tgtttaaaac ttttatcaag cttatcgata
1320ccgtcgacct cgaatgctgc cttctgcggg gcttgccttc tggccatgcc
cttcttctct 1380cccttgcacc tgtacctctt ggtctttgaa taaagcctga
gtaggaagtg agggtctaga 1440actagtgtcg acgcaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1500aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1560aaaaaaaaaa aaaa
1574872414DNAHomo sapiens 87gggttggacc ctcgtacaga agctaatacg
actcactata gggaaataag agagaaaaga 60agagtaagaa gaaatataag agccaccccg
cggtggcggc cgctctagaa ctagtggatc 120ccccgggctg caggaattcg
ataaaagcga tcgcccatca caagtttgta caaaaaagca 180ggctccacca
tggtttctaa actgagccag ctgcagacgg agctcctggc ggccctgctg
240gagtcagggc tgagcaaaga ggcactgctc caggcactgg gtgagccggg
gccctacctc 300ctggctggag aaggccccct ggacaagggg gagtcctgcg
gcggcggtcg aggggagctg 360gctgagctgc ccaatgggct gggggagact
cggggctccg aggacgagac ggacgacgat 420ggggaagact tcacgccacc
catcctcaaa gagctggaga acctcagccc tgaggaggcg 480gcccaccaga
aagccgtggt ggagaccctt ctgcaggagg acccgtggcg tgtggcgaag
540atggtcaagt cctacctgca gcagcacaac atcccacagc gggaggtggt
cgataccact 600ggcctcaacc agtcccacct gtcccaacac ctcaacaagg
gcactcccat gaagacgcag 660aagcgggccg ccctgtacac ctggtacgtc
cgcaagcagc gagaggtggc gcagcagttc 720acccatgcag ggcagggagg
gctgattgaa gagcccacag gtgatgagct accaaccaag 780aaggggcgga
ggaaccgttt caagtggggc ccagcatccc agcagatcct gttccaggcc
840tatgagaggc agaagaaccc tagcaaggag gagcgagaga cgctagtgga
ggagtgcaat 900agggcggaat gcatccagag aggggtgtcc ccatcacagg
cacaggggct gggctccaac 960ctcgtcacgg aggtgcgtgt ctacaactgg
tttgccaacc ggcgcaaaga agaagccttc 1020cggcacaagc tggccatgga
cacgtacagc gggccccccc cagggccagg cccgggacct 1080gcgctgcccg
ctcacagctc ccctggcctg cctccacctg ccctctcccc cagtaaggtc
1140cacggtgtgc gctatggaca gcctgcgacc agtgagactg cagaagtacc
ctcaagcagc 1200ggcggtccct tagtgacagt gtctacaccc ctccaccaag
tgtcccccac gggcctggag 1260cccagccaca gcctgctgag tacagaagcc
aagctggtct cagcagctgg gggccccctc 1320ccccctgtca gcaccctgac
agcactgcac agcttggagc agacatcccc aggcctcaac 1380cagcagcccc
agaacctcat catggcctca cttcctgggg tcatgaccat cgggcctggt
1440gagcctgcct ccctgggtcc tacgttcacc aacacaggtg cctccaccct
ggtcatcggc 1500ctggcctcca cgcaggcaca gagtgtgccg gtcatcaaca
gcatgggcag cagcctgacc 1560accctgcagc ccgtccagtt ctcccagccg
ctgcacccct cctaccagca gccgctcatg 1620ccacctgtgc agagccatgt
gacccagagc cccttcatgg ccaccatggc tcagctgcag 1680agcccccacg
ccctctacag ccacaagccc gaggtggccc agtacaccca cacaggcctg
1740ctcccgcaga ctatgctcat caccgacacc accaacctga gcgccctggc
cagcctcacg 1800cccaccaagc aggtcttcac ctcagacact gaggcctcca
gtgagtccgg gcttcacacg 1860ccggcatctc aggccaccac cctccacgtc
cccagccagg accctgccgg catccagcac 1920ctgcagccgg cccaccggct
cagcgccagc cccacagtgt cctccagcag cctggtgctg 1980taccagagct
cagactccag caatggccag agccacctgc tgccatccaa ccacagcgtc
2040atcgagacct tcatctccac ccagatggcc tcttcctccc agttgtgagc
ggccgcaccc 2100agctttcttg tacaaagtgg tgatggccgc tgtttaaaac
ttttatcaag cttatcgata 2160ccgtcgacct cgaatgctgc cttctgcggg
gcttgccttc tggccatgcc cttcttctct 2220cccttgcacc tgtacctctt
ggtctttgaa taaagcctga gtaggaagtg agggtctaga 2280actagtgtcg
acgcaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
2340aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2400aaaaaaaaaa aaaa
241488320PRTHomo sapiens 88Met Glu Leu Leu Ser Pro Pro Leu Arg Asp
Val Asp Leu Thr Ala Pro1 5 10 15Asp Gly Ser Leu Cys Ser Phe Ala Thr
Thr Asp Asp Phe Tyr Asp Asp 20 25 30Pro Cys Phe Asp Ser Pro Asp Leu
Arg Phe Phe Glu Asp Leu Asp Pro 35 40 45Arg Leu Met His Val Gly Ala
Leu Leu Lys Pro Glu Glu His Ser His 50 55 60Phe Pro Ala Ala Val His
Pro Ala Pro Gly Ala Arg Glu Asp Glu His65 70 75 80Val Arg Ala Pro
Ser Gly His His Gln Ala Gly Arg Cys Leu Leu Trp 85 90 95Ala Cys Lys
Ala Cys Lys Arg Lys Thr Thr Asn Ala Asp Arg Arg Lys 100 105 110Ala
Ala Thr Met Arg Glu Arg Arg Arg Leu Ser Lys Val Asn Glu Ala 115 120
125Phe Glu Thr Leu Lys Arg Cys Thr Ser Ser Asn Pro Asn Gln Arg Leu
130 135 140Pro Lys Val Glu Ile Leu Arg Asn Ala Ile Arg Tyr Ile Glu
Gly Leu145 150 155 160Gln Ala Leu Leu Arg Asp Gln Asp Ala Ala Pro
Pro Gly Ala Ala Ala 165 170 175Ala Phe Tyr Ala Pro Gly Pro Leu Pro
Pro Gly Arg Gly Gly Glu His 180 185 190Tyr Ser Gly Asp Ser Asp Ala
Ser Ser Pro Arg Ser Asn Cys Ser Asp 195 200 205Gly Met Met Asp Tyr
Ser Gly Pro Pro Ser Gly Ala Arg Arg Arg Asn 210 215 220Cys Tyr Glu
Gly Ala Tyr Tyr Asn Glu Ala Pro Ser Glu Pro Arg Pro225 230 235
240Gly Lys Ser Ala Ala Val Ser Ser Leu Asp Cys Leu Ser Ser Ile Val
245 250 255Glu Arg Ile Ser Thr Glu Ser Pro Ala Ala Pro Ala Leu Leu
Leu Ala 260 265 270Asp Val Pro Ser Glu Ser Pro Pro Arg Arg Gln Glu
Ala Ala Ala Pro 275 280 285Ser Glu Gly Glu Ser Ser Gly Asp Pro Thr
Gln Ser Pro Asp Ala Ala 290 295 300Pro Gln Cys Pro Ala Gly Ala Asn
Pro Asn Pro Ile Tyr Gln Val Leu305 310 315 32089237PRTHomo sapiens
89Met Pro Ala Arg Leu Glu Thr Cys Ile Ser Asp Leu Asp Cys Ala Ser1
5 10 15Ser Ser Gly Ser Asp Leu Ser Gly Phe Leu Thr Asp Glu Glu Asp
Cys 20 25 30Ala Arg Leu Gln Gln Ala Ala Ser Ala Ser Gly Pro Pro Ala
Pro Ala 35 40 45Arg Arg Gly Ala Pro Asn Ile Ser Arg Ala Ser Glu Val
Pro Gly Ala 50 55 60Gln Asp Asp Glu Gln Glu Arg Arg Arg Arg Arg Gly
Arg Thr Arg Val65 70 75 80Arg Ser Glu Ala Leu Leu His Ser Leu Arg
Arg Ser Arg Arg Val Lys 85 90 95Ala Asn Asp Arg Glu Arg Asn Arg Met
His Asn Leu Asn Ala Ala Leu 100 105 110Asp Ala Leu Arg Ser Val Leu
Pro Ser Phe Pro Asp Asp Thr Lys Leu 115 120 125Thr Lys Ile Glu Thr
Leu Arg Phe Ala Tyr Asn Tyr Ile Trp Ala Leu 130 135 140Ala Glu Thr
Leu Arg Leu Ala Asp Gln Gly Leu Pro Gly Gly Gly Ala145 150 155
160Arg Glu Arg Leu Leu Pro Pro Gln Cys Val Pro Cys Leu Pro Gly Pro
165 170 175Pro Ser Pro Ala Ser Asp Ala Glu Ser Trp Gly Ser Gly Ala
Ala Ala 180 185 190Ala Ser Pro Leu Ser Asp Pro Ser Ser Pro Ala Ala
Ser Glu Asp Phe 195 200 205Thr Tyr Arg Pro Gly Asp Pro Val Phe Ser
Phe Pro Ser Leu Pro Lys 210 215 220Asp Leu Leu His Thr Thr Pro Cys
Phe Ile Pro Tyr His225 230 23590272PRTHomo sapiens 90Met Phe Val
Lys Ser Glu Thr Leu Glu Leu Lys Glu Glu Glu Asp Val1 5 10 15Leu Val
Leu Leu Gly Ser Ala Ser Pro Ala Leu Ala Ala Leu Thr Pro 20 25 30Leu
Ser Ser Ser Ala Asp Glu Glu Glu Glu Glu Glu Pro Gly Ala Ser 35 40
45Gly Gly Ala Arg Arg Gln Arg Gly Ala Glu Ala Gly Gln Gly Ala Arg
50 55 60Gly Gly Val Ala Ala Gly Ala Glu Gly Cys Arg Pro Ala Arg Leu
Leu65 70 75 80Gly Leu Val His Asp Cys Lys Arg Arg Pro Ser Arg Ala
Arg Ala Val 85 90 95Ser Arg Gly Ala Lys Thr Ala Glu Thr Val Gln Arg
Ile Lys Lys Thr 100 105 110Arg Arg Leu Lys Ala Asn Asn Arg Glu Arg
Asn Arg Met His Asn Leu 115 120 125Asn Ala Ala Leu Asp Ala Leu Arg
Glu Val Leu Pro Thr Phe Pro Glu 130 135 140Asp Ala Lys Leu Thr Lys
Ile Glu Thr Leu Arg Phe Ala His Asn Tyr145 150 155 160Ile Trp Ala
Leu Thr Glu Thr Leu Arg Leu Ala Asp His Cys Gly Gly 165 170 175Gly
Gly Gly Gly Leu Pro Gly Ala Leu Phe Ser Glu Ala Val Leu Leu 180 185
190Ser Pro Gly Gly Ala Ser Ala Ala Leu Ser Ser Ser Gly Asp Ser Pro
195 200 205Ser Pro Ala Ser Thr Trp Ser Cys Thr Asn Ser Pro Ala Pro
Ser Ser 210 215 220Ser Val Ser Ser Asn Ser Thr Ser Pro Tyr Ser Cys
Thr Leu Ser Pro225 230 235 240Ala Ser Pro Ala Gly Ser Asp Met Asp
Tyr Trp Gln Pro Pro Pro Pro 245 250 255Asp Lys His Arg Tyr Ala Pro
His Leu Pro Ile Ala Arg Asp Cys Ile 260 265 27091214PRTHomo sapiens
91Met Thr Pro Gln Pro Ser Gly Ala Pro Thr Val Gln Val Thr Arg Glu1
5 10 15Thr Glu Arg Ser Phe Pro Arg Ala Ser Glu Asp Glu Val Thr Cys
Pro 20 25 30Thr Ser Ala Pro Pro Ser Pro Thr Arg Thr Arg Gly Asn Cys
Ala Glu 35 40 45Ala Glu Glu Gly Gly Cys Arg Gly Ala Pro Arg Lys Leu
Arg Ala Arg 50 55 60Arg Gly Gly Arg Ser Arg Pro Lys Ser Glu Leu Ala
Leu Ser Lys Gln65 70 75 80Arg Arg Ser Arg Arg Lys Lys Ala Asn Asp
Arg Glu Arg Asn Arg Met 85 90 95His Asn Leu Asn Ser Ala Leu Asp Ala
Leu Arg Gly Val Leu Pro Thr 100 105 110Phe Pro Asp Asp Ala Lys Leu
Thr Lys Ile Glu Thr Leu Arg Phe Ala 115 120 125His Asn Tyr Ile Trp
Ala Leu Thr Gln Thr Leu Arg Ile Ala Asp His 130 135 140Ser Leu Tyr
Ala Leu Glu Pro Pro Ala Pro His Cys Gly Glu Leu Gly145 150 155
160Ser Pro Gly Gly Ser Pro Gly Asp Trp Gly Ser Leu Tyr Ser Pro Val
165 170 175Ser Gln Ala Gly Ser Leu Ser Pro Ala Ala Ser Leu Glu Glu
Arg Pro 180 185 190Gly Leu Leu Gly Ala Thr Phe Ser Ala Cys Leu Ser
Pro Gly Ser Leu 195 200 205Ala Phe Ser Asp Phe Leu 21092356PRTHomo
sapiens 92Met Thr Lys Ser Tyr Ser Glu Ser Gly Leu Met Gly Glu Pro
Gln Pro1 5 10 15Gln Gly Pro Pro Ser Trp Thr Asp Glu Cys Leu Ser Ser
Gln Asp Glu 20 25 30Glu His Glu Ala Asp Lys Lys Glu Asp Asp Leu Glu
Ala Met Asn Ala 35 40 45Glu Glu Asp Ser Leu Arg Asn Gly Gly Glu Glu
Glu Asp Glu Asp Glu 50 55 60Asp Leu Glu Glu Glu Glu Glu Glu Glu Glu
Glu Asp Asp Asp Gln Lys65 70 75 80Pro Lys Arg Arg Gly Pro Lys Lys
Lys Lys Met Thr Lys Ala Arg Leu 85 90 95Glu Arg Phe Lys Leu Arg Arg
Met Lys Ala Asn Ala Arg Glu Arg Asn 100 105 110Arg Met His Gly Leu
Asn Ala Ala Leu Asp Asn Leu Arg Lys Val Val 115 120 125Pro Cys Tyr
Ser Lys Thr Gln Lys Leu Ser Lys Ile Glu Thr Leu Arg 130 135 140Leu
Ala Lys Asn Tyr Ile Trp Ala Leu Ser Glu Ile Leu Arg Ser Gly145 150
155 160Lys Ser Pro Asp Leu Val Ser Phe Val Gln Thr Leu Cys Lys Gly
Leu 165 170 175Ser Gln Pro Thr Thr Asn Leu Val Ala Gly Cys Leu Gln
Leu Asn Pro 180 185 190Arg Thr Phe Leu Pro Glu Gln Asn Gln Asp Met
Pro Pro His Leu Pro 195 200 205Thr Ala Ser Ala Ser Phe Pro Val His
Pro Tyr Ser Tyr Gln Ser Pro 210 215 220Gly Leu Pro Ser Pro Pro Tyr
Gly Thr Met Asp Ser Ser His Val Phe225 230 235 240His Val Lys Pro
Pro Pro His Ala Tyr Ser Ala Ala Leu Glu Pro Phe 245 250 255Phe Glu
Ser Pro Leu Thr Asp Cys Thr Ser Pro Ser Phe Asp Gly Pro 260 265
270Leu Ser Pro Pro Leu Ser Ile Asn Gly Asn Phe Ser Phe Lys His Glu
275 280 285Pro Ser Ala Glu Phe Glu Lys Asn Tyr Ala Phe Thr Met His
Tyr Pro 290 295 300Ala Ala Thr Leu Ala Gly Ala Gln Ser His Gly Ser
Ile Phe Ser Gly305 310 315 320Thr Ala Ala Pro Arg Cys Glu Ile Pro
Ile Asp Asn Ile Met Ser Phe 325 330 335Asp Ser His Ser His His Glu
Arg Val Met Ser Ala Gln Leu Asn Ala 340 345 350Ile Phe His Asp
35593382PRTHomo sapiens 93Met Leu Thr Arg Leu Phe Ser Glu Pro Gly
Leu Leu Ser Asp Val Pro1 5 10 15Lys Phe Ala Ser Trp Gly Asp Gly Glu
Asp Asp Glu Pro Arg Ser Asp 20 25 30Lys Gly Asp Ala Pro Pro Pro Pro
Pro Pro Ala Pro Gly Pro Gly Ala 35 40 45Pro Gly Pro Ala Arg Ala Ala
Lys Pro Val Pro Leu Arg Gly Glu Glu 50 55 60Gly Thr Glu Ala Thr Leu
Ala Glu Val Lys Glu Glu Gly Glu Leu Gly65 70 75 80Gly Glu Glu Glu
Glu Glu Glu Glu Glu Glu Glu Gly Leu Asp Glu Ala 85 90 95Glu Gly Glu
Arg Pro Lys Lys Gly Gly Pro Lys Lys Arg Lys Met Thr 100 105 110Lys
Ala Arg Leu Glu Arg Ser Lys Leu Arg Arg Gln Lys Ala Asn Ala 115 120
125Arg Glu Arg Asn Arg Met His Asp Leu Asn Ala Ala Leu Asp Asn Leu
130 135 140Arg Lys Val Val Pro Cys Tyr Ser Lys Thr Gln Lys Leu Ser
Lys Ile145 150 155 160Glu Thr Leu Arg Leu Ala Lys Asn Tyr Ile Trp
Ala Leu Ser Glu Ile 165 170 175Leu Arg Ser Gly Lys Arg Pro Asp Leu
Val Ser Tyr Val Gln Thr Leu 180 185 190Cys Lys Gly Leu Ser Gln Pro
Thr Thr Asn Leu Val Ala Gly Cys Leu 195 200 205Gln Leu Asn Ser Arg
Asn Phe Leu Thr Glu Gln Gly Ala Asp Gly Ala 210 215 220Gly Arg Phe
His Gly Ser Gly Gly Pro Phe Ala Met His Pro Tyr Pro225 230 235
240Tyr Pro Cys Ser Arg Leu Ala Gly Ala Gln Cys Gln Ala Ala Gly Gly
245 250 255Leu Gly Gly Gly Ala Ala His Ala Leu Arg Thr His Gly Tyr
Cys Ala 260 265 270Ala Tyr Glu Thr Leu Tyr Ala Ala Ala Gly Gly Gly
Gly Ala Ser Pro 275 280 285Asp Tyr Asn Ser Ser Glu Tyr Glu Gly Pro
Leu Ser Pro Pro Leu Cys 290 295 300Leu Asn Gly Asn Phe Ser Leu Lys
Gln Asp Ser Ser Pro Asp His Glu305 310 315 320Lys Ser Tyr His Tyr
Ser Met His Tyr Ser Ala Leu Pro Gly Ser Arg 325 330 335Pro Thr Gly
His Gly Leu Val Phe Gly Ser Ser Ala Val Arg Gly Gly 340 345 350Val
His Ser Glu Asn Leu Leu Ser Tyr Asp Met His Leu His His Asp 355 360
365Arg Gly Pro Met Tyr Glu Glu Leu Asn Ala Phe Phe His Asn 370 375
38094632PRTHomo sapiens 94Met Val Ser Lys Leu Ser Gln Leu Gln Thr
Glu Leu Leu Ala Ala Leu1 5 10 15Leu Glu Ser Gly Leu Ser Lys Glu Ala
Leu Leu Gln Ala Leu Gly Glu 20 25 30Pro Gly Pro Tyr Leu Leu Ala Gly
Glu Gly Pro Leu Asp Lys Gly Glu 35 40 45Ser Cys Gly Gly Gly Arg Gly
Glu Leu Ala Glu Leu Pro Asn Gly Leu 50 55 60Gly Glu Thr Arg Gly Ser
Glu Asp Glu Thr Asp Asp Asp Gly Glu Asp65 70 75 80Phe Thr Pro Pro
Ile Leu Lys Glu Leu Glu Asn Leu Ser Pro Glu Glu 85 90 95Ala Ala His
Gln Lys Ala Val Val Glu Thr Leu Leu Gln Glu Asp Pro 100 105 110Trp
Arg Val Ala Lys Met Val Lys Ser Tyr Leu Gln Gln His Asn Ile 115 120
125Pro Gln Arg Glu Val Val Asp Thr Thr Gly Leu Asn Gln Ser His Leu
130 135 140Ser Gln His Leu Asn Lys Gly Thr Pro Met Lys Thr Gln Lys
Arg Ala145 150 155 160Ala Leu Tyr Thr Trp Tyr Val Arg Lys Gln Arg
Glu Val Ala Gln Gln 165 170 175Phe Thr His Ala Gly Gln Gly Gly Leu
Ile Glu Glu Pro Thr Gly Asp 180 185 190Glu Leu Pro Thr Lys Lys Gly
Arg Arg Asn Arg Phe Lys Trp Gly Pro 195 200 205Ala Ser Gln Gln Ile
Leu Phe Gln Ala Tyr Glu Arg Gln Lys Asn Pro 210 215 220Ser Lys Glu
Glu Arg Glu Thr Leu Val Glu Glu Cys Asn Arg Ala Glu225 230 235
240Cys Ile Gln Arg Gly Val Ser Pro Ser Gln Ala Gln Gly Leu Gly Ser
245 250 255Asn Leu Val Thr Glu Val Arg Val Tyr Asn Trp Phe Ala Asn
Arg Arg 260 265 270Lys Glu Glu Ala Phe Arg His Lys Leu Ala Met Asp
Thr Tyr Ser Gly 275 280 285Pro Pro Pro Gly Pro Gly Pro Gly Pro Ala
Leu Pro Ala His Ser Ser 290 295 300Pro Gly Leu Pro Pro Pro Ala Leu
Ser Pro Ser Lys Val His Gly Val305 310 315 320Arg Tyr Gly Gln Pro
Ala Thr Ser Glu Thr Ala Glu Val Pro Ser Ser 325 330 335Ser Gly Gly
Pro Leu Val Thr Val Ser Thr Pro Leu His Gln Val Ser 340 345 350Pro
Thr Gly Leu Glu Pro Ser His Ser Leu Leu Ser Thr Glu Ala Lys 355 360
365Leu Val Ser Ala Ala Gly Gly Pro Leu Pro Pro Val Ser Thr Leu Thr
370 375 380Ala Leu His Ser Leu Glu Gln Thr Ser Pro Gly Leu Asn Gln
Gln Pro385 390 395 400Gln Asn Leu Ile Met Ala Ser Leu Pro Gly Val
Met Thr Ile Gly Pro 405 410 415Gly Glu Pro Ala Ser Leu Gly Pro Thr
Phe Thr Asn Thr Gly Ala Ser 420 425 430Thr Leu Val Ile Gly Leu Ala
Ser Thr Gln Ala Gln Ser Val Pro Val 435 440 445Ile Asn Ser Met Gly
Ser Ser Leu Thr Thr Leu Gln Pro Val Gln Phe 450 455 460Ser Gln Pro
Leu His Pro Ser Tyr Gln Gln Pro Leu Met Pro Pro Val465 470 475
480Gln Ser His Val Thr Gln Ser Pro Phe Met Ala Thr Met Ala Gln Leu
485 490 495Gln Ser Pro His Ala Leu Tyr Ser His Lys Pro Glu Val Ala
Gln Tyr 500 505 510Thr His Thr Gly Leu Leu Pro Gln Thr Met Leu Ile
Thr Asp Thr Thr 515 520 525Asn Leu Ser Ala Leu Ala Ser Leu Thr Pro
Thr Lys Gln Val Phe Thr 530 535 540Ser Asp Thr Glu Ala Ser Ser Glu
Ser Gly Leu His Thr Pro Ala Ser545 550 555 560Gln Ala Thr Thr Leu
His Val Pro Ser Gln Asp Pro Ala Gly Ile Gln 565 570 575His Leu Gln
Pro Ala His Arg Leu Ser Ala Ser Pro Thr Val Ser Ser 580 585 590Ser
Ser Leu Val Leu Tyr Gln Ser Ser Asp Ser Ser Asn Gly Gln Ser 595 600
605His Leu Leu Pro Ser Asn His Ser Val Ile Glu Thr Phe Ile Ser Thr
610 615 620Gln Met Ala Ser Ser Ser Gln Leu625 630
* * * * *