NZ711775B2 - Genetic modification of rats - Google Patents
Genetic modification of rats Download PDFInfo
- Publication number
- NZ711775B2 NZ711775B2 NZ711775A NZ71177514A NZ711775B2 NZ 711775 B2 NZ711775 B2 NZ 711775B2 NZ 711775 A NZ711775 A NZ 711775A NZ 71177514 A NZ71177514 A NZ 71177514A NZ 711775 B2 NZ711775 B2 NZ 711775B2
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- rat
- cells
- cell
- lif
- genetic modification
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Abstract
Compositions and methods are provided for making rat pluripotent and totipotent cells, including rat embryonic stem (ES) cells. Compositions and methods for improving efficiency or frequency of germline transmission of genetic modifications in rats are provided. Such methods and compositions comprise an in vitro culture comprising a feeder cell layer and a population of rat ES cells or a rat ES cell line, wherein the in vitro culture conditions maintain pluripotency of the ES cell and comprises a media having mouse leukemia inhibitor factor (LIF) or an active variant or fragment thereof. Various methods of establishing such rat ES cell lines are further provided. Methods of selecting genetically modified rat ES cells are also provided, along with various methods to generate a transgenic rat from the genetically modified rat ES cells provided herein. Various kits and articles of manufacture are further provided. e an in vitro culture comprising a feeder cell layer and a population of rat ES cells or a rat ES cell line, wherein the in vitro culture conditions maintain pluripotency of the ES cell and comprises a media having mouse leukemia inhibitor factor (LIF) or an active variant or fragment thereof. Various methods of establishing such rat ES cell lines are further provided. Methods of selecting genetically modified rat ES cells are also provided, along with various methods to generate a transgenic rat from the genetically modified rat ES cells provided herein. Various kits and articles of manufacture are further provided.
Description
GENETIC MODIFICATION OF RATS
FIELD
Non-human pluripotent, totipotent, and embryonic stem (ES) cells, in particular
rat otent, totipotent, and/or rat ES cells, and methods of making them. Methods for
making rat pluripotent, totipotent, and ES cells are provided. Methods for targeting rat
pluripotent, totipotent, and/or ES cells are provided. Methods for achieVing germline
transmission of a genetic modification in a rat cell are provided. Media for deriving,
growing, and maintaining rat otent, totipotent, and ES cells are provided.
NCE TO A SEQUENCE LISTING SUBMITTED AS
A TEXT FILE VIA EFS-WEB
The official copy of the sequence listing is submitted concurrently with the
cation as a text file Via EFS-Web, in compliance with the American Standard
Code for Information Interchange (ASCII), with a file name of seqlist.txt, a
creation date of ry 20, 2014 and a size of 2 Kb. The sequence listing filed Via
EFS-Web is part of the specification and is hereby incorporated in its entirety by
reference herein.
OUND
The rat has been a valuable model for many applications, including, but not
limited to, applications in drug discovery. The usefulness of the rat has been mitigated
somewhat by difficulty in obtaining genetically modified rats, in ular, in
developing methods for genetically modifying rats, and generating useful rat cells that
can be used in genetic modification ols, including but not limited to protocols that
result in germline transmission of a genetic modification in a rat genome.
There is a need in the art for rat cells (e. g., embryonic stem cells) that can be
genetically modified such that the genetic modification can be transmitted h the
germline. There is a need in the art for improved frequency of germline ission of
genetic modifications in rats.
There is a need in the art for donor rat pluripotent, totipotent, and/or ES cells
from various strains of rat that are capable of generating F0, or wholly donor cell-
derived, F0 rats. There is a need in the art for donor rat pluripotent, totipotent, and/or ES
cells that are capable of generating rats that comprise a germline genetic modification.
SUMMARY
Compositions and methods are provided for making rat pluripotent and/or
totipotent cells, including rat embryonic stem (ES) cells. Compositions and methods for
improving ncy or frequency of germline transmission of genetic modifications in
rats are provided. In various s, the s and compositions comprise an in vitro
culture comprising a feeder cell layer and a tion of rat ES cells or a rat ES cell
line, wherein the in vitro culture ions allow nance of pluripotency of the rat
ES cell. Various methods of ishing rat ES cell lines are further provided. Method
of selecting genetically modified rat ES cells are also provided, along with various
methods to generate a transgenic rat from the genetically modified rat ES cells are
ed herein. Various kits and articles of manufacture are further provided.
Non-limiting embodiments are as follows:
1. An ed rat ES cell of a strain selected from ACI or DA, wherein the isolated
rat ES cell is and capable of transmitting its genome through the germline.
2. The isolated rat ES cell of embodiment 1, wherein the cell is derived from an
ACI rat.
3. The isolated rat ES cell of embodiment l or 2, wherein the cell is derived from a
Dark Agouti (DA) rat.2.
4. The isolated rat ES cell of embodiment l, 2, or 3, wherein the cell is euploid and
capable of transmitting a ed genetic modification through the ne.
. The isolated rat ES cell of embodiment 4, wherein the rat ES cell comprises a
germline transmission efficiency of the targeted genetic modification of at least 3%.
6. The isolated rat ES cell of embodiment 4, wherein the rat ES cell has a germline
transmission efficiency of the targeted genetic ation of at least 60%.
7. The isolated rat ES cell of any one of embodiments 1-6, wherein the rat ES cell
exhibits a targeting efficiency of homologous recombination of at least 2%.
8. The isolated rat ES cell of any one of embodiments 1-8, wherein the rat ES cell is
capable of transmitting a targeted genetic modification into progeny ing a
successive round of electroporation.
9. The isolated rat ES cells of any one of embodiments 1-8, wherein the rat ES cell
comprises one or more, two or more, or three or more targeted genetic modification.
. The isolated rat ES cell of any one of embodiments 4-9, wherein the targeted
genetic modification comprises an insertion, a deletion, a ut, a knockin, a point
mutation, or a ation thereof.
11. The isolated rat ES cell of embodiment 9, wherein the targeted genetic
ation comprises at least one insertion of a heterologous polynucleotide into a
genome of the cell.
12. The ed rat ES cell of embodiment 11, wherein the heterologous
polynucleotide comprises a selection marker.
13. The isolated rat ES cell of embodiment 12, wherein (a) the selection marker
comprises a non-attenuated ion marker gene ly linked to a promoter; or (b)
the rat ES cell comprises at least 2 copies of the polynucleotide encoding the selection
marker.
14. The isolated rat ES cell of embodiment 12, wherein the selection marker has an
sed actiVity compared to a wild type selection marker.
15. The isolated rat ES cell of any one of embodiments 1-14, wherein the rat ES cell
forms a sphere-like colony when plated on a feeder cell layer in culture comprising a LIF
polypeptide, a GSK3 inhibitor, and a MEK inhibitor.
16. The isolated rat ES cell of any one of ments 1-15, wherein the rat ES cell,
when cultured in vitro, loosely adhere to the feeder cell layer.
17. The isolated rat ES cell of any one of embodiments 1-16, wherein the cell does
not require ine LIF signaling for maintenance of pluripotency.
18. The isolated rat ES cell of any one of embodiments 1-17, wherein the cell is a
male (XY) rat ES cell.
19. The isolated rat ES cell of any one of embodiments 1-19, wherein the cell is a
female (XX) rat ES cell.
. The isolated rat ES cell of any one of embodiments 1-19, wherein the rat ES cell
can be passaged up to at least 11 times in a medium comprising a GSK3 inhibitor and a
MEK inhibitor without decreasing its ing efficiency or ne transmission
efficiency of a targeted genetic modification.
21. The isolated rat ES cell of any one of embodiments 1-20, wherein the rat ES cells
express at least one pluripotency marker selected from Dnmt3 L, Eras, Err-beta, FbxolS,
Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, Utf1, or a
combination thereof
22. The isolated rat ES cell of any one of embodiments l-2l, wherein the rat ES cells
do not express one or more pluripotency markers selected from c-Myc, Ecatl, Rexol, or
a combination f.
23. The isolated rat ES cell of any one of embodiments l-22, wherein the rat ES cells
do not express one or more mesodermal markers ed from Brachyury, Bmpr2, or a
combination thereof.
24. The ed rat ES cell of any one of embodiments l-23, wherein the rat ES cells
do not express one or more endodermal markers selected from Gata6, Soxl7, Sox7, or
combination thereof;
25. The isolated rat ES cell of any one of embodiments l-24, wherein the rat ES cells
do not express one or more neural markers selected from Nestin, Pax6, or combination
thereof
26. The isolated rat ES cell of any one of embodiments 1-25, wherein the cell
expresses a pluripotency marker comprising Oct-4, Sox2, ne phosphatase, or a
combination thereof
27. The isolated rat ES cell of any one of embodiments 1-26, wherein the rat ES cell
is characterized by the expression of one or more of a rat ESC-speciflc gene selected
from one or more of s Junctions Associate Protein (Aj apl), Claudin 5 (Cldn5),
Cdc42 guanine nucleotide ge factor 9 (Arhgef9), Calcium/calmodulin-dependent
protein kinase IV (Camk4), ephrin-Al (Efnal), EPH receptor A4 (Epha4), gap junction
protein beta 5 (Gij), Insulin-like growth factor binding protein-like l (Igfbpll),
Interleukin 36 beta(Illf8), Interleukin 28 receptor, alpha (Il28ra), left-right
determination factor 1 (Leftyl), ia inhibitory factor receptor alpha (Lifr),
Lysophosphatidic acid receptor 2 (Lpar2), Neuronal pentraxin receptor (Ntm), Protein
tyrosine phosphatase non-receptor type 18 (Ptpnl 8), Caudal type homeobox 2 ,
Fibronectin type III and ankyrin repeat domains 1 (Fankl), Forkhead box El id
transcription factor 2) (Foxel), Hairy/enhancer-of-split related with YRPW motif 2
(Hey2), Forkhead box El (thyroid transcription factor 2) (Foxel), Hairy/enhancer-ofsplit
related with YRPW motif 2 (Hey2), Lymphoid er-binding factor 1 (Lefl),
ke 3 (Drosophila) (Sall3), SATB homeobox l (Satbl), 2, or a combination
thereof
28. An isolated tion of rat ES cells, wherein at least 70% of the rat ES cells are
euploid and form sphere-like colonies when plated on a feeder cell layer in vitro.
29. The isolated population of rat ES cells of embodiment 28, wherein the rat ES
cells are d from an AC1 rat.
. The isolated population of rat ES cells of embodiment 28, wherein the rat ES
cells are derived from a Dark Agouti (DA) rat.
31. The ed population of rat ES cells of any one of embodiments 28-30,
wherein the rat ES cells are capable of transmitting their genome through the germline.
32. The isolated population of rat ES cells of any one of embodiments 28-3l,
wherein the rat ES cells have a germline transmission ncy of the targeted genetic
modification of at least 3%.
33. The isolated population of rat ES cells of any one of embodiments 28-3l,
wherein the rat ES cells have a germline transmission efficiency of the targeted genetic
modification of at least 60%.
34. The ed population of rat ES cells of any one of embodiments 28-3l,
wherein the rat ES cells exhibit a targeting efficiency of homologous recombination of at
least 2%.
. The isolated population of rat ES cells of any one of embodiments 28-34,
wherein the rat ES cells are capable of transmitting a ed genetic modification into
progeny following a successive round of oporation.
36. The isolated population of any one embodiments 28-35, wherein the rat ES cells
comprise one or more, two or more, or three or more targeted genetic modification and
can transmit the targeted genetic modification through the germline.
37. The isolated population of rat ES cells of embodiment 36, wherein the targeted
genetic modification is at the rat Rosa26 locus.
38. The isolated population of rat ES cells of embodiment 36, n the targeted
genetic ation comprises an insertion, a deletion, a knockout, a knockin, a point
mutation, or a combination thereof.
39. The isolated population of rat ES cells of embodiment 36, wherein the targeted
genetic modification comprises at least one insertion of a heterologous polynucleotide
into a genome of the cell.
40. The isolated population of rat ES cells of embodiment 39, n the
logous polynucleotide comprises a selection .
41. The ed population of rat ES cells of embodiment 40, wherein
(a) the selection marker comprises a non-attenuated selection marker gene operably
linked to a promoter; or
(b) the rat ES cell comprises at least 2 copies of the polynucleotide encoding the
selection .
42. The isolated population of rat ES cells of embodiment 40, wherein the selection
marker has an increased activity compared to a wild type selection marker
43. The isolated population of rat ES cells of any one of embodiments 28-42,
wherein the cells form a sphere-like colony when plated on a feeder cell layer in culture
comprising a LIF polypeptide, a GSK3 tor, and a MEK tor.
44. The isolated population rat ES cells of any one of embodiments 28-43, wherein
the cells, when cultured in vitro, loosely adhere to the feeder cell layer.
45. The isolated population of rat ES cells of any one of embodiments 28-44,
n the cells do not require paracrine LIF ing for maintenance of pluripotency.
46. The isolated population of rat ES cells of any one of embodiments 28-44,
wherein the rat ES cells are a male (XY) rat ES cells.
47. The isolated population of rat ES cells of any one of embodiments 28-44,
wherein the rat ES cells are female (XX) rat ES cells.
48. The isolated population of rat ES cells of any one of ments 28-47,
wherein the rat ES cells can be passaged up to at least ll times in a medium comprising
a GSK3 inhibitor and a MEK inhibitor without decreasing its ing efficiency or
germline transmission efficiency of a targeted genetic modification.
49. The isolated population of rat ES cells of any one of embodiments 28-48,
wherein the rat ES cells express at least one pluripotency marker selected from Dnmt3 L,
Eras, Err-beta, Fbxol5, Fgf4, Gdf3, Klf4, Lefl, LIF receptor, Lin28, Nanog, Oct4,
Soxl5, Sox2, Utfl, or a combination thereof.
50. The isolated population of rat ES cells of any one of embodiments 28-49,
wherein the rat ES cells do not s one or more pluripotency markers selected from
c-Myc, Ecatl , Rexol, or a combination thereof
51. The ed population of rat ES cells of any one of embodiments 28-50,
wherein the rat ES cells do not express one or more mesodermal markers selected from
Brachyury, Bmpr2, or a combination thereof
52. The isolated population of rat ES cells of any one of embodiments 28-51,
n the rat ES cells do not express one or more endodermal markers selected from
Gata6, Soxl7, Sox7, or combination thereof;
53. The isolated population of rat ES cells of any one of embodiments 28-52,
wherein the rat ES cells do not express one or more neural markers selected from Nestin,
Pax6, or combination thereof.
54. The isolated population of rat ES cells of any one of embodiment 28-53, wherein
the rat ES cells ses a pluripotency marker comprising Oct-4, Sox2, alkaline
phosphatase, or a combination thereof.
55. The isolated population of rat ES cells of any one of embodiments 28-54,
wherein the rat ES cells are characterized by the expression of one or more of a rat ESC-
specific gene selected from one or more of Adheres Junctions Associate Protein (Aj apl),
Claudin 5 (Cldn5), Cdc42 guanine nucleotide exchange factor 9 f9),
Calcium/calmodulin-dependent protein kinase IV (Camk4), ephrin-Al (Efnal), EPH
receptor A4 (Epha4), gap junction protein beta 5 (Gij), Insulin-like growth factor
binding n-like l (Igfbpll), Interleukin 36 beta(Illf8), Interleukin 28 receptor,
alpha (Il28ra), left-right determination factor 1 (Leftyl), Leukemia inhibitory factor
receptor alpha (Lifr), osphatidic acid receptor 2 ), Neuronal pentraxin
receptor (Ntm), Protein tyrosine phosphatase non-receptor type 18 (Ptpn18), Caudal type
homeobox 2 (Cdx2), Fibronectin type III and ankyrin repeat domains 1 ),
Forkhead box El (thyroid transcription factor 2) (Foxel), Hairy/enhancer-of-split related
with YRPW motif 2 (Hey2), Forkhead box El (thyroid ription factor 2) ),
Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Lymphoid enhancer-binding
factor 1 , Sal-like 3 (Drosophila) (Sall3), SATB homeobox l (Satbl), miR-632, or
a ation thereof
56. The isolated population of rat ES cells of any one of embodiments 28-55
wherein the population comprises at least 104 cells.
57. The isolated population of rat ES cells of any one of embodiments 28-56,
wherein the rat ES cells have one or more characteristic comprising:
a. at least 90% of the rat ES cells are euploid;
b. at least 70% of the rat ES cells express at least one pluripotency ; wherein
the at least one pluripotency marker comprises Oct-4, Sox2, alkaline phosphatase, or a
combination thereof;
c. a cell from the rat ES cell population, when ed with a rat host embryo
transmits the genome of the rat ES cell line into an offspring;
d. the rat ES cells when cultured in vitro loosely adhere to a feeder cell layer;
e. the rat ES cells form sphere-like colonies when plated on a feeder cell layer in
vitro; (f) the rat ES cells maintain pluripotency when cultured in vitro in a media
comprising an GSK3 inhibitor, a MEK inhibitor, LIF and a feeder cell layer that is not
genetically modified to express LIF;
f. the rat ES cell exhibits a targeting efficiency of homologous recombination of at
least 2%;
g. the rat ES cells maintain pluripotency in vitro without requiring ine LIF
signaling;
h. at least 70% of the rat ES cells are euploid and form sphere-like colonies when
plated on a feeder cell layer in vitro;
i. the rat ES cells express at least one otency marker selected from Dnmt3 L,
Eras, Err-beta, FbxolS, Fgf4, Gdf3, Klf4, Lefl, LIF receptor, Lin28, Nanog, Oct4,
SoxlS, Sox2, Utfl, or a combination thereof;
j. the rat ES cells do not express one or more differentiation markers selected from
c-Myc, Ecatl, Rexol;
k. the rat ES cells do not express one or more mesodermal markers selected from
Brachyury, Bmpr2, or a combination f;
1. the rat ES cells do not express one or more rmal s ed from
Gata6, Soxl7, Sox7, or combination thereof; and/or
m. the rat ES cells do not express one ore more neural markers selected from
Nestin, Pax6, or combination thereof
58. The isolated tion of rat ES cells of any one of embodiments 28-57,
wherein (a) the rat ES cells is derived from a rat blastocyst; (b) the rat ES cell is derived
from a rat morula stage embryo; and/or, (c) the rat ES cell line is derived from a
superovulated rat.
59. An in vitro culture comprising a feeder cell layer, the population of rat embryonic
stem (ES) cells, and a medium sing a Leukemia Inhibitory Factor (LIF), GSK3
inhibitor, and a MEK inhibitor, n at least 70% of the rat ES cells are euploid and
the rat ES cell forms a sphere-like colony.
60. The in vitro culture of embodiment 59 or 60, wherein the rat ES cell, loosely
adhere to the feeder cell layer.
61. The in vitro culture of embodiment 59, 60, or 61, wherein the rat ES cells are
capable of transmitting their genome through the germline.
62. The in vitro culture of embodiment 59, 60 or 61, wherein the rat ES cells are
derived from an AC1 rat.
63. The in vitro culture of ment 59, 60 or 61, wherein the rat ES cells are
derived from a Dark Agouti (DA) rat.2.
64. The in vitro culture of any one of embodiments 59-63, wherein the rat ES cells
are capable of transmitting a targeted genetic modification through the germline.
65. The in vitro culture of embodiment 64, wherein the rat ES cells comprise a
ne transmission efficiency of the targeted genetic modification of at least 3%.
66. The in vitro culture of ment 64, wherein the rat ES cells have a germline
transmission efficiency of the targeted genetic modification of at least 60%.
67. The in vitro culture of any one of embodiments 59-66, wherein the rat ES cells
exhibit a targeting ncy of gous recombination of at least 2%.
68. The in vitro culture of any one of embodiments 59-67, wherein the rat ES cell is
capable of transmitting a ed genetic modification into progeny following a
successive round of electroporation.
69. The in vitro culture of any one of embodiments 59-68, wherein the rat ES cell
comprises one or more, two or more, or three or more targeted genetic modification.
70. The in vitro e of embodiment 69, wherein the targeted genetic modification
comprises an insertion, a deletion, a knockout, a knockin, a point mutation, or a
combination thereof
71. The in vitro e of embodiment 69, wherein the targeted genetic modification
comprises at least one insertion of a heterologous polynucleotide into a genome of the
cell.
72. The in vitro culture of ment 71, wherein the heterologous polynucleotide
comprises a selection marker.
73. The in vitro culture of embodiment 72, wherein (a) the selection marker
comprises a non-attenuated ion marker gene operably linked to a promoter; or (b)
the rat ES cell comprises at least 2 copies of the polynucleotide encoding the ion
marker.
74. The in vitro culture of embodiment 72, n the selection marker has an
increased activity compared to a wild type selection marker.
75. The in vitro culture of any one of embodiments 59-74, wherein the cell does not
require paracrine LIF signaling for maintenance of pluripotency.
76. The in vitro culture of embodiment of any one of embodiments 59-75, wherein
the cell is a male (XY) rat ES cell.
77. The in vitro culture of embodiment of any one of embodiments 59-75, wherein
the cell is a female (XX) rat ES cell.
78. The in vitro culture of embodiment of any one of ments 59-77, n
the rat ES cell can be passaged up to at least ll times in a medium comprising a GSK3
inhibitor and a MEK tor without decreasing its targeting efficiency or germline
transmission efficiency of a targeted genetic modification.
79. The in vitro culture of any one of embodiments 59-78, wherein the rat ES cells
s at least one pluripotency marker selected from Dnmt3 L, Eras, Err-beta, FbxolS,
Fgf4, Gdf3, Klf4, Lefl, LIF receptor, Lin28, Nanog, Oct4, SoxlS, Sox2, Utfl, or a
combination thereof
80. The in vitro culture of any one of embodiments 59-79, wherein the rat ES cells
do not express one or more pluripotency s selected from c-Myc, Ecatl, Rexol, or
a combination thereof
81. The in vitro culture of any one of ments 59-80, wherein the rat ES cells do
not express one or more mesodermal markers ed from Brachyury, Bmpr2, or a
combination thereof
82. The in vitro culture of any one of embodiments 59-81, wherein the rat ES cells do
not express one or more endodermal markers selected from Gata6, Soxl7, Sox7, or
combination thereof;
83. The in vitro culture of any one of embodiments 59-82, wherein the rat ES cells do
not express one or more neural markers ed from Nestin, Pax6, or combination
thereof
84. The in vitro culture of any one of embodiments 59-83, wherein the cell expresses
a pluripotency marker comprising Oct-4, Sox2, alkaline phosphatase, or a combination
thereof
85. The in vitro culture of any one of embodiments 59-84, wherein the rat ES cells
are characterized by the expression of one or more of a rat ESC-specific gene selected
from one or more of s Junctions Associate Protein (Aj apl), Claudin 5 (Cldn5),
Cdc42 guanine nucleotide exchange factor 9 (Arhgef9), Calcium/calmodulin-dependent
protein kinase IV (Camk4), ephrin-Al ), EPH receptor A4 (Epha4), gap junction
protein beta 5 (Gij), Insulin-like growth factor binding protein-like l (Igfbpll),
Interleukin 36 beta(Illf8), Interleukin 28 receptor, alpha (Il28ra), left-right
ination factor 1 (Leftyl), Leukemia inhibitory factor or alpha (Lifr),
Lysophosphatidic acid receptor 2 (Lpar2), Neuronal pentraxin receptor (Ntm), n
tyrosine phosphatase non-receptor type 18 (Ptpnl 8), Caudal type homeobox 2 ,
Fibronectin type III and ankyrin repeat domains 1 (Fankl), Forkhead box El (thyroid
transcription factor 2) (Foxel), Hairy/enhancer-of-split related with YRPW motif 2
(Hey2), Forkhead box El (thyroid transcription factor 2) (Foxel), Hairy/enhancer-of—
split related with YRPW motif 2 (Hey2), Lymphoid enhancer-binding factor 1 (Lefl),
Sal-like 3 (Drosophila) (Sall3), SATB homeobox l ), miR-632, or a combination
thereof
86. The in vitro e of any one of embodiments 59-84, wherein the concentration
of LIF is 50 U/ml to 150 U/ml.
87. The in vitro culture of any one of embodiments 59-85, wherein the concentration
of LIF is 100 U/ml.
88. The in vitro culture of any one of embodiments 59-87, wherein the LIE is
from mouse or comprises at least 92% sequence identity to SEQ ID NO: 1.
89. The in vitro culture of any one of embodiments 59-88, wherein the rat ES cell is
capable of maintaining a pluripotency without requiring a paracrine LIF signaling.
90. The in vitro culture of any one of embodiments 59-89, wherein the feeder cell
layer is not genetically modified to express LIF.
91. The in vitro culture of any one of embodiments 59-90, wherein the feeder cell
layer comprises a monolayer of mitotically vated mouse embryonic fibroblasts
(MEFs)
92. The in vitro culture of any one of embodiments 59-91, wherein the MEK
inhibitor comprises PD032590l.
93. The in vitro culture of any one of embodiment 59-92, n the GSK-3
inhibitor comprises CHIR9902l.
94. The in vitro culture of any one of embodiments 59-93, wherein the population of
rat ES cells is derived from a rat blastocyst-stage embryo or a rat morula-stage .
95. The in vitro culture of embodiment 94, wherein the blastocyst-stage or the
morula-stage rat embryo r comprises an outgrowth of an amorphous
undifferentiated mass of rat ES cells.
96. The in vitro culture of of embodiment 94, wherein the tion of rat ES cells
comprises an isolated outgrth of an amorphous undifferentiated mass of rat ES cells.
2014/017452
97. A method for generating a rat embryonic stem (ES) cell line comprising:
(a) culturing in vitro a first feeder cell layer and a morula or a blastocyst-stage rat
embryo, wherein the zona pellucida of the morula or blastocyst-stage rat embryo has
been removed, and n the culture ions maintain pluripotency of a rat ES cell
and comprise a medium having mouse leukemia inhibitor factor (LIF) or a sequence
having at least 91% sequence identity to SEQ ID NO:1 and having LIF activity, and a
GSK3 inhibitor, and a MEK inhibitor; and, (b) erring an outgrowth of an
amorphous undifferentiated mass of rat ES cells to an in vitro e well comprising a
second feeder cell layer and culturing the outgrth under conditions comprising the
medium having the mouse LIF or an active variant of the mouse LIF, and thereby
maintaining pluripotency of the rat ES cells; and, establishing a rat ES cell line
therefrom.
98. The method of embodiment 97, wherein the rat ES cell line is passaged at
least 5 times.
99. The method of embodiment 97 or 98, wherein the rat ES cell line is
passaged at least 10 times.
100. The method of embodiment 97, 98, or 99, wherein the medium comprises
about 50 U/ml to about 150 U/ml of mouse LIF.
101. The method of any one of embodiments , wherein the medium
comprises about 100 U/ml ofmouse LIF.
102. The method of any one of embodiments 97-101, wherein the feeder cell
layer is not genetically modified to express LIF.
103. The method of any one of embodiments 97-102, wherein the feeder cell
layer comprises a monolayer of mitotically inactivated mouse embryonic fibroblasts
(MEFs).
104. The method of any one of embodiments 97-103, wherein the MEK
tor comprises PD0325901.
105. The method of any one of embodiments 97-104, wherein the GSK-3
inhibitor comprises CHIR99021.
106. The method of any one of embodiment 97-105, wherein (a) the rat ES cell
line is derived from an AC1 rat or derived from a Dark Agouti (DA) rat; (b) the rat ES
cell line is derived from a -stage or a blastocyst-stage rat embryo; and/or, (c) the
rat ES cell line is derived from a morula-stage or a blastocyst-stage embryo from a
superovulated rat.
107. The method of any one of embodiments 97-106, wherein the medium
r comprises at least one of an FGF receptor inhibitor, a ROCK inhibitor, or an
ALK inhibitor.
108. The method of embodiment 107, wherein the FGF receptor inhibitor
comprises PD184352, the ROCK inhibitor comprises Y-27632, or the ALK inhibitor
comprises A01.
109. The method any one of embodiments 97-108, wherein at least one rat ES
cell has a germline transmission efficiency of the targeted c modification is at least
110. The method of embodiment any one of embodiments 97-109, wherein the
germline transmission efficiency of the ed genetic modification is at least 60%.
111. A method of selecting a rat embryonic stem (ES) cells having stably
incorporated into its genome a heterologous polynucleotide comprising: (a) ing an
in vitro tion of rat ES cells; (b) introducing into at least one rat ES cell a
heterologous polynucleotide comprising a selection marker operably linked to a
promoter active the rat ES cell; and, (c) culturing in vitro the rat ES cell population in an
alternating first and second e media, wherein the first culture medium comprises an
effective amount of a selection agent for a first time period and the second culture
medium does not comprise the selection agent, wherein the in vitro culture conditions are
sufficient to maintain pluripotency; thereby selecting the rat ES cell having stably
integrated into its genome the heterologous polynucleotide.
112. The method of embodiment 111, n the first and second culture
media are alternated every 24 hours.
113. The method of embodiment 111 or 112, wherein the selection marker
imparts ance to an antibiotic.
114. The method of any one of embodiments 111-113, wherein the antibiotic
comprises G418.
115. The method of any one of embodiment 111-114, wherein the ion
marker comprises neomycin phosphotransferase (neor), hygromycin B
phosphotransferase (hygr), puromycin-N-acetyltransferase (puror) blasticidin S
deaminase (bsrr), xanthine/guanine phosphoribosyl transferase (gpt), and herpes simplex
virus thymidine kinase (HSV-k), or a ation thereof.
116. The method of any one of ments 111-115, wherein (a) the
selection marker has an increased activity compared to the wild type ion marker;
and/or (b) multiple copies of the selection marker are stably incorporated into the
genome of the rat ES cell.
117. The method of embodiment 116, wherein the selection marker is a non-
attenuated selection marker.
118. A method for genetically modifying an isolated rat embryonic stem (ES)
cell comprising ucing into the genome of an isolated rat ES cell of any one of
embodiment 1-58 a heterologous polynucleotide to form a genetically modified rat ES
cell.
119. A method ofmaking a genetically modified rat comprising:
(a) introducing into the genome of the isolated rat embryonic stem (ES) cell of
any one of embodiments 1-58, a heterologous polynucleotide to form a rat ES cell having
a genetic modification;
(b) introducing at least one of the rat ES cells comprising the targeted genetic
modification into a rat host embryo to produce an F0 embryo;
(c) implanting the F0 embryo into a surrogate mother;
(d) gestating the F0 embryo in the surrogate mother to term; and, (e) identifying
an F0 rat haVing the targeted genetic modification.
120. The method of embodiment 119, r comprising ng a male F0
rat with a wild type female rat to produce an Fl progeny that is heterozygous for the
targeted c modification.
121. The method of embodiment 120, further comprising breeding a male F0
rat with a wild type female rat to e an Fl y that is heterozygous for the
targeted genetic modification.
122. The method of embodiment 119, further comprising breeding a male rat
of the F1 progeny with a female rat of the F1 progeny to obtain an F2 progeny that is
homozygous for the c modification.
123. The method of any one of embodiments 119-122, wherein at least 3% of
the F0 rats haVing the c modification transmit the genetic modification to the F1
progeny.
124. The method of any one of embodiments 119-123, wherein at least 10% of
the F0 rats haVing the c modification transmit the genetic modification to the F1
progeny.
125. The method of any one of ments 119-124, wherein at least 60% of
the F0 rats having the genetic modification transmit the genetic modification to the F1
progeny.
126. The method of any one of embodiments 119-125, wherein the genetically
modified rat ES cell is from the same rat strain as the rat host embryo.
127. The method of any one of embodiments 119-127, n the genetically
modified rat ES cell is from a different rat strain as the rat host embryo.
128. The isolated population of rat ES cells of any of the preceding claims, the
in Vitro culture of any of the preceding claims, or the method of any of the preceding
claims, wherein the rat ES cells in the population se:
(a) at least 90% of the rat ES cells are euploid;
(b) at least 70% of the rat ES cells express at least one pluripotency
marker; wherein the at least one pluripotency marker comprises Oct-4, Sox2, alkaline
phosphatase, or a combination thereof;
(c) a cell from the rat ES cell population, when combined with a rat host
embryo transmits the genome of the rat ES cell line into an offspring;
(d) the rat ES cells when cultured in Vitro loosely adhere to a feeder cell
layer;
(e) the rat ES cells form sphere-like colonies when plated on a feeder cell
layer in Vitro;
(f) the rat ES cells maintain pluripotency when cultured in Vitro in a
media comprising an GSK3 inhibitor, a MEK inhibitor, LIF and a feeder cell layer that is
not cally modified to express LIF;
(g) the rat ES cell exhibits a targeting efficiency of homologous
recombination of at least 2%;
(h) the rat ES cells maintain pluripotency in Vitro without ing
paracrine LIF signaling;
(i) at least 70% of the rat ES cells are euploid and form sphere-like
colonies when plated on a feeder cell layer in Vitro;
(j) the rat ES cells s at least one pluripotency marker ed from
Dnmt3L, Eras, ta, Fbxol5, Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog,
Oct4, Sox15, Sox2, Utf1, or a ation thereof;
(k) the rat ES cells do not express one or more differentiation markers
selected from c-Myc, Ecat1, Rexol.
(l) the rat ES cells do not express one or more mesodermal markers
selected from Brachyury, Bmpr2, or a combination thereof;
(m) the rat ES cells do not express one or more endomermal markers
selected from Gata6, Soxl7, Sox7, or combination thereof; and/or
(11) the rat ES cells do not express one or more neural markers ed
from Nestin, Pax6, or combination thereof.
BRIEF DESCRIPTION OF THE FIGURES
The patent or application file contains at least one drawing executed in color.
Copies of this patent or patent application publication with color g(s) will be
ed by the Office upon request and payment of the necessary fee.
depicts rESCs, which grow as compact spherical colonies that routinely
detach and float in the dish.
through D depicts various pluripotency markers expressed by rESCs: A
depicts Oct-4 (green); B depicts Sox-2 (red); C s DAPI (blue); D depicts an
y of pluripotency markers expressed by rESCs.
depicts that the rESCs express light levels of alkaline phosphatase (a
pluripotency ) (left), and the ype for line DA.2B is 42X,Y (right).
Karyotyping was done because rESCs often become tetraploid; lines were thus pre-
screened by counting ase chromosome spreads, and lines with mostly normal
counts were then formally karyotyped.
depicts a closer view of a rESC of
depicts production of chimeras by blastocyst injection and transmission of
the rESC genome through the germline; chimeras produced by blastocyst ion using
parental ACI.Gl rESC; high percentage chimeras usually have albino snouts.
depicts Fl agouti pups with albino littermates, sired by ACI/SD chimera
labeled with an asterisk (*) in
Panel A depicts targeting of the rat Rosa 26 locus, which lies between the
Setd5 and Thumpd3 genes as in mouse, with the same spacing. Panel A shows the
structure of the mouse Rosa 26 locus. mRosa26 transcripts consist of 2 or 3 exons.
Panel B depicts the structure of the rRosa26 locus; the rat locus ns a second exon 1
(Exlb) in addition to the homologous exon to mouse exonl (Exla); no third exon has
been identified in rat. Panel C depicts a targeted rRosa26 allele; homology arms of Skb
each were cloned by PCR using genomic DNA from DA rESC; the ed allele
contains a SA-lacZ-hUB-neo cassette ing a ll7bp deletion in the rRosa26 .
depicts a control brain of a l4-week-old wild type rat, which was treated
with X-gal. The control brain showed a low level of ound staining for LacZ
(dorsal view).
depicts LacZ expression in the brain of an rRosa26 heterozygous rat (14-
week old). The lacZ reporter was expressed ubiquitously throughout the brain of the
rRosa26 heterozygote.
depicts a control heart and thymus (inset) of a l4-week-old wild type rat,
which were treated with X-gal. The control heart and thymus showed a low level of
background staining for LacZ.
depicts LacZ expression in the heart and thymus (inset) of a 14-week-
old rRosa26 heterozygous rat. The lacZ reporter was expressed ubiquitously throughout
the heart and thymus of the rROSA26 heterozygote.
depicts a l lung of a l4-week-old wild type rat, which were treated
with X-gal. The control lung showed a low level of background staining for LacZ.
depicts LacZ expression in the lung of a l4-week-old rRosa26
heterozygote rat. The lacZ reporter was expressed ubiquitously throughout the lung of
the rRosa26 heterozygote.
and H depict LacZ expression in e12.5 embryos. In contrast to the wild-
type control embryo (H), which shows a low level of background LacZ staining, the
rRosa26 heterozygous embryo exhibited ubiquitous expression of the LacZ reporter
throughout the embryo.
and J depict LacZ expression in el4.5 s. In st to the wild-
type control embryo (J), which shows a low level of background LacZ staining, the
rRosa26 zygous rat embryo exhibited ubiquitous expression of the LacZ reporter
throughout the embryo.
Figure 9A-B provides a photograph showing the analysis of the chromosome
number of the ACI.Gl rat ES cell line.
Figure lOA-B provides a photograph showing the analysis of the chromosome
number of the DA.2B rat ES cell line.
Figure llA-B provides a photograph showing the is of the chromosome
number of the DA.C2 rat ES cell line.
DETAILED DESCRIPTION
The t methods and compositions now will be described more fully
hereinafter with reference to the accompanying drawings, in which some, but not all
embodiments of the methods and compositions are shown. Indeed, these methods and
compositions may be embodied in many different forms and should not be construed as
limited to the embodiments set forth herein; rather, these embodiments are provided so
that this disclosure will satisfy able legal requirements. Like numbers refer to like
elements throughout.
Many modifications and other embodiments of the s and compositions set
forth herein will come to mind to one skilled in the art to which this methods and
compositions pertains having the benefit of the teachings presented in the foregoing
descriptions and the associated drawings. Therefore, it is to be understood that the
methods and compositions are not to be limited to the specific embodiments disclosed
and that modifications and other embodiments are included within the scope of the
appended claims. Although specific terms are employed herein, they are used in a
c and descriptive sense only and not for purposes of tion.
I. Overview
The rat has long been the preferred rodent model organism for several fields of
biomedical research such as cardiovascular e, metabolism, toxicology,
neurobiology and behavior. Hundreds of strains of rat have been developed; some are
excellent models for x human diseases such as hypertension, diabetes and cancer.
However, progress in understanding the genetics of these models has been severely
hampered by the lty of modifying the rat genome in a controlled manner. Through
the use of pecific endonucleases it is le to produce mutations in a gene of
interest, but this method s imprecise and expensive. Targeting and germline
transmission of rat ES cells remains a difficult task to achieve.
Isolation of rat ES cells (rESC) from two inbred s of rat is described herein.
rESC from the DA and ACI strains were derived. These cells express pluripotency
markers and exhibit a normal 42X,Y karyotype. High percentage chimeras have been
produced, by microinjection into SD host embryos at the blastocyst stage, and
transmission of the rESC genome has been demonstrated through the germline for both
strains. Using plasmid targeting vectors, we have produced targeted ons in the rat
lent of the ROSA26 locus, and we have achieved germline transmission of the
targeted allele in both strains. These heterozygous animals express lacZ in all tissues at
all stages examined.
In various aspects, ES cells were derived from the ACI strain in order to obtain a
favorable number of male progeny from ACI donor ES cells. In one ment, the
amount of male progeny is about 50%.
In various aspects, ES cells were derived from the DA strain in order to obtain
primarily female progeny.
II. Rat Embryonic Stem (ES) Cells
Various compositions and methods are provided herein which comprise
nic stem (ES) cells from rat. Stem cells are a cell population possessing the
capability to self-renew indefinitely and are pluripotent. An "embryonic stem cell" or an
“ES cell” comprises a stem cell obtained from an embryo or a fetus. The various rat ES
cells provided herein can have one or more of any of the following properties:
(a) have germ-line competency, meaning when the rat ES cell is implanted
into a rat host embryo, the genome of the rat ES cell line is transmitted into an
offspring;
(b) have germ-line competency following at least one targeted genetic
ation, meaning when the rat ES cell having the targeted genetic
modification is implanted into a rat host embryo, the targeted c
modification within the genome of the rat ES cell line is transmitted into an
offspring;
(c) have pluripotency in vitro;
(d) have totipotency in vitro;
(e) when cultured in vitro loosely adhere to a feeder cell layer;
(f) when cultured in vitro form sphere-like es when plated on a feeder
cell layer in vitro;
(g) maintain pluripotency when cultured in vitro under conditions comprising
a feeder cell layer that is not genetically modified to express leukemia inhibitor
factor (LIF), wherein the culture media ses a ent concentration of
LIF;
(h) maintain pluripotency when cultured in vitro under conditions comprising
a feeder cell layer, wherein the culture media comprises mouse LIF or an active
t or nt thereof;
WO 30706
(i) comprise a molecular signature that is characterized by
i) the sion of one or more of rat ES cell-specific genes comprising
Adheres Junctions Associate Protein (Aj apl), Claudin 5 (Cldn5), Cdc42 guanine
nucleotide exchange factor 9 (Arhgef9), Calcium/calmodulin-dependent protein kinase
IV (Camk4), ephrin-Al (Efnal), EPH receptor A4 (Epha4), gap junction protein beta 5
(Gij), Insulin-like growth factor binding n-like l (Igfbpll), Interleulin 36
beta(Illf8), Interleukin 28 receptor, alpha (Il28ra), left-right determination factor 1
l), Leukemia inhibitory factor receptor alpha (Lifr), Lysophosphatidic acid
receptor 2 (Lpar2), Neuronal xin receptor (Ntm), Protein tyrosine phosphatase
non-receptor type 18 (Ptpnl8), Caudal type homeobox 2 (Cdx2), Fibronectin type III and
ankyrin repeat domains 1 (Fankl), Forkhead box El (thyroid transcription factor 2)
(Foxel), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Forkhead box El
id transcription factor 2) (Foxel), Hairy/enhancer-of-split related with YRPW
motif 2 (Hey2), Lymphoid enhancer-binding factor 1 (Lefl ), Sal-like 3 phila)
(Sall3), SATB homeobox l (Satbl), miR-632, or a combination thereof;
ii) the expression ofat least 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, l2, l3, 14, 15, l6,
17, 18, 19, 20, 21, 22, 23, 24, 25 or more of the rat ES cell-specific genes comprising
Adheres Junctions Associate Protein (Aj apl), Claudin 5 (Cldn5), Cdc42 guanine
nucleotide exchange factor 9 (Arhgef9), Calcium/calmodulin-dependent n kinase
IV (Camk4), ephrin-Al (Efnal), EPH receptor A4 (Epha4), gap junction protein beta 5
(Gij), n-like growth factor binding n-like l (Igfbpll), Interleulin 36
beta(Illf8), Interleukin 28 receptor, alpha (Il28ra), left-right determination factor 1
(Leftyl), Leukemia inhibitory factor receptor alpha (Lifr), Lysophosphatidic acid
receptor 2 (Lpar2), Neuronal pentraxin receptor (Ntm), Protein tyrosine phosphatase
non-receptor type 18 (Ptpnl8), Caudal type homeobox 2 (Cdx2), Fibronectin type III and
ankyrin repeat domains 1 (Fankl), Forkhead box El id transcription factor 2)
(Foxel), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Forkhead box El
(thyroid transcription factor 2) (Foxel), Hairy/enhancer-of-split d with YRPW
motif 2 (Hey2), id enhancer-binding factor 1 (Lefl ), Sal-like 3 (Drosophila)
(Sall3), SATB ox l (Satbl), 2, or a combination thereof;
iii) at least a 20-fold increase in the expression of one or more of the rat
ES cell-specific genes as set forth in Table 14 when compared to a F1H4 mouse ES cell;
iv) at least a 20-fold increase in the expression of at least 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more ofthe rat ES
cell-specific genes as set forth in Table 14 when compared to a F1H4 mouse ES cell;
V) the expression of one or more of rat ES pecific genes as set forth
in Table 13;
vi) the expression ofat least 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more ofthe rat ES cell-specific
genes as set forth in Table 13;
vii) at least a 20-fold increase in the expression of one or more of the rat
ES cell-specific genes as set forth in Table 13 when compared to a F1H4 mouse ES cell;
viii) at least a 20-fold se in the expression of at least 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or
more of the rat ES cell-specific genes as set forth in Table 13 when ed to a F1H4
mouse ES cell;
ix) at least a 20-fold decrease in the expression of one or more of the rat
ES cell-specific genes as set forth in Table 12 when ed to a F1H4 mouse ES cell;
and/or
x) at least a 20-fold decrease in the sion of at least 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or
more of the rat ES cell-specific genes as set forth in Table 12 when compared to a F1H4
mouse ES cell;
xi) any combination of sion of the rat ES cell-specific genes of
parts (i)-(X);
xii) a relative expression level of pluripotency markers as shown in Table
15 for at least 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16,17 or 18 ofthe listed
pluripotency markers. See, otency ranking column of Table 15 for relative
expression levels;
xiii) a relative expression level of the mesodermal markers as shown in
Table 15 for at least 2, 3, or 4 of the listed mesodermal markers. See, mesodermal
ranking column in Table 15 for relative expression levels;
xiv) a relative expression level of endodermal markers as shown in Table
for at least 2, 3, 4, 5, or 6 of the listed endodermal markers. See, endodermal ranking
column in Table 15 for relative expression levels;
xv) a relative sion level of neural markers as shown in Table 15 for
at least 2 and 3 of the listed neural markers. See, neural ranking column in table 15 for
relative expression levels;
xvi) a relative expression level of ctoderm markers as shown in
Table 15 for the listed trophectoderm markers. See, trophectoderm ranking column in
table 15 for relative expression levels;
xvii) any relative expression level of one or more (2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,2 4, 25, 26, 27, 28, 29 or 30) ofthe
pluripotency markers, mesodermal markers, endodermal markers, neural markers and/or
trophectoderm markers set forth in Table 15;
xviii) the ve expression level of each of the markers set forth in
table 15;
xix) any combination of the signatures set forth in xii-xiix; and/or
xx) any combination of the signature set forth in i-xiix;
(j) have the ability to produce a F0 rat;
(k) are capable of being subcultured and maintaining the undifferentiated
state;
(1) have the same number of somes as a normal rat cell;
(m) maintain pluripotency in vitro without requiring paracrine LIF signaling;
and/or
(11) have self renewal, meaning they divide indefinitely while maintaining
otency.
One or more of the characteristics outlined in (a)-(n) can be present in an isolated
rat ES cell, a rat ES cell population or a rat ES cell line provided herein, wherein the rat
ES cells have not undergone a ed genetic modification. In other embodiments, one
or more of the characteristics outlined in (a)-(n) can be t in an ed rat ES cell,
a rat ES cell population or a rat ES cell line provided herein that has one or more targeted
genetic modifications. A targeted genetic modification ses an alteration in the
genome of the rat ES cell and includes, for example, an insertion, a deletion, a knockout,
a knockin, a mutation, or a combination thereof. In other instances, the targeted genetic
modification ses at least one insertion of a logous polynucleotide into the
genome of the rat ES cell. A fiarther description of such targeted genetic modifications
are discussed elsewhere herein.
In c embodiments, the various rat ES cells and cell lines provided herein
are germ-line ent, g when the rat ES cell is implanted into a rat host
embryo, the genome of the rat ES cell is transmitted into an offspring. Such transmission
into the offspring (i.e., the F1 population) can occur when the rat ES cell has not
undergone a targeted genetic modification. In addition, a rat ES cell haVing a targeted
genetic modification are also germ-line ent, meaning when the rat ES cell haVing
the targeted genetic ation is implanted into a rat host embryo, the targeted genetic
modification of the rat ES cell is transmitted to the ing (i.e., the F1 population.)
Thus, in various aspects, the rat ES cells and methods described herein are employed to
obtain a high frequency, or high efficiency, of germline transmission of a a rat cell
genome from both rat ES cells that have not undergone a targeted genetic modification
and also from rat ES cells that have undergone a targeted genetic modification. In
s embodiments, the ncy of germline transmission is greater than 1:600,
greater than 1:500, greater than 1:400, greater than 1:300, greater than 1:200, and greater
than 1:100. In various embodiments, the ncy of germline transmission is greater
than 1%, greater than 2%, greater than 3%, r than 4%, greater than 5%, greater
than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, up to
about 16%, greater than 25%, greater than 50%, greater than 60%, greater than 65%,
greater than 70%, greater than 75% or greater. In various embodiments, the frequency of
germline transmission ranges from 9% to 16%. In various aspects, percent of donor
rESC-derived progeny in the F1 generation is 1% or more, 2% or more, 3% or more,
% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more,
from 3% to about 10% or more; from 3% or more to about 63%, from about 10% to
about 30%, from about 10% to about 50%, from about 30% to about 70%, from about
30% to about 60%, from about 20% to about 40%, from about 20% to 65%, or from
about 40% to 70%. Thus, a rat ES cell provided herein that has not undergone a
targeted genetic modification or, alternatively, a rat ES cell that has a targeted genetic
modification have the ability to transmit their genome into the F1 population.
A rat ES cell that has not undergone a targeted genetic modification or a rat ES
cell that has a targeted genetic modification can be pluripotent and/or totipotent.
“Pluripotent” or “pluripotency” refers to a stem cell that has the potential to differentiate
into any of the three germ layers: endoderm, mesoderm, or rm. Cell y is a
l term which describes a cell's ability to differentiate into other cell types. See, for
example, Hans et al. (2007). "The Potential of Stem Cells: An Inventory". Human
biotechnology as Social Challenge. Ashgate Publishing, Ltd. p. 28, herein incorporate by
reference. The term “totipotency” or “totipotent” is the ability of a single cell to divide
and produce all of the differentiated cells in an organism. See, for example, Western P
(2009). Int. J. Dev. Biol. 53 (2—3): 393—409, herein incorporated by reference. In
specific embodiments, the various ES cells disclosed herein can be either pluripotent
and/or totipotent.
s methods can be used to determine if a rat ES cell is pluripotent. For
e, the ES cell can be assayed for the expression of s pluripotent markers
ing, but not limited to, Oct-4, Sox2, alkaline atase, or a combination
thereof See, for example, Okamoto, K. et al., Cell, 60: 461-472 (1990), Scholer, H. R.
et al., EMBO J. 9: 2185-2195 (1990)) and Nanog (Mitsui, K. et al., Cell, 113: 631-642
(2003), Chambers, I. et al., Cell, 113: 643-655 (2003) for various methods of assaying
for the presence or the level of such markers. See, also Figures 2 and 3 provided herein.
Other otency markers include, for example, the presence of at least 1, 2, 3, 4, or 5
pluripotency marker comprising Nanog, Klf4, Dppa2, Fgf4, Rex1, Eras, ta and/or
Sall3. Other pluripotency markers include, for example, the absence of at least 1, 2, 3, 4,
, 6, 7, 8, 9 or 10 pluripotency marker comprising T/Brachyury, Flk1, Nodal, Bmp4,
Bmp2, Gata6, Sox17, Hhex1, Sox7, and/or Pax6.
In specific embodiments, the expression and/or the level of expression of these
markers can be determined using RT-PCR. Various kits are available to determine the
level and/or presence of alkaline phosphatase, including, for example, an ALP tissue
staining kit (Sigma) and Vector Red Alkaline Phosphatase Substrate Kit I (Funakoshi)
and the like. Additional assays e in situ hybridization, immunohistochemistry,
immunofiuorescence. In specific embodiments, the rat ES cell is characterized by
expression of at least one pluripotency marker, including for example expression of Oct-
4, Sox2, alkaline phosphatase, or a combination f, and preferably all three of these
markers.
The various rat ES cell provided herein (i.e. rat ES cells that have not undergone
a targeted c modification and/or rat ES cells that have a targeted genetic
modification) are capable of maintaining pluripotency and/or totipotency while being
maintained in in vitro culturing conditions. Thus, the s rat ES cells provide herein
can, in some embodiments, be subcultured while still maintaining the undifferentiated
state. s methods of culturing the rat ES cells are discussed in further detail
elsewhere herein.
The rat embryonic stem cells ed herein have been isolated from the rat
embryo employing various isolation, purification, and culture ion techniques
which are sed in detail elsewhere . The term "cell" as used herein refers to
individual cells, cell lines, or cultures derived from such cells. An “isolated” rat ES cell
or rat embryo has been removed from its natural environment. The term ted” can
mean free from 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the constituents with
which a component is found in its l state. As used herein, a rat ES “cell line”
comprises a population of isolated rat cells that were developed from a single rat ES cell
and therefore the population efcells within a given cell line have a unifei‘m genetic
makeup ether than fer mutations er haryetjypic changes eccurring during propagation or
during targeted genetic modificatiens, For example, as indicated elsewhere, the
disclesed rat ES cells are characterized by a, high level (if euploidy. Nevertheless, in
seine cell lines the level efeuplcidy is less than 100% due to hai'yetypie changes in
prepagatien of the line frem a single cell. Moreover, a given population of rat ES cells
can se at least 10 exp 3, 10 exp4, 10x104, 10x105, 10x106, 10x107, 10x108,
10x109, or 10x1010 cells or greater. Some cell populations have sufficient cells to permit
selection of a desired d cell but not an excessively greater number so as to reduce
the possibility of mutations or karyotypic changes ping in the cell line. For
example, some cell populations have 10exp3 to 10exp6 cells.
As discussed elsewhere herein, various s are provided for the targeted
c modification of a rat ES cell line. When such methods are carried out, at least
one cell within a rat ES cell line contains the targeted genetic modification. Through
various culturing and/or ion techniques rat ES cell lines having one or more desired
targeted genetic modifications are produced.
In specific embodiments, a rat ES cell, a population of rat ES cell or a rat ES cell
line (that have not undergone a targeted c modification and/or have a targeted
genetic modification) are euploid, and thus have a chromosome number that is an exact
multiple of the haploid number. In further embodiment, a rat ES cell, a population of rat
ES cells or a rat ES cell line (that have not undergone a targeted genetic modification
and/or have a targeted genetic modification) are diploid, and thus have two haploid sets
of homologous chromosomes. When referring to a rat ES cell population or a population
of cells from a given population of rat ES cells or a rat ES cell line (that have not
undergone a targeted c modification and/or have a targeted genetic modification),
at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% ofthe cells with the given
population or euploid and/or diploid. In other ces, when referring to a rat ES cell
population or a population of cells from a given rat ES cell line (that have not undergone
a targeted genetic modification and/or have a targeted genetic modification), at least
about 50% to 95%, about 60% to 90%, about 60% to 95%, about 60% to 85%, about
60% to 80%, about 70% to 80%, about 70% to 85%, about 70% to about 90%, about
70% to about 95%, about 70% to about 100%, about 80% to about 100%, about 80% to
about 95%, about 80% to about 90%, about 90% to about 100%, about 90% to about
99%, about 90% to about 98%, about 90% to about 97%, about 90% to about 95% of the
cells within the given tion are euploid and/or diploid.
In still r embodiments, a rat ES cell, a population of rat ES cells or a rat ES
cell line (that have not undergone a targeted genetic modification and/or have a targeted
genetic modification) have 42 chromosomes. When referring to a rat ES cell tion
or a population of cells from a given rat ES cell line (that have not undergone a targeted
genetic modification and/or have a targeted genetic modification) at least about 50%,
60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or 100% of the cells with the given population have 42
chromosomes. In other instances, when referring to a rat ES cell population or a
population of cells from a given rat ES cell line (that have not undergone a targeted
genetic modification and/or have a targeted genetic modification) at least about 50% to
95%, about 60% to 90%, about 60% to 95%, about 60% to 85%, about 60% to 80%,
about 70% to 80%, about 70% to 85%, about 70% to about 90%, about 70% to about
95%, about 70% to about 100%, about 80% to about 100%, about 80% to about 95%,
about 80% to about 90%, about 90% to about 100%, about 90% to about 99%, about
90% to about 98%, about 90% to about 97%, about 90% to about 95% of the cells within
the given population have 42 chromosomes.
In further embodiments, a rat ES cell, a population of rat ES cells or a rat ES cell
line (that have not undergone a ed genetic modification and/or have a targeted
genetic modification) provided herein form sphere-like colonies when plated on a feeder
cell layer in vitro. The "sphere-like" morphology refers to the shape of rat ES cell
colonies in culture, rather than the shape of individual ES cells. The rat ES cell colonies
are spherical-like. Colonies which are loosely attached to the feeder cells appear ar
(have a circular-like logy). Free-floating colonies are spherical-like. The rat ES
cell colonies are spherical-like and very compact, g: the boundaries between cells
are very hard to see. The edge of the colony appears bright and sharp. Individual nuclei
are difficult to distinguish because the cells are very small (so that the nucleus takes up
most of the volume of the cell). Mouse ES Cells form elongated colonies and attach
strongly to feeder cells. mESC morphology can vary with strain; e. g. B6 colonies are
rounder and more domed than FlH4 colonies but are still more elongated than rESC.
Human ES cell colonies are flatter and more spread out than mESC colonies. The instant
rat ES colonies are not flat and do not resemble human ES cell colonies.
In still further embodiments, a rat ES cell, a population of rat ES cells or a rat ES
cell line (that have not undergone a targeted c modification and/or have a targeted
genetic modification) have a circular morphology. A morphology scale for a circle is
ed below, where a score of a 10 represents a perfect circle and a score of a 1
represents an ellipse.
Morphology scale of a circle:
=A circle with a structure having a longitudinal axis and a vertical axis that run
through the center of the structure and are of equal length.
9 = A structure having a longitudinal axis and vertical axis that run through the
center of the structure, wherein one of the axis is between 0.9999 to 0.9357 the
length of the other axis.
8 = A structure having a longitudinal axis and al axis that run h the
center of the structure, wherein one of the axis is n 0.9357 to 0.875 the
length of the other axis.
7 = A ure having a longitudinal axis and vertical axis that run through the
center of the structure, wherein one of the axis is between 0.875 to about 0.8125
the length of the other axis.
6 = A structure having a longitudinal axis and vertical axis that run h the
center of the structure, wherein one of the axis is between 0.8125 to 0.750 the
length of the other axis.
= A structure having a longitudinal axis and vertical axis that run through the
center of the ure, wherein one of the axis is between 0.750 to 0.6875 the
length of the other axis.
4 =A structure having a longitudinal axis and vertical axis that run through the
center of the structure, wherein one of the axis is between 0.6875 to 0.625 the
length of the other axis.
WO 30706
3 =A structure having a longitudinal axis and vertical axis that run h the
center of the structure, wherein one of the axis is between 0.625 to 0.5625 the
length of the other axis.
2: A structure having a longitudinal axis and vertical axis that run h the
center of the circle, wherein one of the axis is between 0.5625 to 0.523 the length
of the other axis.
1: An e is defined as having a longitudinal axis and al axis that run
through the center of the structure, wherein one of the axis is between 0.523 to
0.500 the length of the other axis.
In one non-limiting embodiment, the rat ES cell population or a population of
cells from a given rat ES cell line (that have not undergone a targeted genetic
modification and/or have a targeted genetic modification) have at least about 50%, 60%,
65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100% of the cells with the given population have a circular
morphology score of a 10, 9 or 8. In other embodiments, the rat ES cell population or a
population of cells from a given rat ES cell line (that have not undergone a targeted
genetic modification and/or have a targeted genetic modification) have at least about
50% to 95%, about 60% to 90%, about 60% to 95%, about 60% to 85%, about 60% to
80%, about 70% to 80%, about 70% to 85%, about 70% to about 90%, about 70% to
about 95%, about 70% to about 100%, about 80% to about 100%, about 80% to about
95%, about 80% to about 90%, about 90% to about 100%, about 90% to about 99%,
about 90% to about 98%, about 90% to about 97%, about 90% to about 95% of the cells
within the given tion have a circular morphology score of a 10, 9, or 8.
In another non-limiting embodiment, the rat ES cell population or a population of
cells from a given rat ES cell line (that have not undergone a targeted genetic
modification and/or have a targeted genetic modification) have at least about 50%, 60%,
65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100% of the cells with the given population have a circular
morphology score of a 7, 6, 5, 4 or 3. In other non-limiting embodiments, the rat ES cell
population or a population of cells from a given rat ES cell line (that have not one
a targeted genetic modification and/or have a targeted genetic modification) have at least
about 50% to 95%, about 60% to 90%, about 60% to 95%, about 60% to 85%, about
60% to 80%, about 70% to 80%, about 70% to 85%, about 70% to about 90%, about
70% to about 95%, about 70% to about 100%, about 80% to about 100%, about 80% to
about 95%, about 80% to about 90%, about 90% to about 100%, about 90% to about
99%, about 90% to about 98%, about 90% to about 97%, about 90% to about 95% of the
cells within the given population have a circular morphology score of a 7, 6, 5, 4, or 3.
In still further embodiments, -like colonies form when the rat ES cells (that
have not undergone a targeted genetic modification and/or have a targeted genetic
modification) are plated on a feeder cell layer in vitro. A morphology scale for a sphere
is ed below, where a score of a 10 represents a perfect sphere and a score of a 1
represents a three dimensional elliptical structure.
Morphology scale of a sphere-like ure:
10=A sphere is a structure haVing an X-axis and a Y-axis and a Z-axis each of
which runs through the center of the structure and are of equal .
9 = A ure haVing an X axis and a Y-axis and a Z-axis that run through the
center of the structure, wherein one of the axis is between 0.9999 to 0.9357 the
length of at least one of the other axes.
8 = A structure haVing an X axis and a Y-axis and a Z-axis that run through the
center of the structure, wherein one of the axis is between 0.9357 to 0.875 the
length of at least one or both of the other axes.
7 = A ure haVing an X axis and a Y-axis and a Z-axis that run through the
center of the structure, wherein one of the axis is between 0.875 to 0.8125 the
length of at least one or both of the other axes.
6 =A structure haVing an X axis and a Y-axis and a Z-axis that run through the
center of the structure, wherein one of the axis is between 0.8125 to 0.750 the
length of at least one or both of the other axes.
= A structure haVing an X axis and a Y-axis and a Z-axis that run through the
center of the structure, wherein one of the axis is 0.750 to 0.6875 the length of at
least one or both of the other axes.
4 = A structure haVing an X axis and a Y-axis and a Z-axis that run through the
center of the structure, wherein one of the axis is 0.6875 to 0.625 the length of at
least one or both of the other axes.
3 =A structure haVing an X axis and a Y-axis and a Z-axis that run through the
center of the structure, wherein one of the axis is between 0.625 to 0.5625 the
length of at least one or both of the other axes.
2: A structure having an X axis and a Y-axis and a Z-axis that run through the
center of the structure, wherein one of the axis is between 0.5625 to 0.523 the
length of at least one or both of the other axes.
1: A structure having an X axis and a Y-axis and a Z-axis that run through the
center of the ure, wherein one of the axis is between 0.523 to 0.500 the
length of at least one or both of the other axes.
In one non-limiting embodiment, the rat ES cell population or a population of
cells from a given rat ES cell line (that have not undergone a targeted genetic
ation and/or have a targeted genetic ation) have at least about 50%, 60%,
65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100% of the colonies that form when the cells are plated on a
feeder cell layer in vitro have a sphere-like morphology of a 10, 9 or 8. In other
embodiments, the rat ES cell population or a population of cells from a given rat ES cell
line (that have not one a targeted genetic modification and/or have a targeted
genetic modification) have at least about 50% to 95%, about 60% to 90%, about 60% to
95%, about 60% to 85%, about 60% to 80%, about 70% to 80%, about 70% to 85%,
about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about
80% to about 100%, about 80% to about 95%, about 80% to about 90%, about 90% to
about 100%, about 90% to about 99%, about 90% to about 98%, about 90% to about
97%, about 90% to about 95% of the colonies that form when the cells are plated on a
feeder cell layer in vitro have a sphere-like morphology of a 10, 9 or 8.
In another non-limiting embodiment, the rat ES cell population or a population of
cells from a given rat ES cell line (that have not undergone a targeted genetic
modification and/or have a targeted genetic modification) have at least about 50%, 60%,
65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100% of the colonies that form when the cells are plated on a
feeder cell layer in vitro have a sphere-like logy of a 7, 6, 5, 4, or 3. In other
embodiments, the rat ES cell population or a population of cells from a given rat ES cell
line (that have not one a targeted genetic modification and/or have a targeted
genetic modification) have at least about 50% to 95%, about 60% to 90%, about 60% to
95%, about 60% to 85%, about 60% to 80%, about 70% to 80%, about 70% to 85%,
about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about
80% to about 100%, about 80% to about 95%, about 80% to about 90%, about 90% to
about 100%, about 90% to about 99%, about 90% to about 98%, about 90% to about
97%, about 90% to about 95% of the colonies that form when the cells are plated on a
feeder cell layer in vitro have a sphere-like logy of a 7, 6, 5, 4, or 3.
A given rat ES cell, a tion of rat ES cells or a rat ES cell line provided
herein can be a male (XY) rat ES cell, a male (XY) population of rat ES cells, or a male
(XY) rat ES cell line. In other embodiments, a population of rat ES cells or a rat ES cell
line provided herein can be a female (XX) rat ES cell, a female (XX) population of rat
ES cells, or a female (XX) rat ES cell line. Any such rat ES cell, population of rat ES
cells or rat ES cell line can comprise the dy and/or diploidy as described above.
The various rat ES cell provided herein can be from any rat strain, including but
not limited to, an ACI rat strain, a Dark Agouti (DA) rat strain, a Wistar rat strain, a LEA
rat strain, a Sprague Dawley (SD) rat strain, or a Fischer rat strain such as Fisher F344 or
Fisher F6. The various rat ES cells can also be obtained from a strain derived from a mix
of two or more strains recited above. In one embodiment, the rat ES cell is derived from
a strain selected from a DA strain and an ACI strain. In a specific embodiment, the rat
ES cell is d from an ACI strain. The ACI rat strain is are characterized as having
black agouti, with white belly and feet and an RT1 “V1 haplotype. Such strains are
available from a variety of sources including Harlan Laboratories. In other
embodiments, the various rat ES cells are from a Dark Agouti (DA) rat strain which is
characterized as having an agouti coat and an RT1 “V1 haplotype. Such rats are ble
from a variety of source including Charles River and Harlan Laboratories. In a r
embodiment, the various rat ES cells provided herein are from an inbred rat strain.
In specific embodiments the rat ES cell line is from an ACI rat and comprises the
ACI.Gl rat ES cell as described herein. In another embodiment, the rat ES cell line is
from a DA rat and comprises the DA.2B rat ES cell line or the DA.2C rat ES cell line as
bed herein.
A given rat ES cell provided herein can be obtained from a rat embryo at any stage of rat
embryo development. Representative stages of rat embryo development are outline
below in Table l. The rat embryos employed to derive the rat ES cells can be a -
stage embryo, a blastocyst-stage , or a rat embryo at a developmental stage
between a morula-stage embryo and a blastocyst-stage embryo. Thus, in specific
embodiments, the rat embryo employed is at or between the Witschi stages of 5 and 7.
In other embodiments, the rat embryo employed is at the Witschi stage 5, 6, or 7.
In one embodiment, the rat ES cell is obtained from a rat blastocyst. In other
embodiments, the rat ES cell is obtained from a cyst from a superovulated rat. In
other embodiments, the rat ES cells are obtained from an 8-cell stage embryo which is
then cultured in vitro until it develops into a morula-stage, blastocyst stage, an embryo
between the Witschi stages 5 and 7, or into an embryo at the Witschi stage 5, 6, or 7. At
which time the embryos are then plated. Morula-stage s comprise a t ball
of cells with no internal cavity. Blastocyst-stage embryos have a visible internal cavity
(the coel) and contain an inner cell mass (ICM). The ICM cells form ES cells.
Table 1. Stages of Rat Embryo pment
Standard Stages Age
Identification of Stages. .
(Witschi) (days)
Cleavage and Blastula
l l 1 cell (in oviduct)
2 2 2 cells (in oviduct)
3 3 4 cells (in oviduct)
4 3.25 8-12 cells (in oviduct)
3.5 Morula (in uterus)
6 4 Early blastocyst (in uterus)
7 5 Free blastocyst (in uterus)
Gastrula
Implanting blastocyst, with trophoblastic cone and inner
8 6
cell mass; outgrowth of endoderm (hypoblast)
rophoblast; inner cell mass nt), covered with
9 6.75
endoderm
Near complete implantation; pendant begins differentiation
1 0 7 .25 . . .
1nto embryonic and extra-embryonic parts
Completion of implantation; primary amniotic cyst;
11 7 75'
ectoplacental cone
Further provided are various rat ES cells (that have not undergone a ed
genetic modification and/or have a targeted genetic modification) which are
characterized by
The rat ES cell is characterized by:
i) the sion of one or more of rat ES cell-specific genes comprising
Adheres Junctions Associate Protein (Aj apl), Claudin 5 (Cldn5), Cdc42 guanine
nucleotide exchange factor 9 (Arhgef9), Calcium/calmodulin-dependent protein kinase
IV (Camk4), ephrin-Al (Efnal), EPH receptor A4 ), gap junction protein beta 5
(Gjb5), Insulin-like growth factor binding protein-like l (Igfbpll), Interleulin 36
beta(Illf8), Interleukin 28 or, alpha (Il28ra), left-right determination factor 1
(Leftyl), Leukemia inhibitory factor receptor alpha (Lifr), Lysophosphatidic acid
receptor 2 (Lpar2), Neuronal pentraxin receptor (Ntm), Protein tyrosine phosphatase
non-receptor type 18 (Ptpnl8), Caudal type homeobox 2 (Cdx2), ectin type III and
ankyrin repeat domains 1 (Fankl), Forkhead box El (thyroid transcription factor 2)
(Foxel), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), ad box El
(thyroid transcription factor 2) (Foxel), Hairy/enhancer-of-split related with YRPW
motif 2 , Lymphoid enhancer-binding factor 1 (Lefl ), Sal-like 3 (Drosophila)
(Sall3), SATB homeobox l (Satbl), miR-632, or a combination f;
ii) the expression ofat least 2, 3, 4, 5, 6, 7, 8, 9,10,ll,l2,l3,l4,l5,l6,l7,l8,
19, 20, 21, 22, 23, 24, 25 or more of the rat ES cell-specific genes comprising Adheres
ons Associate Protein (Ajapl), Claudin 5 (Cldn5), Cdc42 guanine tide
exchange factor 9 (Arhgef9), Calcium/calmodulin-dependent protein kinase IV (Camk4),
ephrin-Al (Efnal), EPH receptor A4 (Epha4), gap junction protein beta 5 (Gjb5),
n-like grth factor binding protein-like l (Igfbpll), Interleulin 36 beta(Illf8),
Interleukin 28 or, alpha (Il28ra), left-right determination factor 1 (Leftyl),
Leukemia inhibitory factor receptor alpha (Lifr), Lysophosphatidic acid receptor 2
(Lpar2), al pentraxin receptor (Ntm), Protein tyrosine phosphatase non-receptor
type 18 (Ptpnl8), Caudal type homeobox 2 (Cdx2), Fibronectin type III and ankyrin
repeat domains 1 ), ad box El (thyroid transcription factor 2) (Foxel),
Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Forkhead box El (thyroid
transcription factor 2) (Foxel), enhancer-of-split d with YRPW motif 2
(Hey2), Lymphoid enhancer-binding factor 1 (Lefl), Sal-like 3 (Drosophila) (Sall3),
SATB homeobox l (Satbl), miR-632, or a combination thereof;
iii) at least a 20-fold increase in the expression of one or more of the rat ES cell-
specific genes as set forth in Table 14 when compared to a FlH4 mouse ES cell;
iV) at least a 20-fold increase in the expression of at least 2, 3, 4, 5, 6, 7, 8, 9, 10,
ll, l2, l3, l4, l5, l6, l7, l8, 19, 20, 21, 22, 23, 24, 25 or more ofthe rat ES cell-specific
genes as set forth in Table 14 when compared to a FlH4 mouse ES cell;
V) the expression of one or more of rat ES cell-specific genes as set forth in
Table 13;
Vi) the sion ofat least 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, l2, l3, l4, l5, l6, l7,
l8, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more ofthe rat ES cell-specific genes
as set forth in Table 13;
vii) at least a 20-fold increase in the sion of one or more of the rat ES cell-
specific genes as set forth in Table 13 when compared to a F1H4 mouse ES cell;
viii) at least a 20-fold increase in the sion of at least 2, 3, 4, 5, 6, 7, 8, 9,
, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more of
the rat ES cell-specific genes as set forth in Table 13 when compared to a F1H4 mouse
ES cell;
ix) at least a 20-fold decrease in the sion of one or more of the rat ES cell-
specific genes as set forth in Table 12 when compared to a F1H4 mouse ES cell; and/or
x) at least a 20-fold decrease in the sion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more ofthe
rat ES cell-specific genes as set forth in Table 12 when compared to a F1H4 mouse ES
cell;
xi) any ation of expression of the rat ES cell-specific genes of parts (i)-
(X);
xii) a relative expression level of pluripotency markers as shown in Table 15 for
at least 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16,17 or 18 ofthe listed otency
markers. See, otency ranking column of Table 15 for relative expression levels;
xiii) a relative expression level of the mesodermal markers as shown in Table 15
for at least 2, 3, or 4 of the listed mesodermal markers. See, mesodermal ranking column
in Table 15 for relative expression levels;
xiv) a relative expression level of endodermal markers as shown in Table 15 for
at least 2, 3, 4, 5, or 6 of the listed endodermal markers. See, endodermal ranking
column in Table 15 for relative expression levels;
xv) a relative expression level of neural s as shown in Table 15 for at least
2 and 3 of the listed neural markers. See, neural ranking column in table 15 for relative
expression levels;
xvi) a relative expression level of trophectoderm markers as shown in Table 15
for the listed trophectoderm markers. See, trophectoderm ranking column in table 15 for
relative expression levels;
xvii) any relative expression level of one or more (2, 3, 4, 5, 6, 7, 8, 9, 10, ll, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,2 4, 25, 26, 27, 28, 29 or 30) ofthe pluripotency
s, mesodermal markers, endodermal markers, neural markers and/or
trophectoderm markers set forth in Table 15;
xviii) the relative expression level of each of the markers set forth in table 15;
xix) any combination of the signatures set forth in xii-xiix; and/or
xx) any combination of the signature set forth in i-xiix.
In one embodiment, when transplanted into a pre-morula stage rat embryo, the rat
ES cell (that have not undergone a ed genetic modification and/or have a targeted
genetic modification) can contribute to at least 90% of the cells in an F0 generation,
contribute to at least 95% of the cells in an F0 generation, bute to at least 96% of
the cells in an F0 generation, contribute to at least 97% of the cells in an F0 generation,
contribute to at least 98% of the cells in an F0 generation, or bute to at least 99% of
the cells in an F0 generation.
III. Derivation and Propagation ofRat Embryonic Stem (ES) Cells
Various s are provided for obtaining the rat ES cells disclosed . In
specific embodiments, such methods comprise (a) providing an in vitro culture
comprising a feeder cell layer and a population of isolated rat embryonic stem (ES) cells;
(b) culturing in vitro under conditions which are sufficient to maintain pluipotency
and/or totipotency of the isolated rat ES cell. Such methods thereby allow for the
propagation of a rat ES cell population and/or a rat ES cell line.
In one embodiment, a method for culturing a rat embryonic stem cell line is
provided. Such methods comprises culturing in vitro a feeder cell layer and a rat ES cell
line, wherein the culture conditions maintain pluripotency of the rat ES cells and
comprise a media having mouse leukemia inhibitor factor (LIF) or an active variant or
fragment thereof. The various methods fiarther se passaging and culturing in vitro
the cells of the rat ES cell line, wherein each subsequent in vitro culturing ses
ing the rat ES cells on the feeder cell layer under conditions that maintain
pluripotency of the rat ES cells and comprises a media having mouse LIF or an active
variant or fragment thereof.
i. Culture Conditions
The culture media ed in the various methods and compositions will
maintain the rat ES cells. The terms "maintaining" and "maintenance" refer to the stable
preservation of at least one or more of the characteristics or ypes of the rat ES
cells outline herein. Such phenotypes can include maintaining pluripotency and/or
totipotency, cell morphology, gene expression s and the other filnctional
teristics of the rat stem cells described herein. The term "maintain" can also
encompass the propagation of stem cells, or an increase in the number of stem cells
being cultured. The term filrther contemplates culture conditions that permit the stem
cells to remain pluripotent, while the stem cells may or may not continue to divide and
increase in number.
The term "feeder cell" or “feeder cell layer” refers to a e of cells that grow
in vitro and secrete at least one factor into the culture medium that is used to support the
growth of another cell of interest in the culture. The feeder cells employed herein aid in
maintaining the otency of the rat ES cells, and in specific embodiments, one or
more of the other characteristics or phenotypes described . Various feeder cells
can be used including, for example, mouse embryonic asts, including mouse
embryonic fibroblasts obtained between the 12th and 16th day of pregnancy. In specific
embodiments, feeder cell layer comprises a monolayer of mitotically inactivated mouse
embryonic fibroblasts (MEFs).
The in vitro cultures of the rat ES cells fiarther comprise an effective amount of
Leukemia Inhibitor Factor (LIF) or an active variant or fragment thereof. Leukemia
inhibitory factor (LIF) belongs to the IL-6 receptor family. LIF binds to a heterodimeric
membrane receptor made up of a LIF-specific subunit, gp190 or LIFR, and the subunit
gp130, which is shared with the other members of the IL-6 family. LIF inhibits the
differentiation of embryonic stem cells in mice and contribute to stem cell self-renewal.
Human and mouse LIF share 79% sequence homology and exhibit cross-species activity.
Rat LIF (rtLIF) is a 22.1 kDa protein containing 202 amino acid es that exhibits
91% amino acid sequence identity with murine LIF ama et al. 1998). There are
six possible asparagine-linked glycosylation (N-glycosylation) sites which are conserved
among the LIF ptide from the various species and an additional site of Asn150
which is specific for rat LIF. The tertiary structure of the mouse LIF and its filnction is
bed in further detail in Aikawa et al. (1998) Biosci. Biotechnol. m. 62 1318-
1325 and k et al. (2005) Immunology nancy, editor Gil Mor., US Patent
No. 5,750,654 and D P g (1987) EMBO Journal 198720, each ofwhich is
herein incorporated by reference in their entirety. A partial mouse LIF sequence is
reported on the SwissProt website under the accession number P09056.
Mouse LIF activity is assessed by its ability to induce differentiation of M1
myeloid leukemia cells. The specific activity is 1 x 106 units/ml (Cat. No. 03-0011 from
Stemgent) and 1 x 107 units/ml (Cat. No. 03100 from Stemgent), where 50 units is
defined as the amount of mouse LIF required to induce differentiation in 50% of the M1
colonies in 1 ml of medium. See, also, Williams, R.L. et al. (1988) Nature 336: 684-
687.; Metcalf, D. et al. (1988) Leukemia 2: 1; Niwa, H. et al. (2009) Nature 460:
118-122; Xu, J. et al. (2010) Cell Biol Int. 34: 791-797; Fukunaga, N. et al. (2010) Cell
Reprogram. 12: 369-376; and, Metcalf D. (2003) Stem Cells 21: 5-14, each of which is
herein incorporated by reference in their entirety. An “effective amount of LIF”
comprises a concentration of LIF that allows the rat ES cells of an in vitro e to
remain in an erentiated gv'lui'lpetent state. Vatli‘ms markers that can be used, t0
assay for the cells remaining in a plurlpetent state are sed ere herein,
The Lil: petypeptlde en'ipleyed in the writing i'netheds and een‘zpi‘mitiens
previded herein can be from any organism, including from a mammal, a rodent, a
human, a rat or a mouse. In one embodiment, the LIE polypeptide is from a mouse. In
still r embodiments, the mouse LIF polypeptide comprises the amino acid sequence
set forth in SwissProt Accesion number: P09056, which is herein incorporated by
reference in its entirety and is also set forth in SEQ ID NO: 1.
In other embodiments, an active t or fragment of the mouse LIF
ptide as set forth in SEQ ID NO: 1 or in SwissProt Accesion number: P09056 can
be used. Such active variants and fragments (including active variants having at least
75%, 80%, 85% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to SEQ ID NO: 1) are discussed in further detail elsewhere .
LIF polypeptide or the active t or fragment thereof can be provided to the
in vitro culture in a variety of ways. In one embodiment, the effective amount of the LIE
polypeptide or the active variant or fragment thereof is added to the culture media. In
other ments, the feeder cells have been genetically modified to press the
LIE polypeptide or the active variant or fragment thereof. Such feeder cells include
feeder cells prepared from gamma—irradiated or mlten‘ayelwl? treated BIA—M meme
fibroblasts that express metrixnasseeieted LIE. Method of generating and using such
genetically modified feeder cells can be found, for example, in See, Buelir er al. 2003)
Bit)! Repma’ 682222429, Rathjen er a}. (T1990) Cell 62 llllfiwl l 15, and Buehr er al.
(2008) Cell 1315:12874298“, each el‘whlcb is herein ineerpm‘eted by reference The
heterologous LIF expressed in the feeder cells can be from the same organism as the
feeder cells or from an organism that is different from that of the feeder cell. In addition,
the heterologous LIF expressed in the feeder cells can be from the same or from a
different organism than the ES cells the feeder layer is supporting.
In still other embodiments, the feeder cells employed in the various methods
disclosed herein are not genetically modified to express a heterologous LIF polypeptide
or an active variant or fragment thereof. Thus, in particular embodiments, the monolayer
of mitotically inactivated mouse embryonic fibroblast employed in the methods has not
been cally modified to express a heterologous LIF ptide.
In other embodiments, the LIE polypeptide or the active variant or fragment
thereof is added to the culture media. When LIF is added to the culture media, the LIE
can be from any organism, ing from a mammal, a rodent, a human, a rat or a
mouse. In one embodiment, the LIE present in the e media is from a mouse. In
still fiarther embodiments, the mouse LIF polypeptide comprises the amino acid sequence
set forth in SEQ ID NO: 1. In other embodiments, an active variant or fragment of the
mouse LIF polypeptide as set forth in SEQ ID NO:l can be used. Such active variants
and fragments ding active ts having at least 75%, 80%, 85% 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1) are
discussed in further detail elsewhere herein.
In specific embodiments, the rat ES cells and rat ES cell lines provided herein
maintain pluripotency in vitro Without requiring paracrine LIF signaling.
In specific embodiments, LIF or an active variant or fragment thereof is present
in the culture media at any concentration that maintains the rat ES cells. LIF polypeptide
or active variant or fragment thereof is present in the culture media at about 25U/ml to
about 50U/ml, at about 50U/ml to about lOOU/ml, at about lOOU/ml to about 125U/ml,
at about 125U/ml to about l, at about l50U/ml to about l75U/ml, at about
l to about 200U/ml, at about 200U/ml to about 225U/ml, at about 225U/ml to
about 250U/ml, at about 250U/ml to about 300U/ml, to about 300U/ml to about
325U/ml, at about l to about 350U/ml, at about 350U/ml to about 400U/ml, at
about 400U/ml to about 425U/ml, at about 425U/ml to about 450U/ml, at about 450U/ml
to about 475U/ml, at about 475U/ml to about 500U/ml, at about 75U/ml to about
500U/ml or greater. In other embodiments, LIF polypeptide or active variant or
fragment thereof is present in the e media at about 25U/ml to about 50U/ml, at
about 25U/ml to about lOOU/ml, at about 75U/ml to about 125U/ml, at about 50U/ml to
about l50U/ml, at about 90U/ml to about 125U/ml, at about 90U/ml to about llOU/ml,
at about 80U/ml to about l50U/ml, at about 80U/ml to about 125U/ml. In a c
embodiment, LIF ptide or active variant or fragment f is present in the
culture media at about lOOU/ml.
When mouse LIF is employed, the mouse LIF polypeptide or active variant or
fragment thereof is present in the culture media at any concentration that maintains the
rat ES cells. Mouse LIF polypeptide or active variant or fragment f is present at
about 25U/ml to about 50U/ml, at about 50U/ml to about lOOU/ml, at about lOOU/ml to
about 125U/ml, at about 125U/ml to about l50U/ml, at about l50U/ml to about
l75U/ml, at about l75U/ml to about 200U/ml, at about 200U/ml to about 225U/ml, at
about 225U/ml to about 250U/ml, at about 250U/ml to about l, to about 300U/ml
to about 325U/ml, at about l to about 350U/ml, at about l to about
400U/ml, at about 400U/ml to about 425U/ml, at about 425U/ml to about 450U/ml, at
about 450U/ml to about 475U/ml, at about l to about 500U/ml, at about 75U/ml
to about l or greater. In other embodiments, mouse LIF polypeptide or active
variant or fragment thereof is present at about 25U/ml to about 50U/ml, at about 25U/ml
to about lOOU/ml, at about 75U/ml to about 125U/ml, at about 50U/ml to about
l50U/ml, at about 90U/ml to about 125U/ml, at about 90U/ml to about llOU/ml, at
about 80U/ml to about l50U/ml, at about 80U/ml to about l. In a specific
embodiment, mouse LIF polypeptide or active variant or fragment thereof is present in
the culture media at about l.
The culture media employed maintains rat ES cells. As such, in specific
embodiments, the culture media employed in the various method and compositions will
maintain the pluripotency of all or most of (i.e., over 50%) of the rat ES cells in a cell
line for a period of a at least 5, 10 or 15 passages. In one embodiment, the culture media
comprises one or more compounds that assist in maintaining pluripotency. In one
embodiment, the culture media comprises a MEK pathway inhibitor and a glycogen
synthase kinase-3 ) inhibitor. The media can further comprise additional
ents that aid in maintaining the ES cells, including for e, FGF or
inhibitors, ROCK inhibitors, and/or ALK (TGFb receptor) inhibitors. A non-limiting
example of an FGF receptor inhibitors includes PD184352. A non-limiting example of a
ROCK inhibitor includes Y-27632, and non-limiting example of an ALK (TGFb
receptor) inhibitor includes AOl. In specific embodiments, 2i media (table 2) is used
with 10 uM ROCKi when thawing cryopreserved rESC or when re-plating rESC after
dissociation with trypsin.
In other embodiments, the media ses a combination of inhibitors
consisting of a MEK pathway inhibitor and a glycogen synthase kinase-3 )
inhibitor.
In one non-limiting embodiment, the culture media comprises a GSK-3 inhibitor
comprising CHIR99021 and/or comprises a MEK inhibitor comprising 901. In
other embodiments, the media comprises a combination of inhibitors consisting of
CHIR99021 and PD0325901. Either of these compounds can be obtained, for example,
from Stemgent. In specific embodiments, CHIR99021 is present in the culture media at
a tration of about 0.5 u to about 3 uM, about 0.5u to about 3.5 uM, about 0.5 uM
to about 4 uM, about 0.5uM to about 1 uM, about 1 uM to about l.5uM, about l.5uM to
about 2 uM, about 2 uM to about 2.5 uM about 2.5 to about 3 uM, 3 uM to about 3.5
uM. In further embodiments, CHIR99021 is present in the culture media at a
tration of about 3 uM. In other embodiments, PD0325901 is present in the
culture media at a concentration of about 0.4 uM to about luM, about 0.4 uM to about
1.5 uM, about 0.4 uM to about 2 uM, about 0.4 uM to about 0.8 uM, 0.8uM to about 1.2
uM, about 1.2 to about 1.5 uM. In further ments, PD0325901 is present in the
culture media at a concentration of about luM. In specific embodiments, CHIR99021 is
present in the culture media at a concentration of about 3 uM and PD0325901 is present
at a concentration of about luM.
In one non-limiting embodiment, the culture media employed in the various
methods and itions sed herein is set forth in Table 2. Within the context of
this application, the media described in Table 2 is referred to as 2i media.
Table 2: Non-limiting rat ES culture media.
Reagent
DMEM/F l2 basal media
asal media
Penicillin/streptomycin
L-Glutamine
2-Mercaptoethanol
N2 supplement
B27 supplement
LIF 100 U/ml
PD0325901 (MEK inhibitor).
CHIR99021 (GSK inhibitor).
Additional media that can be employed e those disclosed in Li et al. (2008)
Cell 135:1299-1310, Yamamoto et al. (2012) Transgenic Rats 21:743-755, Ueda et al.
(2008) PLoS ONE 2800, Meek et al. (2010) PLoS ONE 4 (l2): el4225; Tong et al.
(2010) Nature 467:211-213; US Patent Publication 2012/0142092, Buehr et al. (2008)
Cell l35:l287-l298, Li et al. (135) Cell 1299-1310, each ofwhich is herein incorporated
by reference in their entirety. When employing such media, the concentration and the
source of LIF can be d as outlined herein. In c ments, the various
culture medias are used in combination with mouse LIF or an active variant or fragment
thereof, and in even further embodiments, the s culture medias comprise a mouse
LIF or an active variant or fragment thereof at a concentration of about 50U/ml to about
100U/ml, about 50U/ml to about l, or about 100U/ml.
The temperature of the cultures of rat ES cells, both for the production of the ES
cell line and for the culturing and maintaining of the ES line it typically carried out at
about 35°C to about 37.50 C. In specific embodiment, the temperature is 370°C. The
culture is typically carried out at 7.5% C02.
ii. Establishing a rat ES Cell Line
Methods for generating a rat embryonic stem (ES) cell line are provided. Such
methods comprises (a) culturing in vitro a first feeder cell layer and a morula-stage
embryo, a blastocyst-stage , or a rat embryo at a developmental stage between a
-stage embryo and a blastocyst-stage embryo, wherein the zona pellucida of the
rat embryo has been removed and wherein the culture ions maintain pluripotency
of the rat ES cell and comprises a media having mouse leukemia inhibitor factor (LIF) or
an active variant or fragment thereof; and, (b) transferring an outgrowth of an amorphous
undifferentiated mass of rat ES cells to an in vitro culture well comprising a second
feeder cell layer and culturing the outgrowth under conditions that maintain pluripotency
of the rat ES cells and comprises a media having mouse LIF or an active variant or
fragment thereof, and thereby establishing a rat ES cell line. The various methods r
comprise passaging and ing in vitro the cells of the rat ES cell line, wherein each
uent in vitro culturing comprises culturing the rat ES cells on the feeder cell layer
under conditions that maintain pluripotency of the rat ES cells and comprises a media
having mouse LIF or an active variant or fragment thereof Rat ES cell lines made by
such methods are also provided.
Non-limiting examples of methods to establish a rat ES cell line having the
s characteristics discussed herein are set forth in Example 3. Briefly, a rat embryo
(i.e., a morula-stage embryo, a blastocyst-stage embryo, or a rat embryo at a
developmental stage between a morula-stage embryo and a blastocyst-stage embryo) is
2014/017452
flushed from the uteruses of a female rat. In specific embodiments, a blastocyst or an 8
cell embryo is obtained. The zona pellucida is removed and the rat embryos are cultured
on feeder cells (as sed elsewhere herein) which, in specific embodiments, comprise
a monolayer of cally inactivated mouse embryonic fibroblasts (MEFs). The cells
of the morula-stage embryo, the cyst-stage embryo, or the rat embryo at a
developmental stage between a morula-stage embryo and a blastocyst-stage embryo are
cultured in vitro under conditions that maintain the ES rat cells and thereby are sufficient
to maintain pluripotency and/or totipotency of the ES cells. Various medias can be
employed at this stage, ing any of the various medias discussed above which have
LIF, including mouse LIF or an active t or fragment thereof, in the media.
The cultures are monitored for the presence of an outgrowth (an amorphous
undifferentiated mass of . Once the outgrth reaches an appropriate size, a given
outgrowth is transferred to new feeder plate and cultured. Transfer is accompanied by
enzymatic dissociation using trypsin in order to produce le colonies. This transfer
is commonly referred to as “passage 1”. The speed at which each line expands varies.
The media is changed as needed in order to maintain the pluripotency or totipotency of
the rat ES cells. The culture is monitored for the presence of colonies having embryonic
stem cell morphology. Such morphology includes one or more of the following
characteristics: (a) round, circular mounds that rise above the monolayer of feeder cells;
(b) cells that are packed tightly together such that cell boarders are difficult to see; (c)
smaller cell size; (d) small amount of cytoplasm and enlarged nucleus, (e) form sphere-
like colonies when plated on feeder cells in vitro. Once such colonies appear, the
culturing can continue until reaching approximately 50% ncy. The colonies are
then transferred to a new feeder plate. er is accompanied by enzymatic
dissociation using trypsin in order to expand the number of colonies. This is referred to
as ge 2”. The cells are continued to be cultured with feeder cells until they reach
approximately 50% confluent, at which point the cells can undergo r passages to
maintain the cell lines or the lines can be frozen. See, also Tong et al. (2010) Nature 467
(9):2l l-2l5; Li et al. (2008) Cell 135 : 1299-13 10, and Buehr et al. (2008) Cell
135: 1287-1298, each of which is herein incorporated by reference. Thus, in specific
embodiments, the various rat ES cells, cell lines and cell populations disclosed herein are
capable of being subcultured and maintaining the undifferentiated state.
In one miting embodiment, the derivation of the rat ES cells occurs as
follows. At day 0, female rats are euthanized and the ts and uterine horns are
dissected out and place into a tissue culture dish containing warm N2B27 media. Media
is flushed through the uterine horns and oviducts to eject blastocysts into the media. The
blastocysts are collected and transfer to embryo culture dish containing KSOM + 2i
(1 uMPD0325901, 3 uM CHIR9902l). KSOM can be sed from Millipore, catalog
number is MRD. The 2i media referred to herein comprises the media set forth in
Table 2. The cells are culture overnight at 37° at 7.5% C02.
In other non-limiting embodiments, the rat ES cells are derived from 8-cell
embryos or frozen 8-cell embryos. The s are cultured in M2 medium for 10
s at room temperature and are then transfer to KSOM + 2i and culture overnight.
A non-limiting example for derivation of the rat ES cells is as follows: on Day 1,
transferring cavitated embryos to 2i medium (Table 2) and culture overnight. The
culturing is continued un-cavitated in KSOM +2i. On day 2, all remaining embryos are
transferred to 2i medium, whether or not they have cavitated. Culture continues
ght in 2i medium. On day 3, s are incubated with acid tyrodes to remove
the zona pellucida and washed 3 times in 2i medium to remove the acid tyrodes. Each
embryo is deposited into a separate well feeder plate, in which each well ns a
monolayer of mitotically inactivated mouse embryonic fibroblasts (MEFs). The cells are
cultured ght in 2i medium. On day 4 and 5, the cells plated embryos are
monitored for the presence of an outgrowth, an amorphous undifferentiated mass of
cells. Outgrowths are ready for transfer when they are approximately twice the size of
the plated embryo. Each day, spent media is removed and replaced with fresh 2i media.
The outgrowths are transferred to new feeder wells, and again spent media is removed
and the wells are washed with PBS. The PBS is removed and trypsin is added and
incubated for about 10 minutes. The trypsin reaction is stopped by the addition of 30ul
2i media and 10% PBS. The cells are gently dissociated and the entire content is
erred to a well in a feeder plate. This is referred to as Passage 1 (Pl). The cells are
cultured overnight in 2i medium. On day 5-8, depending on how fast each line expands,
the 2i media is changed each day and e is red for the ce of colonies
with an ESC logy. Such ESC morphology is discussed in detail ere
herein. Culturing continues until colonies expand to about 50% confluency. The
colonies are then trypsinzied and passaged as before into feeder wells. This is referred to
as passage 2. Feeding and monitoring each line is continued until they are approximately
50% confluent. The cells are trypsinized as usual. The trypsin is stopped with 2i media
+ 10% PBS. The cells are ed by centrifilgation, and the cells in 400 ul Freezing
Medium (70% 2i, 20% PBS, 10% DMSO). The cells can then be frozen. This is referred
to passage 3.
iii. Maintaining and Passaging a rat ES cell Line
Further provided are methods for maintaining or culturing a rat embryonic stem
cell line. The method comprises culturing in vitro a feeder cell layer and a rat ES cell
line, n the e conditions maintain pluripotency of a rat embryonic stem (ES)
cell and comprise a media having mouse leukemia inhibitor factor (LIF) or an active
variant or fragment thereof. Such methods employ the culture media and feeder cell
layer as outlined above. In one embodiment, the rat ES cell line can be passaged at least
, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50 or
more times. In specific ments, the rat ES cells can be passaged up to at least 11
times in a medium comprising a GSK3 inhibitor and a MEK inhibitor without decreasing
its targeting efficiency or germline transmission efficiency of a targeted genetic
modification.
The rat ES cell lines are fed and monitored. In specific embodiments, passage
occurs when the culture is approximately 30%, 40%, 50%, or 60% confluent. In other
embodiments, passage occurs when the culture is 50% confluent. ing on how
fast each line expands, the es can occur every 24, 25, 26, 27, 28, 29, 30, 40, 45,
50, 55, 60, 65, 70, 75, 80, 90, 95 or 100 hours. In other embodiments, the time between
passages ranges between 24 hours and 96 hours, between about 30 and 50 hours,
between about 25 and 75 hours, between about 50 and 96 hours, between about 25 and
75 hours, between about 35 and 85 hours, or between about 40 and 70 hours. In one
embodiment, the rat ES cell, cell line or cell population as disclosed herein has a
doubling time ranging from about 24 hours to about 36 hours. In one embodiment, the rat
ES cell has a doubling time of 25 hours.
The s rat ES cell lines when derived and maintained as outlined herein can
have one or more any the following properties:
(a) have germ-line competency, meaning when the rat ES cell is implanted
into a rat host embryo, the genome of the rat ES cell line is transmitted into an offspring;
(b) have germ-line competency following ed genetic modification,
meaning when the rat ES cell is implanted into a rat host embryo, the targeted genetic
modification within the genome of the rat ES cell line, is transmitted into an offspring;
(c) have pluripotency in vitro;
(d) have totipotency in vitro;
(e) when cultured in vitro loosely adhere to a feeder cell layer;
(f) when cultured in vitro form sphere-like colonies when plated on a feeder
cell layer in vitro;
(g) in pluripotency when cultured in vitro under conditions comprising
a feeder cell layer that is not genetically modified to express leukemia inhibitor factor
(LIF), wherein the e media comprises a sufficient concentration of LIF;
(h) maintain pluripotency when cultured in vitro under conditions comprising
a feeder cell layer, wherein the culture media comprises mouse LIF or an active variant
or fragment thereof;
(i) comprise a lar signature characterized by
i) the expression of one or more of rat ES cell-specific genes comprising
Adheres Junctions Associate Protein (Aj apl), Claudin 5 (Cldn5), Cdc42 guanine
nucleotide ge factor 9 (Arhgef9), m/calmodulin-dependent protein kinase
IV (Camk4), ephrin-Al (Efnal), EPH or A4 (Epha4), gap junction protein beta 5
(Gij), Insulin-like growth factor binding n-like l ll), Interleulin 36
beta(Illf8), Interleukin 28 receptor, alpha (Il28ra), left-right determination factor 1
(Leftyl), Leukemia tory factor receptor alpha (Lifr), Lysophosphatidic acid
receptor 2 (Lpar2), Neuronal pentraxin receptor (Ntm), Protein tyrosine phosphatase
non-receptor type 18 (Ptpn18), Caudal type homeobox 2 (Cdx2), Fibronectin type III and
ankyrin repeat s 1 (Fankl), Forkhead box El (thyroid transcription factor 2)
(Foxel), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), ad box El
(thyroid transcription factor 2) ), Hairy/enhancer-of-split related with YRPW
motif 2 (Hey2), Lymphoid enhancer-binding factor 1 (Lefl ), Sal-like 3 (Drosophila)
(Sall3), SATB homeobox l (Satbl), miR-632, or a combination thereof;
ii) the expression ofat least 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, l2, l3, 14, 15, l6,
17, 18, 19, 20, 21, 22, 23, 24, 25 or more of the rat ES cell-specific genes sing
Adheres Junctions Associate Protein (Aj apl), Claudin 5 (Cldn5), Cdc42 guanine
nucleotide exchange factor 9 (Arhgef9), Calcium/calmodulin-dependent protein kinase
IV (Camk4), ephrin-Al ), EPH receptor A4 (Epha4), gap junction protein beta 5
(Gjb5), Insulin-like growth factor binding protein-like 1 (Igfbpll), Interleulin 36
beta(Illf8), Interleukin 28 receptor, alpha (Il28ra), left-right determination factor 1
l), Leukemia inhibitory factor receptor alpha (Lifr), Lysophosphatidic acid
receptor 2 (Lpar2), Neuronal pentraxin receptor (Ntm), Protein tyrosine phosphatase
non-receptor type 18 (Ptpn18), Caudal type homeobox 2 (Cdx2), Fibronectin type III and
ankyrin repeat domains 1 (Fankl), ad box E1 (thyroid transcription factor 2)
(Foxel), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Forkhead box E1
(thyroid transcription factor 2) (Foxel), Hairy/enhancer-of-split related with YRPW
motif 2 , Lymphoid enhancer-binding factor 1 (Lefl ), Sal-like 3 (Drosophila)
(Sall3), SATB homeobox 1 (Satbl), miR-632, or a combination thereof;
iii) at least a 20-fold increase in the expression of one or more of the rat
ES pecific genes as set forth in Table 14 when compared to a FlH4 mouse ES cell;
iV) at least a 20-fold increase in the expression of at least 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more ofthe rat ES
cell-specific genes as set forth in Table 14 when compared to a FlH4 mouse ES cell;
V) the expression of one or more of rat ES cell-specific genes as set forth
in Table 13;
Vi) the expression ofat least 2, 3, 4, 5, 6, 7, 8, 1,12,13,14,15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more ofthe rat ES cell-specific
genes as set forth in Table 13;
Vii) at least a 20-fold increase in the expression of one or more of the rat
ES cell-specific genes as set forth in Table 13 when ed to a FlH4 mouse ES cell;
Viii) at least a 20-fold increase in the sion of at least 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or
more of the rat ES cell-specific genes as set forth in Table 13 when compared to a FlH4
mouse ES cell;
ix) at least a 20-fold decrease in the sion of one or more of the rat
ES cell-specific genes as set forth in Table 12 when compared to a FlH4 mouse ES cell;
and/or
x) at least a 20-fold decrease in the sion of at least 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or
more of the rat ES cell-specific genes as set forth in Table 12 when compared to a FlH4
mouse ES cell;
xi) any combination of expression of the rat ES cell-specific genes of
parts (i)-(X);
xii) a relative expression level of pluripotency markers as shown in Table
for at least 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16,17 or 18 ofthe listed
otency markers. See, otency ranking column of Table 15 for relative
expression levels;
xiii) a relative expression level of the mesodermal markers as shown in
Table 15 for at least 2, 3, or 4 of the listed mesodermal markers. See, mesodermal
ranking column in Table 15 for relative expression levels;
xiv) a relative expression level of endodermal markers as shown in Table
for at least 2, 3, 4, 5, or 6 of the listed endodermal markers. See, endodermal ranking
column in Table 15 for relative expression levels;
xv) a relative expression level of neural markers as shown in Table 15 for
at least 2 and 3 of the listed neural markers. See, neural ranking column in table 15 for
relative expression levels;
xvi) a relative expression level of trophectoderm s as shown in
Table 15 for the listed trophectoderm markers. See, trophectoderm ranking column in
table 15 for relative expression levels;
xvii) any relative expression level of one or more (2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,2 4, 25, 26, 27, 28, 29 or 30) ofthe
otency markers, mesodermal markers, rmal s, neural markers and/or
trophectoderm markers set forth in Table 15;
xviii) the relative expression level of each of the s set forth in
table 15;
xix) any combination of the signatures set forth in xii-xiix; and/or
xx) any combination of the signature set forth in i-xiix;
(j) have the y to produce a F0 rat;
(k) capable of being subcultured and ining the undifferentiated state;
(1) have the same number of chromosomes a normal rat cell; and/or
(m) maintain pluripotency in vitro without ing paracrine LIF signaling.
(11) have self renewal, meaning they divide indefinitely while maintaining
pluripotency.
Such properties of a given rat ES cell line can be present at any one of the
passage, including at passages 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
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22, 23, 24, 25, 30, 40, 50 or later.
Thus, in one non-limiting embodiment, an in vitro culture sing a feeder
cell layer and a population of rat ES cells is provided, wherein the in vitro culture
conditions maintain pluripotency of the rat ES cells and comprises a media having mouse
leukemia inhibitor factor (LIF) or an active variant or fragment thereof In specific
embodiments, the rat ES cells cultured under such conditions maintains pluripotency of
at least 50% of cell population over a period of at least 10 passages, maintains
otency of at least 60% of cell population over a period of at least 10 passages,
maintains pluripotency of at least 70% of cell population over a period of at least 10
passages, maintains pluripotency of at least 75% of cell population over a period of at
least 10 passages, maintains pluripotency of at least 80% of cell population over a period
of at least 10 passages, maintains pluripotency of at least 85% of cell tion over a
period of at least 10 passages, ins pluripotency of at least 90% of cell population
over a period of at least 10 passages, or maintains otency of at least 95% of cell
tion over a period of at least 10 passages.
Further provided herein are in vitro cultures comprising the various rat ES cells,
cell populations and cell lines disclosure herein, as well as, culturing kits for these
various ES cells. For example, as sed above, in specific embodiments, the various
rat ES cells provided herein have a one or more of the following characteristics: (1)
when cultured in vitro loosely adhere to a feeder cell layer; (2) when cultured in vitro
they form sphere-like colonies when plated on a feeder cell layer in vitro; (3) they
maintain pluipotency when cultured in vitro under conditions comprising a feeder cell
layer that is not genetically modified to express ia inhibitor factor (LIF), wherein
the culture media comprises a sufficient concentration of LIF; and/or (4) they capable of
being subcultured and maintaining the undifferentiated state. Moreover, the rat ES cell
populations of any of these in vitro cultures can comprise, for example, a population of
cells in which at least 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or 100% of the cells within the population are euploid, are diploid and/or have
42 chromosomes.
One method of culturing a rat nic stem cells in vitro ses (a)
providing an in vitro culture comprising a feeder cell layer and a population of isolated
rat embryonic stem (ES) cells; and (b) culturing the in vitro culture under ions
which are sufficient to maintain pluipotency or totipotency of the isolated rat embryonic
stem (ES) cell, and wherein the rat ES cells form colonies that loosely adhere to the
feeder cell layer. “Loose adherence” or “adhere loosely” means that, if the culture dish
is urbed for a period of time (minimum 8 hours), some colonies will adhere to the
feeders such that they can maintain adherence if the dish is gently moved. In smaller
wells (where the media moves less), loose adherence can happen faster. In either case,
these colonies can be dislodged by either a) swirling the media in the dish or by gently
pipetting media across the surface of the feeders. The morphology of these loosely
adherent es is still spherical. In such instances the rat ES cells form sphere-like
colonies when plated on a feeder cell layer in vitro. Such sphere-like colonies are shown
for e in Figure 1.
iv. Kits and In Vitro Cultures
The rat ES cells and rat ES cell lines ed herein can be contained within a
kit or an article of manufacture. In specific embodiments, the kit or article of
cture comprises any of the rat ES cell lines or populations disclosed herein. The
kit can further comprise any culture media that maintains the rat ES cell, including media
that ins the pluripotency of the rat ES cells. Such media can comprise culture
media having mouse LIF or an active variant or fragment thereof, as discussed in greater
detail elsewhere herein. The media within the kit can fiarther comprise a MEK inhibitor
and a GSK-3 inhibitor, or alternatively, the media within the kit can r comprise a
combination of inhibitors consisting of a MEK inhibitor and a GSK-3 inhibitor. In
specific embodiments, the media in the kit comprises a MEK inhibitor comprising
PD032590l and/or a GSK-3 inhibitor comprising CHIR99021. Any of the various
medias discussed herein, can be contained within the kit.
r provided is a kit or article of manufacture that comprises any of the rat
ES cell lines or populations disclosed herein, any of the various media disclosed ,
and a population of feeder cells. In one embodiment, the feeder cells in the kit or article
of manufacture are not genetically modified to express LIF and/or the feeder cells
comprises mitotically inactivated mouse embryonic fibroblasts (MEFs). Any of the
other feeder cells disclosed herein can be employed in the kit or article of manufacture.
IV. Genetic Modification ofRat Embryonic Stem (ES) Cells
The s rat ES cells and cell lines sed herein can be modified to contain
at least one targeted genetic modification. Thus, various methods are provided for
genetically modifying an isolated rat embryonic stem (ES) cell as disclosed herein. The
method comprises introducing into the genome of an ed rat ES cell disclosed herein
a targeted genetic modification to form a genetically modified rat ES cell. The targeted
genetic modification can comprise any modification to the genome of rat ES including,
for example, an insertion, a deletion, a knockout, a knockin, a mutation, or a combination
thereof. In one embodiment, the targeted genetic ation comprises insertion of a
heterologous cleotide into the genome of the rat ES cell. As used herein,
“heterologous” in reference to a sequence is a ce that originates from a foreign
species, or, if from the same species, is substantially modified from its native form in
composition and/or genomic locus by deliberate human intervention.
In one aspect, an isolated rat ES cell or rat ES cell line is provided that is capable
of ning pluripotency following one or more genetic modifications in vitro and that
is capable of transmitting a genetically modified genome to a germline of an F1
generation. Thus, the rat ES cell maintains its pluripotency to develop into a plurality of
cell types following the one or more serial genetic modifications in vitro (e.g., two, three,
four, five, or six or more serial c modifications). In other embodiments, le
targeted genetic modifications are made in a given rat ES cell, ing, for example, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 14, 15 or more. As such, multiple heterologous
polynucleotides can also be integrated into the genome, including for example, 2, 3, 4, 5,
6, 7, 8, 9,10,11,12,1314,15 or more.
In one embodiment, following any one of the one to 15 serial genetic
ations, the genetically modified rat ES cells upon exposure to differentiation
medium are capable of differentiation into a plurality of cell types. In one embodiment,
following any one of the one to 15 serial genetic modifications, the cally modified
rat ES cells are capable of being maintained in an undifferentiated state in culture. In
one embodiment, the genetically d and cultured rat ES cells in the
undifferentiated state, when employed as donor cells in a rat host embryo, populate the
embryo and form a blastocyst comprising the one to fifteen genetic modifications. In
one embodiment, the blastocyst, when implanted into a surrogate mother under
conditions suitable for gestation, develops into an F0 rat progeny that comprises the one
to 15 genetic modifications.
Various methods for making targeted genetic ations within a genome of a
rat ES cell can be used. For example, in one instance, the targeted genetic modification
employs a system that will generate a ed genetic modification via a homologous
recombination event. In other instances, the rat ES cells can be modified using nuclease
agents that generate a single or double strand break at a targeted genomic location. The
single or double-strand break is then repaired by the non-homologous end joining
pathway (NHEJ). Such s find use, for example, in generating targeted loss of
function genetic modifications. See, for example, Tesson et al. (2011) Nature
Biotechnology 29:695-696, herein incorporated by reference. Such agents include,
Transcription Activator-Like Effector Nuclease (TALEN) (; zer
et al. (2010) PNAS 10.1073/pnas.1013133107; Scholze & Boch (2010) Virulence 1:428-
432; Christian et al. Genetics (2010) 186:757-761; Li et al. (2010) Nuc. Acids Res.
(2010) doi:10.1093/nar/gkq704; and Miller et al. (2011) Nature Biotechnology 29: 143—
148; US Patent ation No. 2011/0239315 A1, 2011/0269234 A1, 2011/0145940
A1, 2003/0232410 A1, 2005/0208489 A1, 2005/0026157 A1, 2005/0064474 A1,
2006/0188987 A1, and 2006/0063231 A1 (each hereby incorporated by reference); a
zinc-finger nuclease (ZFN) (US20060246567; US20080182332; US20020081614;
US20030021776; WO/2002/057308A2; US20130123484; US20100291048; and,
WO/2011/017293A2, each ofwhich is herein orated by reference); a
meganuclease (see, Epinat et al., (2003) Nucleic Acids Res 31 62; Chevalier et al.,
(2002) Mol Cell -905; Gimble et al., (2003) Mol Biol 334:993-1008; Seligman et
al., (2002) Nucleic Acids Res 0-9; Sussman et al., (2004) JMol Biol 342:31-41;
Rosen et al., (2006) Nucleic Acids Res 34:4791-800; Chames et al., (2005) Nucleic Acids
Res 33:e178; Smith et al., (2006) Nucleic Acids Res 34:e149; Gruen et al., (2002)
Nucleic Acids Res 30:e29; Chen and Zhao, (2005) Nucleic Acids Res 33:e154;
WO2005105989; WO2003078619; WO2006097854; WO2006097853; WO2006097784;
and WO2004031346); and a CAS/CRISPER system (Mali P et al. (2013) Science 2013
Feb 15;339(6121):823-6; Jinek M et al. e 2012 Aug 17;337(6096):816-21; Hwang
WY et al. Nat Biotechnol 2013 Mar;31(3):227-9; Jiang W et al. Nat Biotechnol 2013
Mar;31(3):233-9; and, Cong L et al. Science 2013 Feb 15;339(6121):819-23, each of
which is herein incorporated by reference).
In other ments, the targeted genomic modification can be made by
employing a homologous recombination targeting vector. In such instances, the
targeting vector ses the insert polynucleotide and fiarther comprises an am
and a ream homology arm which flank the insert polynucleotide. The homology
arms which flank the insert cleotide correspond to genomic regions within the
ed genomic locus. For ease of reference, the corresponding genomic regions within
the targeted genomic locus are referred to herein as “target sites”. Thus, in one example,
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a targeting vector can comprise a first insert polynucleotide flanked by a first and a
second homology arm corresponding to a first and a second target site located at the
targeted genomic locus. The targeting vector y aids in the integration of the insert
polynucleotide into the targeted genomic locus through a homologous recombination
event that occurs between the homology arms and the ponding target sites within
the genome of the cell.
As used , a homology arm and a target site spond” or are
“corresponding” to one another when the two regions share a sufficient level of sequence
identity to one another to act as substrates for a homologous recombination reaction. By
“homology” is meant DNA sequences that are either identical or share sequence identity
to a ponding sequence. The sequence identity between a given target site and the
ponding homology arm found on the targeting vector can be any degree of
sequence identity that allows for homologous recombination to occur. For example, the
amount of sequence identity shared by the homology arm of the targeting vector (or a
fragment f) and the target site (or a fragment thereof) can be at least 50%, 55%,
60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% sequence
identity, such that the sequences undergo homologous recombination. Moreover, a
corresponding region of homology between the homology arm and the corresponding
target site can be of any length that is sufficient to promote homologous recombination at
the cleaved recognition site.
In specific embodiments, the isolated rat ES cell, cell line or cell population
exhibits a homologous recombination efficiency of at least 2%, at least 3%, at least 4%,
at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%,
at least 12%, at least 13%, at least 14%, at least 15%, at least 20%, at least 25%, at least
%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at
least 65%, at least 70%, at least 75%, or at least 80%. In one ment, the
homologous recombination efficiency employing the rat ES cell is greater than 4%.
In specific embodiments, a selection marker is employed when generating a
targeted genetic modification in a rat ES cell. The polynucleotide encoding the selection
marker can be present on the ing vector which is designed to uce the targeted
genetic modification into the genome, or it can be found on a separate plasmid or vector.
The polynucleotide ng the selection marker can be contained within in expression
cassette. The various components of such expression cassettes are discussed in r
detail elsewhere herein.
Various selection markers can be used in the methods and compositions disclosed .
Such selection markers can, for example, impart resistance to an antibiotic such as G418,
hygromycin, blastocidin, puromycin or neomycin. Such ion markers include
neomycin phosphotransferase (neor), hygromycin B phosphotransferase (hygr),
puromycin N—acetyl-transferase and cidin S deaminase (bsrr). In still other
embodiments, the selection marker is operably linked to an inducible promoter and the
expression of the selection marker is toxic to the cell. miting examples of such
selection markers include xanthine/guanine phosphoribosyl erase (gpt),
hahypoxanthine-guanine phosphoribosyltransferase (HGPRT) or herpes simplex virus
thymidine kinase K). See, for example, Santerre et al. (1984) Gene 30:147-56;
Joyner (1999) The Practical Approach Series, 293; Santerre et al. (1984) Gene 30: 147-
56; Bernard et al. (1985) Exp Cell Res 158:237-43; Giordano and McAllister (1990)
Gene, 88:285-8; Izumi et al. (1991) Exp Cell Res 197:229-33), each ofwhich is herein
incorporated by reference in their entirety. In specific embodiments, the neoR able
marker is the neomycin phosphotransferase (ne0) gene of Beck et al. (1982) Gene,
19:327-36, which is herein incorporated by reference. The neoR selection maker is that
used in US Patent No, 7,205,148 or 6,596,541, each of which are herein incorporated by
reference.
In specific embodiments, the selection marker employed is a non-attenuated
ion marker. A “non-attenuated selection marker” comprises a selection marker that
retains the ty of the native polypeptide or the selection marker has an increased
activity when compared to the native form of the ptide. An increased in activity
of a ion marker can comprise any statistically significant increase in activity
including, for example, an increase of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70% 80% or higher. Thus, a non-attenuated ion marker when expressed in a host
cell will allow for a higher percentage of the host cells to survive in the presence of a
higher concentration of the selection agent than when employing an attenuated selection
marker. Non-attenuated selection markers include, for example, a neomycin non-
attenuate selection marker. See, for e, Beck et al. (1982) Gene, 19:327-36 or in
US Patent No, 7,205,148 or 6,596,541, each of which are herein incorporated by
reference.
In other ces, the sed activity of a selection marker when compared to
an attenuated selection marker and/or the wild type (native) selection marker can result
from increasing the copy number of either the non-attenuated, attenuated or native
selection marker within the genome of the rat ES cell. As such, a given rat ES cell can
comprise within its genome at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of a given
selection marker (i.e., a non-attenuated selection marker, an attenuated selection marker
or a native (wildtype) selection marker).
The various selection markers ed are encoded by a polynucleotide
operably linked to a promoter active in the rat ES cell. In specific embodiments, the
increase in the activity of the ion marker can result from an increase in expression
of the selection marker. Thus, a er can be employed to elevate the expression
levels of a given ion marker. Promoters of interest e, but are not limited to,
the CMV er, the PGK promoter and the CAG promoter. In one embodiment, the
human Ubiquitin (hUb) promoter is used to express the selection marker. See,
Valenzuela et al. (2003) Nature hnology 21:652-659, herein incorporate by
reference in its entirety.
In one embodiment, the rat ES cell ins its otency to develop into a
plurality of cell types following a single round of electroporation with an exogenous
nucleic acid. In r embodiment, the rat ES cell maintains its pluripotency to
develop into a plurality of cell types following a second round of oporation with an
exogenous nucleic acid, following a third round of electroporation with an exogenous
nucleic acid, following a fourth round of electroporation with an exogenous nucleic acid,
following a fifth round of electroporation with an exogenous nucleic acid, following a
sixth round of electroporation with an exogenous nucleic acid, following a seventh round
of electroporation with an ous nucleic acid, ing an eighth round of
electroporation with an exogenous nucleic acid, following a ninth round of
electroporation with an exogenous nucleic acid, following a tenth round of
electroporation with an exogenous nucleic acid, following an eleventh round of
electroporation with an exogenous nucleic acid, following a twelfth round of
electroporation with an exogenous nucleic acid, following a thirteenth round of
electroporation with an exogenous nucleic acid, following a fourteenth round of
electroporation with an exogenous nucleic acid and/or following a fifteenth round of
electroporation with an exogenous nucleic acid. In other embodiments, the rat ES cell is
capable of transmitting a targeted genetic modification into progeny following a
2014/017452
successive round of electroporation (i.e., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
rounds of electroporation).
1'. Introducing Sequences into a Rat Embryonic Stem Cell
The methods provided herein comprise ucing into a cell with one or more
polynucleotides or polypeptide constructs comprising the various components needed to
make the targeted genomic modification. "Introducing" means presenting to the cell the
sequence (polypeptide or polynucleotide) in such a manner that the sequence gains
access to the interior of the cell. The methods ed herein do not depend on a
particular method for introducing any component of the targeted genomic integration
system into the cell, only that the polynucleotide gains access to the interior of a least
one cell. s for introducing polynucleotides into various cell types are known in
the art and include, but are not limited to, stable transfection methods, transient
transfection methods, and virus-mediated s. Such method include, but are not
limited to, electroporation, ytoplasmic injection, viral infection (including
adenovirus, lentivirus, and retrovirus vectors), transfection, lipid-mediated ection
and/or factionTM. See, for example, Stadtfeld et al. (2009) Nature Methods
6(5):329-330; Yusa et al. (2009) Nat. Methods 6:363-369; Woltjen et al. (2009) Nature
458, 766-770. Such methods include, but are not limited to, direct delivery ofDNA such
as by ex vivo transfection (Wilson et al., Science, 244: 1344-1346, 1989, Nabel and
Baltimore, Nature 326:71 1-713, 1987), optionally with Fugene6 (Roche) or
Lipofectamine (Invitrogen), by injection (US. Pat. Nos. 5,994,624, 5,981,274,
,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each
incorporated herein by reference), including nj ection (Harland and Weintraub, J.
Cell Biol., 101 :1094-1099, 1985; US. Pat. No. 5,789,215, incorporated herein by
reference); by electroporation (US. Pat. No. 5,384,253, incorporated herein by reference;
Tur-Kaspa et al., Mol. Cell Biol., 6:716-718, 1986; Potter et al., Proc. Nat'l Acad. Sci.
USA, 81 :7161-7165, 1984); by calcium ate precipitation (Graham and Van Der
Eb, Virology, -467, 1973; Chen and Okayama, Mol. Cell Biol., 745-2752,
1987; Rippe et al., Mol. Cell Biol., 10:689-695, 1990); by using DEAE-dextran followed
by polyethylene glycol (Gopal, Mol. Cell Biol., 5:1188-1190, 1985); by direct sonic
loading (Fechheimer et al., Proc. Nat'l Acad. Sci. USA, 84:8463-8467, 1987); by
me mediated ection (Nicolau and Sene, Biochim. Biophys. Acta, 721 :185-
190, 1982; Fraley et al., Proc. Nat'l Acad. Sci. USA, 76:3348-3352, 1979; Nicolau et al.,
Methods Enzymol., 149:157-176, 1987; Wong et al., Gene, 10:87-94, 1980; Kaneda et
al., Science, 243:375-378, 1989; Kato et al., Biol. Chem, 266:3361-3364, 1991) and
receptor-mediated transfection (Wu and Wu, Biochemistry, 27:887-892, 1988; Wu and
Wu, J. Biol. Chem., 262:4429-4432, 1987); and any combination of such methods, each
of which is incorporated herein by reference.
In some ments, the cells employed in the methods and compositions have
a DNA construct stably incorporated into their genome. "Stably incorporated" or y
uced" means the introduction of a polynucleotide into the cell such that the
nucleotide sequence integrates into the genome of the cell and is capable of being
inherited by progeny thereof. Any protocol may be used for the stable incorporation of
the DNA constructs or the various components employed to generate the targeted
genomic ation.
Transfection protocols as well as protocols for introducing polypeptides or
polynucleotide sequences into cells may vary. miting transfection methods
include chemical-based transfection methods include the use of mes; rticles;
calcium phosphate (Graham et al. (1973). Virology 52 (2): , Bacchetti et al.
(1977) Proc Natl Acad Sci USA 74 (4): 1590—4 and, Kriegler, M (1991). Transfer and
Expression: A Laboratory Manual. New York: W. H. Freeman and Company. pp. 96—
97); dendrimers; or cationic rs such as DEAE-dextran or polyethylenimine. Non
chemical s include electroporation; Sono-poration; and optical transfection .
Particle-based transfection include the use of a gene gun, magnet assisted ection
(Bertram, J. (2006) Current Pharmaceutical Biotechnology 7, 277—28). Viral methods
can also be used for transfection.
A non-limiting example, of a method of introduction a heterologous
polynucleotide into a rat ES cell follows. The rat ES cells, as described herein, are
passaged for about 24 to about 48 prior to electroporation. About 24 hours prior to
electroporation the media is changed to RVG2i + ROCKi (10uM 2). The rat ES
cells are trypsinized and the rat ES cells are isolated. The rat ES cells are suspended to
achieve a final cell concentration of about 2 x 10A6 to about 10 X 10A6 cells per 75 ul.
About 752 of rat ES cells are added to about 507t DNA comprising the heterologous
polynucleotide and about 125% EP buffer is added. In one non-limiting ment, the
electroporation is carried out with the following parameters: 400V; 400V; 9; 100 uF.
The cells are then cultured in RVG2i and 10uM ROCKi and can be transferred onto
feeder cells.
ii. Selecting Rat Embryonic Stem Cells Having a Targeted Genomic Modification
s method are provided for selecting and maintaining rat ES cells having
stably incorporated into their genome a targeted genetic modification. In one non-
limiting example, when introducing a heterologous polynucleotide into a rat ES cell, the
method can comprise (a) providing an in vitro population of rat ES cells; (b)
introducing into at least one rat ES cell a heterologous polynucleotide comprising a
selection marker operably linked to a promoter active the rat ES cell; and, (c) culturing
in vitro the rat ES cell population in an alternating first and second culture media
wherein the first culture media comprises an effective amount of a selection agent for a
first time period and the second culture media do not comprise the selection agent,
wherein the in vitro culture conditions in pluripotency or totipotency; and thereby
selecting the rat ES cell having stably integrated into its genome the heterologous
polynucleotide. The s methods by which the rat ES cell having the targeted
genetic modification can be selected in a given population can employ an in vitro culture
system which allows the rat ES cells to maintain pluripotency. Thus, any of the in vitro
culture medias and feeder cells discussed herein can be employed.
In specific embodiments, the first and the second culture media are ated
about every 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 hours or more. In a specific embodiment, the
first and second culture media are alternated every 24 hours.
Any appropriate selection marker can be used and the corresponding selection
agent will be present at an ive tration with the culture media. Such selection
markers include any of the native, attenuated or non-attenuated selection marker
discussed herein. In one embodiment, the ion marker employed s resistance
to an antibiotic, including for example, G418. miting selection s comprise
in phosphotransferase II (nptII) or hygromycin phosphotransferase (hpt).
The concentration of the selection agent is such as to allow for the selection of a
rat ES cell having the selection marker while maintaining the pluipotency of the rat ES
cells that are present within the culture. When employing, for example, G418, the
concentration of the G418 in the selection media can range from about 50 11ng to about
125 ug/ml, about 60 ug/ml to about 125ug/ml, about 70 11ng to about ml, about
80ug/ml to about 125ug/ml, about 90ug/ml to about 125ug/ml, about 100ug/ml to about
125 ug/ml, about 110 ug/ml to about 125ug/ml, about 80ug/ml to about 100ug/ml, about
l to about 85ug/ml, about 70ug/ml to about 80ug/ml. In one embodiment, the
concentration of G418 in the culture is 75 ug/ml.
The media employed in the selection allows the rat nic stem cells to retain
pluripotency. Such media is described in detail elsewhere herein.
The selection protocol can be initiated at any time following the introduction of
the polynucleotide encoding the selection marker into the genome of the rat ES cell. In
specific embodiments, the selection protocol begins 10, 15, 20, 24, 30, 35, 40, 50, 60 or
more hours after the introduction of the selection marker into the rat ES cell. In one
embodiment, the selection protocol beings about 2 days following the introduction of the
polynucleotide encoding the ion marker.
A non-limiting selection protocol employing G418 is as follows. Day 2, (211d day
after the introduction of the polynucleotide encoding the selection marker) the population
of rat ES cells is incubated cells in 2i media and G418 at 75 ug/ml. At day 3, the
tion of rat ES cells are incubated cells in 2i media without G418. At day 4, the
population of rat ES cells are incubated in 2i media and G418 at 75 ug/ml. At day 5, the
population of rat ES cells are incubated cells in 2i media t G418. At day 6, the
tion of rat ES cells are ted in 2i media and G418 at 75 ug/ml. At day 7, the
population of rat ES cells is incubated in 2i media without G418. At day 8, the
population of rat ES cells is incubated in in 2i media without G418 and 75 ug/ml. At day
9, the population of rat ES cells is incubated cells in 2i media without G418. At day 10,
the population of rat ES cells is incubated cells in 2i media and G418 at 75 ug/ml. At
day 11, the population of rat ES cells is incubated cells in 2i media without G418. At
day 12, colonies are picked for expansion and screening.
Following the selection of the rat ES cells haVing the selection marker, the
colonies can be expanded. In specific embodiments, the period for expansion can be
about 1, 2, 3, 4, 5, or more days in a e condition that maintains the pluripotency of
the cells. In one miting embodiment, the selected colonies are expanded for 3
days. In a further ment, the media employed is a 2i media. Each clone can then
be passed and fiarther expanded.
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The rat ES cells and cell lines having one or more of the targeted c
modifications can have one or more any the following properties:
(a) have germ-line competency following targeted genetic modification,
meaning when the rat ES cell is implanted into a rat host , the targeted genetic
modification within the genome of the rat ES cell line, is transmitted into an offspring;
(b) have pluripotency in vitro;
(c) have totipotency in vitro;
(d) when cultured in vitro loosely adhere to a feeder cell layer;
(e) when cultured in vitro form sphere-like es when plated on a feeder
cell layer in vitro;
(f) maintain pluripotency when cultured in vitro under conditions comprising
a feeder cell layer that is not genetically modified to express leukemia inhibitor factor
(LIF), wherein the culture media comprises a sufficient concentration of LIF;
(g) maintain pluripotency when cultured in vitro under conditions comprising
a feeder cell layer, wherein the culture media ses mouse LIF or an active variant
or fragment thereof;
(h) comprise a molecular signature characterized by
i) the expression of one or more of rat ES cell-specific genes sing
Adheres Junctions Associate Protein (Aj apl), Claudin 5 (Cldn5), Cdc42 guanine
nucleotide exchange factor 9 (Arhgef9), Calcium/calmodulin-dependent protein kinase
IV (Camk4), ephrin-Al (Efnal), EPH receptor A4 (Epha4), gap junction protein beta 5
(Gij), Insulin-like growth factor binding protein-like l (Igfbpll), Interleulin 36
beta(Illf8), Interleukin 28 receptor, alpha (Il28ra), left-right determination factor 1
(Leftyl), Leukemia inhibitory factor receptor alpha (Lifr), Lysophosphatidic acid
or 2 (Lpar2), Neuronal pentraxin or (Ntm), Protein tyrosine phosphatase
non-receptor type 18 (Ptpn18), Caudal type homeobox 2 (Cdx2), Fibronectin type III and
n repeat domains 1 (Fankl), Forkhead box El (thyroid transcription factor 2)
(Foxel), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Forkhead box El
(thyroid transcription factor 2) (Foxel), Hairy/enhancer-of-split related with YRPW
motif 2 (Hey2), Lymphoid enhancer-binding factor 1 (Lefl ), Sal-like 3 (Drosophila)
), SATB ox l (Satbl), miR-632, or a combination thereof;
ii) the expression ofat least 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,l3,l4,15,l6,
17, 18, 19, 20, 21, 22, 23, 24, 25 or more of the rat ES cell-specific genes comprising
s Junctions Associate Protein (Aj apl), Claudin 5 (Cldn5), Cdc42 guanine
nucleotide exchange factor 9 (Arhgef9), Calcium/calmodulin-dependent protein kinase
IV (Camk4), ephrin-Al (Efna1), EPH receptor A4 (Epha4), gap junction protein beta 5
(Gjb5), Insulin-like growth factor binding protein-like 1 (Igfbpll), Interleulin 36
beta(111f8), Interleukin 28 receptor, alpha (Il28ra), left-right ination factor 1
(Leftyl), Leukemia inhibitory factor receptor alpha (Lifr), Lysophosphatidic acid
receptor 2 (Lpar2), Neuronal pentraxin receptor (Ntm), Protein tyrosine phosphatase
non-receptor type 18 (Ptpn18), Caudal type homeobox 2 (Cdx2), Fibronectin type III and
ankyrin repeat domains 1 (Fankl), Forkhead box E1 (thyroid transcription factor 2)
), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Forkhead box E1
(thyroid transcription factor 2) (Foxel), enhancer-of-split related with YRPW
motif 2 , Lymphoid enhancer-binding factor 1 (Lefl ), Sal-like 3 (Drosophila)
(Sall3), SATB homeobox 1 (Satbl), miR-632, or a combination thereof;
iii) at least a 20-fold increase in the expression of one or more of the rat
ES cell-specific genes as set forth in Table 14 when ed to a F1H4 mouse ES cell;
iV) at least a d increase in the expression of at least 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more ofthe rat ES
pecific genes as set forth in Table 14 when compared to a F1H4 mouse ES cell;
V) the expression of one or more of rat ES cell-specific genes as set forth
in Table 13;
Vi) the expression ofat least 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more ofthe rat ES cell-specific
genes as set forth in Table 13;
Vii) at least a 20-fold increase in the expression of one or more of the rat
ES cell-specific genes as set forth in Table 13 when compared to a F1H4 mouse ES cell;
Viii) at least a 20-fold increase in the expression of at least 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or
more of the rat ES pecific genes as set forth in Table 13 when compared to a F1H4
mouse ES cell;
ix) at least a 20-fold decrease in the expression of one or more of the rat
ES cell-specific genes as set forth in Table 12 when compared to a F1H4 mouse ES cell;
and/or
x) at least a 20-fold decrease in the expression of at least 2, 3, 4, 5, 6, 7,
8, 9,10,11,12,13,14,15,16,17,18,19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or
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more of the rat ES pecific genes as set forth in Table 12 when compared to a F1H4
mouse ES cell;
xi) any combination of expression of the rat ES cell-specific genes of
parts (i)-(X);
xii) a relative expression level of pluripotency markers as shown in Table
for at least 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16,17 or 18 ofthe listed
pluripotency markers. See, pluripotency ranking column of Table 15 for relative
expression levels;
xiii) a relative expression level of the mesodermal markers as shown in
Table 15 for at least 2, 3, or 4 of the listed mesodermal markers. See, rmal
ranking column in Table 15 for relative expression levels;
xiv) a relative sion level of rmal markers as shown in Table
for at least 2, 3, 4, 5, or 6 of the listed endodermal s. See, endodermal ranking
column in Table 15 for relative expression levels;
xv) a relative expression level of neural markers as shown in Table 15 for
at least 2 and 3 of the listed neural markers. See, neural ranking column in table 15 for
relative expression levels;
xvi) a relative expression level of trophectoderm markers as shown in
Table 15 for the listed trophectoderm markers. See, trophectoderm ranking column in
table 15 for ve expression levels;
xvii) any relative expression level of one or more (2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,2 4, 25, 26, 27, 28, 29 or 30) ofthe
pluripotency markers, mesodermal s, endodermal markers, neural markers and/or
trophectoderm markers set forth in Table 15;
xviii) the relative expression level of each of the markers set forth in
table 15;
xix) any combination of the signatures set forth in xii-xiix; and/or
xx) any combination of the signature set forth in i-xiix.
(i) have the y to produce a F0 rat;
(j ) capable of being subcultured and maintaining the undifferentiated state;
(k) having the same number of chromosomes a normal rat cell;
(I) maintain pluripotency in vitro without requiring paracrine LIF signaling;
and/or
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(m) have self renewal, meaning they divide indefinitely while ining
pluripotency.
z'l'l'. Expression Cassettes
The terms “polynucleotide,3, ECpolynucleotide sequence, 3) CEnucleic acid ce,”
and “nucleic acid fragment” are used hangeably herein. These terms encompass
nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA
that is single- or double-stranded, that optionally contains synthetic, non-natural or
altered nucleotide bases. A polynucleotide in the form of a polymer ofDNA may be
comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or
mixtures thereof. Polynucleotides can se deoxyribonucleotides and
cleotides include both naturally occurring molecules and synthetic analogues, and
any combination these. The polynucleotides provided herein also encompass all forms
of sequences including, but not limited to, single-stranded forms, double-stranded forms,
hairpins, stem-and-loop structures, and the like.
Further provided are recombinant polynucleotides. The terms “recombinant
polynucleotide” and “recombinant DNA construct” are used interchangeably herein. A
recombinant construct comprises an artificial or heterologous combination of nucleic
acid sequences, e.g., regulatory and coding sequences that are not found together in
nature. In other embodiments, a recombinant construct may comprise regulatory
sequences and coding sequences that are derived from different sources, or regulatory
sequences and coding sequences derived from the same source, but arranged in a manner
different than that found in . Such a construct may be used by itself or may be
used in conjunction with a vector. If a vector is used, then the choice of vector is
dependent upon the method that is used to transform the host cells as is well known to
those skilled in the art. For example, a plasmid vector can be used. Genetic ts
required to successfully transform, select and propagate host cells and comprising any of
the isolated nucleic acid fragments provided herein. Screening may be accomplished by
Southern analysis of DNA, Northern analysis ofmRNA expression, immunoblotting
analysis of protein expression, or phenotypic analysis, among others.
In specific embodiments, one or more of the components described herein can be
provided in an expression te for expression in a rat cell. The cassette can include 5'
and 3' regulatory sequences ly linked to a polynucleotide ed herein.
“Operably ” means a fianctional linkage between two or more elements. For
e, an operable linkage between a polynucleotide of interest and a regulatory
sequence (i.e., a er) is a fianctional link that allows for expression of the
polynucleotide of interest. Operably linked elements may be contiguous or non-
contiguous. When used to refer to the g of two protein coding regions, operably
linked means that the coding regions are in the same reading frame. In r instance,
a nucleic acid sequence encoding a protein may be operably linked to regulatory
sequences (e.g., promoter, enhancer, silencer sequence, etc.) so as to retain proper
riptional regulation. The cassette may additionally n at least one additional
polynucleotide of st to be co-introduced into the rat ES cell. Alternatively, the
onal polynucleotide of interest can be provided on multiple expression cassettes.
Such an sion cassette is provided with a plurality of restriction sites and/or
recombination sites for insertion of a recombinant polynucleotide to be under the
transcriptional regulation of the regulatory regions. The expression te may
additionally contain selection marker genes.
The expression cassette can include in the 5'-3' direction of transcription, a
transcriptional and ational initiation region (i.e., a promoter), a recombinant
polynucleotide provided , and a transcriptional and translational termination region
(i.e., termination region) functional in mammalian cell or a host cell of interest. The
regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational
termination regions) and/or a polynucleotide provided herein may be native/analogous to
the host cell or to each other. Alternatively, the regulatory regions and/or a
polynucleotide ed herein may be heterologous to the host cell or to each other.
For example, a promoter operably linked to a heterologous polynucleotide is from a
species different from the s from which the polynucleotide was derived, or, if from
the same/analogous species, one or both are substantially modified from their original
form and/or c locus, or the promoter is not the native promoter for the operably
linked polynucleotide. Alternatively, the regulatory regions and/or a recombinant
polynucleotide provided herein may be entirely synthetic.
The termination region may be native with the transcriptional initiation region,
may be native with the ly linked recombinant polynucleotide, may be native with
the host cell, or may be derived from another source (i.e., foreign or heterologous) to the
promoter, the recombinant polynucleotide, the host cell, or any combination thereof
In preparing the expression cassette, the various DNA fragments may be
manipulated, so as to provide for the DNA sequences in the proper orientation. Toward
this end, adapters or linkers may be employed to join the DNA fragments or other
manipulations may be involved to e for convenient restriction sites, removal of
superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro
mutagenesis, primer repair, restriction, annealing, resubstitutions, e. g., transitions and
transversions, may be involved.
A number of promoters can be used in the expression cassettes provided herein.
The promoters can be selected based on the desired outcome. It is recognized that
different applications can be enhanced by the use of ent promoters in the expression
cassettes to te the timing, location and/or level of expression of the
polynucleotide of st. Such expression constructs may also contain, if desired, a
promoter regulatory region (e.g., one conferring inducible, constitutive, environmentally-
or developmentally-regulated, or cell- or tissue-specific/selective expression), a
transcription tion start site, a ribosome binding site, an RNA processing signal, a
transcription termination site, and/or a polyadenylation signal.
iv. Generating F0 Rat Embryos and F1 Progeny Having the Targeted Genetic
ation
The various methods and compositions provided herein can be used to generate a
genetically modified rat. Such methods generally comprise (a) introducing into the
genome of an ed rat ES cell disclosed herein a targeted genetic modification to form
a rat ES cell having a genetic modification; (b) implanting at least one of the cally
modified rat ES cells having the genetic modification into a rat host embryo to produce a
F0 embryo; (c) implanting the F0 embryo into a surrogate mother; (d) gestating the F0
embryo in the surrogate mother to term; and, (e) identifying a F0 rat having the c
modification.
The genetically modified rat ES cells having the genetic modification can be
ted into a rat host embryo that is from the same rat strain or from a different rat
strain. For example, a genetically modified DA rat ES cell can be implanted into a DA
rat host embryo or it can be implanted into an SD host , ACI host embryo or
other heterologous rat host embryo. Similarly, a genetically modified ACI rat ES cell
can be introduced into an ACI rat host embryo or it can be introduced into an SD host
, DA host embryo or other heterologous rat host . Likewise, the surrogate
mother can be from the same rat strain as the genetically modified rat cell and/or the rat
host embryo or the surrogate mother can be from a different rat strain as the genetically
modified rat cell and/or the rat host embryo. In one non-limiting embodiment, the
genetically modified rat cell is from a DA strain, the host rat embryo is from an SD host
embryo and the surrogate mother is from a DA strain. In another non-limiting
embodiment, the cally modified rat cell is from an ACI strain, the host rat embryo
is from an SD strain, and the surrogate mother is from a DA strain.
In still r embodiments, the chimeric rat (F0) can be breed to produce an F1
y that is zygous for the targeted genetic ation. In addition, the male
rat of the F1 progeny can be breed with a female rat of the Fl progeny to obtain an F2
progeny that is homozygous for the genetic modification.
The methods and compositions provided herein allow for at least 1%, 3%, 5%,
%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or
greater of the F0 F0 rats having the genetic modification to transmit the genetic
modification to the F1 progeny.
In some embodiments, the rat ES cells having the targeted genetic modification are
introduced into a pre-morula stage embryo from a corresponding sm, e.g., an 8-
cell stage mouse embryo. See, e.g., US 7,576,259, US 7,659,442, US 7,294,754, and US
2008-0078000 A1, all of which are incorporated by nce herein in their entireties. In
other embodiments, the For, the donor rat ES cells may be implanted at 4 cell stage, 8
cell stage of the host embryo.
The rat embryos comprising the genetically modified rat ES cells is incubated
until the blastocyst stage and then implanted into a surrogate mother to produce an F0.
Rats bearing the genetically modified genomic locus can be identified via modification
of allele (MOA) assay as described herein. The resulting F0 generation derived from the
genetically modified rat ES cells is crossed to a wild-type rat to obtain F1 generation
offspring. Following genotyping with specific primers and/or probes, F1 rats that are
heterozygous for the genetically modified genomic locus are crossed to each other to
produce a rat that is homozygous for the genetically modified genomic locus.
r provided is a F0 rat embryo comprising an inner cell mass having at least
one heterologous stem cell comprising any one of the rat ES cells ed herein. In
other embodiments, progeny of a rat F0 embryo are provided n at least 50%, 60%,
70% or more of the F0 progeny are derived from a genetically modified rat ES cell of as
sed herein.
In one aspect, a method for making a rat ES cell is ed, comprising ng
from a morula-stage rat embryo, a blastocyst-stage rat embryo, or a rat embryo at a
developmental stage between a morula-stage embryo and a blastocyst-stage embryo a rat
cell, and ing the rat cell from the rat embryo under conditions sufficient to maintain
pluripotency and/or totipotency. In on ment, the conditions sufficient to maintain
pluripotency and/or totipotency include 2i media.
In one aspect, a method for making a genetically modified rat is ed,
comprising a step of modifying a rat ES cell genome with a nucleic acid sequence of
interest to form a modified rat ES cell, and employing the modified rat ES cell as a donor
rat ES cell, combining the rat donor ES cell with a rat host embryo, ing the donor
ES cell and rat host embryo, and employing the cultured host embryo to make a
genetically modified rat.
In one , a method for making genetically modified rat Fl progeny is
ed, comprising a step of modifying a rat ES cell genome with a nucleic acid
sequence of interest to form a modified rat ES cell, and employing the modified rat ES
cell as a donor rat ES cell, combining the rat donor ES cell with a rat host embryo,
culturing the donor ES cell and rat host embryo, and employing the cultured host embryo
to make a genetically modified rat, wherein the progeny are about 3%, about 10% or
more, or about 63% or more derived from the genetically modified donor rat ES cell.
In one embodiment, the cultured host embryo is implanted into a surrogate rat
mother, and the cultured host embryo is gestated in the surrogate mother.
In one aspect, a method of transmitting a genetic modification from a rat
pluripotent cell to a rat progeny with high frequency is ed, comprising genetically
modifying a pluripotent rat cell with a nucleic acid sequence of interest on a bacterial
artificial chromosome to form a genetically modified rat pluripotent cell, and employing
the genetically modified rat pluripotent cell with a rat host embryo in a rat surrogate
mother to te a progeny comprising the genetic modification and, optionally,
breeding the progeny.
In one , a method for making a rat ES cell is provided, wherein the method
comprises culturing a frozen 8-cell stage embryo to a blastocyst stage, and deriving from
the cultured blastocyst a rat cell, and culturing the rat cell under conditions sufficient to
maintain otency and/or tency.
V. Variants, Fragments and ce Identity
Active variants and fragments of the disclosed LIF polypeptide, particularly the
mouse LIF polypeptide are provided . “Variants” refer to substantially similar
sequences. As used , a "variant polypeptide” is intended to mean a polypeptide
derived from the native protein by deletion (so-called truncation) of one or more amino
acids at the N—terminal and/or C-terminal end of the native protein; deletion and/or
addition of one or more amino acids at one or more internal sites in the native protein; or
substitution of one or more amino acids at one or more sites in the native protein. Variant
polypeptides continue to possess the desired biological ty of the native polypeptide,
that is, they inhibit the differentiation of rat and/or mouse embryonic stem cells and
contribute to stem cell self-renewal. A variant of a polypeptide or disclosed herein (i.e.
SEQ ID NOS: 1 or rot Accession No. ) will typically have at least about
65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
more sequence identity with the reference sequence.
The term “fragment” refers to a portion of an amino acid comprising a specified
number of contiguous amino acid. In ular embodiments, a fragment of a
polypeptide disclosed herein may retain the biological activity of the full-length
polypeptide and hence inhibit the entiation of rat and/or mouse embryonic stem
cells and contribute to stem cell self-renewal. Fragments of a polypeptide sequence
disclosed herein (i.e. SEQ ID NOS: 1 or SwissProt Accession No. P09056) may
se at least 10, 15, 25, 30, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,
170, 180, 190, 200, contiguous amino acids, or up to the total number of amino acids
present in a full-length n.
As used herein, "sequence identity" or "identity" in the context of two
polynucleotides or ptide sequences makes reference to the residues in the two
sequences that are the same when aligned for maximum correspondence over a specified
comparison window. When percentage of ce identity is used in reference to
proteins it is recognized that residue positions which are not identical often differ by
conservative amino acid substitutions, where amino acid residues are substituted for
other amino acid residues with similar chemical properties (e.g., charge or
hydrophobicity) and therefore do not change the functional properties of the molecule.
When sequences differ in conservative tutions, the percent ce identity may
be adjusted upwards to correct for the conservative nature of the substitution. Sequences
that differ by such conservative substitutions are said to have "sequence similarity" or
"similarity". Means for making this adjustment are well known to those of skill in the
art. Typically this involves scoring a conservative substitution as a partial rather than a
full ch, thereby sing the percentage sequence identity. Thus, for example,
where an identical amino acid is given a score of 1 and a non-conservative substitution is
given a score of zero, a conservative substitution is given a score n zero and 1.
The scoring of conservative tutions is calculated, e.g., as implemented in the
program PC/GENE (Intelligenetics, Mountain View, California).
As used herein, "percentage of sequence ty" means the value determined by
comparing two optimally aligned sequences over a comparison window, wherein the
portion of the polynucleotide sequence in the comparison window may se
ons or deletions (i.e., gaps) as ed to the reference sequence (which does not
comprise additions or deletions) for optimal alignment of the two sequences. The
percentage is calculated by determining the number of positions at which the identical
nucleic acid base or amino acid residue occurs in both sequences to yield the number of
d positions, ng the number of matched positions by the total number of
positions in the window of comparison, and multiplying the result by 100 to yield the
percentage of sequence identity.
Unless otherwise , sequence identity/similarity values provided herein refer
to the value obtained using GAP Version 10 using the following parameters: % identity
and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight
of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino
acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62
scoring matrix; or any equivalent program thereof. "Equivalent m" means any
sequence comparison program that, for any two sequences in question, generates an
alignment having identical nucleotide or amino acid residue matches and an identical
t sequence identity when compared to the corresponding alignment generated by
GAP n 10.
The following examples are offered by way of illustration and not by way of
limitation.
EXAMPLES
Example 1: Rat ES Cell Derivation.
rESC Characterization. As shown in rESC grow as compact spherical colonies
which routinely detach and float in the dish (close-up, . . B, C, F, G: rESC
express pluripotency markers including Oct-4 () and Sox2 (), and
express high levels of alkaline phosphatase ( left . Karyotype for line
DA.2B is 42X,Y( right panel). rESC often become tetraploid; thus, lines were
pre-screened by ng metaphase some spreads; lines with mostly normal
counts were then formally karyotyped.
ACI blastocysts were collected from super-ovulated females obtained commercially; DA
blasts were cultured from frozen 8-cell embryos obtained commercially. Zona idae
were removed with Acid s and blasts were plated onto mitotically inactivated
MEFs. Outgrowths were picked and expanded using standard methods. All blasts were
plated, cultured and expanded using 2i media (Li et al. (2008) Germline competent
embryonic stem cells derived from rat blastocysts, Cell 135: 1299-13 10; incorporated
herein by reference).
Table 3. Rat ES Cell Derivation
Embryo source Blastocysts Frozen 8-cell embryos cultured to
(Superovulation) blastocyst
Outgrowths: 32 (30% of blasts) 10 (45% of blasts)
Lines: 16 (50% of 9 (90% of outgrowths)
wths)
Karyotyped: 3; all 42X,Y 6: 3 42X,X
3 42X,Y
GLT validated: l l) l 42X,X (DA.2C)
1 42X,Y (DA.2B)
Example 2: Rat Production
Chimeric rats were produced by blastocyst injection and transmission of the rESC
genome through the germline. Chimeras produced by blastocyst microinjection using
parental ACI.Gl rESC are shown in Fl agouti pups with albino littermates, sired
by the ACI/SD chimera labeled with an asterisk (*) in are shown in
ne transmission of parental rESC. Three euploid rESC lines were
evaluated for pluripotency by microinjection into albino SD blastocysts. Chimeras were
identified by agouti coat color indicating rESC contribution. For each line, a majority of
chimeras transmitted the rESC genome to F1 offspring (Table 4).
Table 4. Germline Transmission of Parental rESC
Total pups rESC- GLT
Chimeras Germline
Line from GLT derived efficiency
bred transmitters
chimeras pups (%)
ACI.G1 5 3 (60%) 103 11 11
DA.2B 5 4 (80%) 129 11 9
DA.2C
3 2 (66%) 45 7 16
(XX)
Example 3: rESC Targeting: The Rat Rosa 26 Locus.
The rRosa26 locus lies between the Setd5 and Thumpd3 genes as in mouse, with
the same spacing. The rRosa 26 locus ( Panel B) differs from the mRosa 26 locus
( Panel A). The mRosa26 transcripts consist of 2 or 3 exons. The rat locus
contains a 2nd exon 1 (Ex1b) in on to the homologous exon to mouse exon1
(Ex1a). No 3rd exon has been identified in rat. Targeting of a rRosa26 allele is depicted
in Panel C, where homology arms of 5kb each were cloned by PCR using
genomic DNA from DA rESC. The targeted allele ns a Z-hUb-neo cassette
ing a 117bp deletion in the rRosa26 intron.
Targeting efficiency at the rRosa 26 locus was determined (Table 5). Linearized
vector was electroporated into DA or ACI rESC, and ected colonies were cultured
in 2i media + G418, using standard techniques. Individual colonies were picked and
screened using a Loss of Allele (LOA) assay (Valenzuela, D. et al. (2003) High-
throughput engineering of the mouse genome coupled with high-resolution expression
is, Nature Biotech. 21 60, incorporated herein by reference).
Table 5. rRosa26 Targeting Efficiency
Colonies Reconfirmed Targeting efficiency
Cell line
picked. . .
pos1t1ves (%)
ACI.Gl
Chimera production and germline transmission using targeted Rosa26 rESC.
Reconfirmed targeted rRosa26 clones were microinjected into SD cysts, which
were then transferred to pseudopregnant DS recipient females, using standard
techniques. Chimeras were identified by coat color; male F0 chimeras were bred to SD
females. ne (agouti) Fl pups were genotyped for the presence of the targeted
Rosa26 allele; nine of 22 agouti pups genotyped as heterozygous at the Rosa26 locus
(Table 6).
Table 6. Germline Transmission Using Targeted Rosa26 rESC
R26 Clones Germline rESC- dErSiEe-d
Cell line clones producing Transmitting derived
. . . pups
1n] ected Ch1meras Clones Pups
((y )0
AH7: 64 AH7: 4l AH7: 63
AE3: ll2 AE3: 6 AE3: 3
DE9: 4 10
Exam le 3: Derivation of Rat Embr onic Stem Cells.
Superovulation protocol, rats
Day 0: ed with pregnant mare serum: IP, 20 U (0.4 ml).
Day 1 : no action
2014/017452
Day 2: (46 hr. later): injected with hCG, IP, 50 U (1 ml).
- set up single female matings.
Day 3: checked plugs. Females were d. This is day 0.5.
Day 6 (e35): Euthanized s and flushed embryos.
ES Cell derivation ol gsuperovulationz
Day 0:
1) Euthanized female rat with C02.
2) Swabbed ventral abdomen with 70% ethanol; using scissors, opened the
ventral body wall to expose the viscera.
3) Dissected out the oviducts and uterine horns and placed them into a tissue
e dish containing warm N2B27 media. Washed out as much blood
as possible and transferred to a new dish with N2B27.
4) Using a 1 ml syringe and a blunt 27g , flushed media through the
uterine horns and oviducts to eject blastocysts into the media.
) ted the blastocysts with a mouth pipet and transfer to embryo
culture dish containing KSOM + 2i (1 uMPD032590l, 3 uM
CHIR99021). KSOM is a culture medium produced by Millipore. Catalog
number is MR-lO6-D.
6) Cultured overnight at 37°; 7.5% C02.
ES Cell derivation protocol gfrozen embryos!
Day 0:
1) Thawed frozen 8-cell embryos (commercially obtained) into M2 medium.
Cultured 10 minutes at room temperature.
2) Transferred to KSOM + 2i and culture overnight.
ES Cell derivation protocol gsame for both)
Day 1:
1) Transferred cavitated embryos to 2i medium & culture overnight.
2) Continued culturing un-cavitated embryos in KSOM +2i
Day 2:
2014/017452
1) Transferred all remaining embryos to 2i medium (whether or not they've
cavitated).
2) Cultured overnight; continued culturing earlier embryos in 2i medium.
Day 3:
1) Transferred embryos for 30 — 60 s with Acid Tyrodes to remove
the zona pellucida.
2) Washed embryos 3X in 2i medium to remove Acid Tyrodes.
3) ted each embryo into a separate well of a 96-well feeder plate (the
well contains a monolayer of mitotically vated mouse embryonic
fibroblasts (MEFs).
4) Cultured overnight in 2i medium.
Day4—5:
1) Monitored plated embryos for the presence of an wth (an
amorphous undifferentiated mass of . Outgrowths are ready for
transfer when they are approximately twice the size of the plated embryo.
2) Each day: remove spent media with a mircropipet and replace with fresh
2i media.
3) Transferred outgrowths to new feeder wells:
a. Removed spent media and gently wash well with PBS.
b. Removed PBS and add 30 ul 0.05% trypsin; incubate for 10
minutes.
c. Stopped trypsin reaction by adding 30 ul 2i + 10% FBS.
d. Gently iated the cells with a micropipettor and transferred
entire contents of the well to a new well in a 24-well feeder plate.
This was Passage 1 (Pl).
e. Cultured overnight in 2i medium.
Day 5 — 8: (timing depends on how fast each line expands)
1) Changed media each day (2i media) and monitored for the presence of
colonies with an ESC morphology.
2) When colonies , continued culturing until colonies expand to ~
50% confluency.
3) Tryspinzed and passaged colonies as before; plated on feeders, 1 well per
line, in a 6-well dish. This was Passage 2 (P2).
Ongoing:
1) Continued feeding and monitoring each line until approximately 50%
2) Trypsinized cells as usual.
3) stopped trypsin with 2i + 10% FBS; ed the cells by centrifiJgation
(5’, 1200 rpm in Beckman-Coulter tabletop centrifuge).
4) Aspirated the supernatant and gently resuspend the cells in 400 pl
Freezing Medium (70% 2i, 20% PBS, 10% DMSO).
) Distributed the cells into 2 Vials and freeze at -80°. This was Passage 3
(P3).
6) For erm storage, transferred the Vials to liquid N2 storage.
The 2i media was prepared as follows in Table 7.
Reagent Vendor Concentration
DMEM/F l2 basal media InVitrogen/Life lx
Technologies
Neurobasal media Invitrogen/Life lx
Technologies
Penicillin/streptomycin InVitrogen/Life
Technologies
L-Glutamine InVitrogen/Life
logies
aptoethanol InVitrogen/Life
Technologies
N2 supplement Invitrogen/Life lx
Technologies
B27 supplement InVitrogen/Life lx
Technologies
LIF Millipore 100 U/ml
PD0325901 (MEK Stemgent 1 uM
inhibitor) .
CHIR9902l (GSK Stemgent 3 uM
inhibitor) .
Materials: Pregnant Mare’s Serum Gonadotropin (PMSG)
Human Pregnancy Urine Chorionic Gonadotropin (HCG)
Female Rats (5-12 weeks old)
Male rats (12 wks. to 8 mos. old), one per cage
Syringes/needles
Animal room with lights on 6:00-18:00
Procedure:
Day 1: 8:00-10:00 AM
Inject females with 20 IU PMSG (0.4 ml), IP
Discard unused PMSG.
Day 3: 8:00-10:00 AM (48 hours after PMSG injection)
Inject females with 50 IU HCG (1 ml), IP
Place one female per male in mating cage.
Discard unused HCG.
Day 4: 8:00-10:00 AM (24 hrs. after HCG injection)
Check females for plugs.
Hormone ers
PMSG: Sigma #G-4877 (1000 IU). Resuspend in PBS to a final [ ] of 50 IU/ml.
Store at -20° in 1 ml ts.
HCG: Sigma #CG-5 (5000 IU). end in PBS to a final [ ] of 50 IU/ml.
Store at -20° in 1 ml aliquots.
Example 4: yping of Rat Embryonic Stem Cell Lines
The rat ES cell line generated herein were karyotyped, and the results are
summarized in Tables 8-1 1.
Table 8
ACI.G1 Kar 0 in_ Results Number of cells
Number of cells karyotyped 7
Number of cells analyzed 20
Number of 42, XY cells 18
Number of abnormal cells 2
40, XY, -5, -9 1
41, XY, -14 1
42, XY 18
Other notes:
Two analyzed cells were missing
different autosomes, which may
be a sporadic occurrence due to
technical ct. 90% of
analyzed cells had a normal male
42, XY karyotype.
Figure 9 provides a photograph
showing the analysis of the
chromosome number of the
ACI.Gl rat ES cell line.
Table 9
DA.2B Kar 0 in_ Results Number of cells
Number of cells karyotyped 6
Number of cells analyzed 20
Number of 42, XY cells 20
Number of abnormal cells 0
42, XY 20
Other notes:
All analyzed cells had a normal
diploid 42, XY karyotype.
Figure 10 provides a photograph
g the analysis of the
chromosome number of the
DA.2B rat ES cell line.
Table 10
DA.C2 Kar 0t in_ Results Number of cells
Number of cells karyotyped 5
Number of cells analyzed 20
Number of 42, XY cells 20
Number of abnormal cells 0
42, XX
Other notes:
100% of analyzed cells had
normal female XX rat ype.
Figure 11 provides a photograph
showing the analysis of the
chromosome number of the
DA.C2 rat ES cell line.
Table 11
Lines Lines
Blastocyst Karyotype
s plated estabdlishe Karyotypes
BN x SD all lines were high % complex
F1 8 (20%) polyploid
G1: 90% 42 XY; others were 70-85%
16W) euploid
-—-—2B: 100% 42 XY; 2C: 100% 42 ers were 95-100% euploid
-—--_—
Totals 92 34 (37%)
Example 5: Electroporation of Vector into Rat Embryonic Stem Cell
1. Passaged rat ES cells 24-48 hrs prior to electroporation.
2. Changed media to RVG2i + ROCKi (10uM 2) 24 hr. prior to
electroporation
3. Changed media 30’ prior to trypsinization.
. Aliquoted DNA to be electroporated.
d DNA to warm at RT for >10 min.
. Heated DNA for 5’ @ 62°C. Place DNA on ice.
Trypsinized cells:
a. Collected floating colonies. Washed plate to collect as many floaters as
possible.
Pelleted colonies: 3’ @ 750 rpm.
Washed pellet 1X with 5-10m1 PBS and re-spin/pe11et
ted supernatant; add 500% trypsin, 0.05% + 1% chicken serum.
i. Did not p001 more than 1 10 cm plate of colonies per tube. If there
are too many colonies packed into the bottom of the tube during
trypsinization they will clump and most of the cells will be lost.
6. 4’ @ 37°. Pipeted colonies l times to minimize clumping.
f. Repeated steps 1-2 X: 4’ @ 37°.
g. Stopped trypsin with 500% RVG2i + 10% PBS.
8. Pelleted cells: 5’ @ 1200 rpm.
9. Resuspend cells in 10 m1 PBS. Count two 20% aliquots to determine total cell
number.
. Pelleted cells (5’/1200rpm); calculate total cell number and total resuspension
volume to achieve correct cell concentration (target #/75 ul EP buffer).
11. Resuspend in a minimal volume of EP buffer; measure total volume and adjust
to target volume with EP . Electroporation buffer is sold by Millipore. The catalog
# is ESD. See, Valenzuela et al. (2003) Nature Biotechnology 21 :652-659, which
is herein incorporated by reference.
12. Add 751 cells to 507t DNA; transfer the 125K ce11s/DNA solution to one well of
a BTX l cuvette.
a. Filled the empty wells in the same column with 1251 EP .
13. Pulsed the cuvette once in the BTX electroporator:
WO 30706
a. Settings: 400V; 9; 100 uF (settings may vary)
14. Placed cuvette on ice for 15’ to r.
. Removed cells into 5 ml RVG2i + 10uM ROCKi.
16. Added to 15 cm plate with 20 ml RVG2i + 10uM ROCKi. Plate has 2X neoR
MEFs (or other MEFs depending on project). The neoR selectable marker is the
neomycin phosphotransferase (neo) gene of Beck et al. (1982) Gene, 19:327-36 or in US
Patent No, 148 or 6,596,541, each of which are herein incorporated by reference.
17. Incubated @ 37°. Begin selection 48hrs later.
ROCK inhibitor used was Y-27632.
Example 6: Selecting Targeted Genetic Modification in a Rat Embryonic Stem
1. Passaged cells for 24-48 hrs prior to electroporation.
2. Changed media to RVG2i + ROCKi (10uM Y-27632) 24 hr. prior to
electroporation
3. Changed media 30’ prior to nization.
9.09:“ Aliquoted DNA to be electroporated.
Allowed DNA warm at RT for >10 min.
Heated DNA for 5’ @ 62°C. Place DNA on ice.
Trypsinized cells:
h. Collected g colonies. Washed plate to collect as many floaters as
possible.
i. Pelleted colonies: 3’ @ 750 rpm.
j. Washed pellet 1X with 5-10ml PBS and re-spin/pellet
k. Aspirated supernatant; add 500% trypsin, 0.05% + 1% chicken serum.
i. Did not pool more than 1 10 cm plate of colonies per tube. If there
are too many colonies packed into the bottom of the tube during
trypsinization they will clump and most of the cells will be lost.
1. 4’ @ 37°. Pipeted colonies several times to minimize clumping
m. Repeated 1-2 X: 4’ @ 37°.
11. Stopped trypsin with 500k RVG2i + 10% PBS.
8. Pelleted cells: 5’ @ 1200 rpm.
9. ended cells in 10 ml PBS. Count two 20% aliquots to determine total cell
number.
. Pelleted cells (5’/1200rpm); calculate total cell number and total resuspension
volume to achieve correct cell concentration t #/75 ul EP buffer).
11. Resuspend in a minimal volume of EP buffer; measured total volume and
adjusted to target volume with EP buffer.
12. Added 75% cells to 50% DNA; transfer the 125k cells/DNA solution to one well
of a BTX 48-well cuvette.
a. Filled the empty wells in the same column with 1251 EP buffer.
13. Pulsed the cuvette once in the BTX electroporator:
a. Settings: 400V; 400V; 9; 100 uF (settings may vary)
14. Placed cuvette on ice for 15’ to recover.
. Removed cells into 5 ml RVG2i + 10uM ROCKi.
16. Added to 15 cm plate with 20 ml RVG2i + 10uM ROCKi. Plate had 2X neoR
MEFs (or other MEFs depending on project).
17. Incubated @ 37°. Began ion 48hrs later.
18. G418 ion protocol was as follows:
a. Day 2 (211d day after EP): incubated cells in 2i media + G418, 75 ug/ml.
b. Day 3: incubated cells in 2i media without G418
c. Day 4: incubated cells in 2i media + G418, 75 ug/ml.
d. Day 5: incubated cells in 2i media t G418
e. Day 6: incubated cells in 2i media + G418, 75 ug/ml.
f. Day 7: incubated cells in 2i media without G418
g. Day 8: incubated cells in 2i media + G418, 75 ug/ml.
h. Day 9: incubated cells in 2i media without G418
i. Day 10: incubated cells in 2i media + G418, 75 ug/ml.
j. Day 11: incubated cells in 2i media without G418
k. Day 12: picked colonies to expand for screening. Each colony was
dissociated in 0.05% trypsin + 1% chicken serum for 10 minutes and then
plated into 1 well of a 96-well feeder plate.
19. Expanded colonies for 3 days in 2i media.
. ed clones 1:1 to new 96-well feeder plates.
WO 30706
21. Expanded clones for 3 days in 2i media.
22. For each clone, dissociated colonies in trypsin. Froze 2/3 of each clone and store
at -80°; plated the remaining 1/3 onto laminin plates (96-well plates coated with 10
ug/ml laminin).
23. When the laminin plates were confluent, passed off to the screening lab for
genotyping of the clones.
Example 7. Molecular Signature of the Rat Embryonic Stem Cells
The genes listed in Table 13 were expressed at levels 20-fold higher in rat ES
cells than the corresponding genes in mouse ES cells. The genes listed in Table 12 were
expressed at d lower in rat ES cells than the corresponding genes in mouse ES
cells.
The microarray data in Tables 12 and 13 was generated as follows. Rat ES cells
(ACI.G2 and DA.2B) and mouse ES cells (FlH4) were cultured in 2i media for 3
passages until confluent. FlH4 cells were cultured on gelatin-coated plates in the
absence of feeders. FlH4 mouse ES cells were derived from 129S6/SvaTac and
C57BL/6NTac heterozygous embryos (see, e.g., US Pat. No. 7,294,754 and Poueymirou,
W.T., Auerbach, W., Frendewey, D., Hickey, J.F., Escaravage, J.M., Esau, L., Dore,
A.T., Stevens, 8., Adams, N.C., Dominguez, M.G., Gale, N.W., Yancopoulos, G.D.,
DeChiara, T.M., Valenzuela,D.M. (2007), incorporated by nce herein in its
entirety).
The following protocol was used for sample prep:
Materials included TRIzol Reagentp; RNA Lysis Buffer (Zymo Kit); and 1.5 mL
orf tubes.
The 1.5mL Eppendorf tubes were labeled with the Sample ID. Cells grown on a
plate were rinsed in 37C PBS. PBS was removed and 300 ul of Trizol was added. A
scraper was used to break the cells in Trizol. The lysed cells were collected in Trizol in a
1.5mL orf tube. For cells grown on suspension, the cells were rinsed in 37C PBS.
The cells were collected in a 1.5mL tube, the cells were spun down, PBS was d
and 300 ul of Trizol was added. The cells were pipeted up and down to break the cells.
Samples were sorted for FACS with 101 to 105 cells, the volume was trated to
less than 100uL. 4 volumes ofRNA Lysis buffer was added and mix by pipetting. For
, 320uL RNA Lysis buffer was added to 80uL sample. Samples were stored at —
°C.
RNA-Seq was used to measure the expression level of mouse and rat genes.
Sequencing reads were mapped to mouse and rat reference genome by Tophat, and
RPKM (fragments per kilobase of exon per million fragments mapped) were calculated
for mouse and rat genes. gy genes based on gene symbol were selected, and then
used t-test to e gene's expression level between mouse and rat.
miR-632 is in the top 10 highest expressed in rat ESCs but they were not
sed in mouse ES cells. There is ore no comparative data for these genes.
Based on the levels of expression compared to other genes and their known function in
the embryonic pment, the expression of miR-632 were used as a marker for rat ES
cells.
Table 12. The genes listed were expressed at levels 20-fold lower in rat ES cells than the
corresponding genes in mouse ES cells.
ID Notes Symbol Type<s> Dmg<s>
ATP-binding
cassette, sub-
family B
Abcb (MDR/TAP), Plasma
lb Abcb lb member 1B Membrane transporter
smooth ,
Acta2 ACTA2 aorta Cytoplasm other
smooth muscle,
Actg2 ACTG2 enteric Cytoplasm other
1 AEBPl 1 Nucleus peptidase
W ANG”_-
l2 angiopoietin-like 2 Space other
ankyrin repeat
Ankrd ANKRD domain 1 (cardiac transcriptio
l 1 muscle) Cytoplasm n regulator
Anxa Plasma
1 ANXAl annexin A1 Membrane other hydrocortisone
6 ANXA6_-annexin A6 Membrane other
ANXA8
Anxa (includes Plasma
8 others) annexin A8-like 2 Membrane other
Rho guanine
nucleotide
Arhge ARHGE exchange factor
£25 F25 (GEF) 25 Cytoplasm other
Axl -_-AXL tyrosine kinase Membrane kinase cabozantinib
Baspl transcriptio
signal protein 1
Bgn BGN _-biglycan Space other
bone marrow
stromal cell antigen Plasma
Bst2 BST2 2 Membrane other
transcriptio
Btf3 -—BTF3 factor 3 Nucleus n regulator
BTG family, transcriptio
Btg2 BTGZ member 2 Nucleus n regulator
Capsl CAPSL hosine-like Other other
-caveolin l, transmemb
caveolae protein, Plasma rane
CaVl CAVl 22kDa ne receptor
Ccdc8
-_CCDC80 containing 80 A5:0~('D5:m other
Ccnd
A5:O~(I)5:m other
Cd24
CD248 endosialin VIembrane other
CD44 molecule
(Indian blood Plasma
Cd44 CD44 group) VIembrane enzyme
G-protein
coupled
Cd97 --CD97 CD97 molecule VIembrane receptor
CDC42 effector
Cdc4 CDC42E protein (Rho
2ep5 P5 GTPase binding) 5 asm other
cadherin 11, type 2,
thl OB-cadherin Plasma
CDHl l (osteoblast) Membrane other
Cdkn transcriptio
A kinase inhibitor 2A Nucleus n regulator
Cdo l -mCDOl diox enase t .e l C o .lasm enz me
CAP-GLY domain
containing linker -
Clip3 -CLIP3 protein 3 Cytoplasm other
lipofuscinosis,
Cln5 CLN5 neuronal 5 Cytoplasm other
calponin 1, basic,
Cnnl CNNl smooth muscle Cytoplasm other
collagenase
Colla collagen, type I, Extracellular clostridium
COLlAl alpha 1 Space other histolyticum
collagenase
Colla collagen, type I, Extracellular idium
COLlAZ alpha 2 Space other histolyticum
collagenase
Col3a collagen, type III, Extracellular clostridium
COL3A1 al .ha 1 S 0 ace other histol icum
collagenase
ColSa en, type V, Extracellular clostridium
COLSAZ alpha 2 Space other histolyticum
Col6a collagen, type VI, Extracellular collagenase
COL6A2 alpha 2 Space other clostridium
histolyticum
CRYAB c stallin, alnha B other
colony stimulating
factor 1 Extracellular
Csfl CSFl (macrophage) Space cytokine
cystathionase
(cystathionine
Cth O mT gamma-lyase) Cytoplasm enzyme
collagen triple
Cthrc helix repeat Extracellular
CTHRCl containing 1 Space other
Ctsc CTSC Cytoplasm peptidase
cysteine-rich,
angiogenic inducer, Extracellular
CYR6 l 6 1 Space other
DEAD (Asp-Glu-
Ala-Asp) box
DDX58 polypeptide 58 asm enzyme
dickkopfWNT
signaling pathway
DKK3 inhibitor 3 S ace c okine
DMCl —-recombinasel Nucleus enzyme
DPYSL3 —-e-like3 Cytoplasm enzyme
DSE _-epimerase Cytoplasm enzyme
dual specificity - phosphatas
DUSPl atase 1 Nucleus e
dual specificity
phosphatase 27 phosphatas
DUSP27 (putative) Other e
dual specificity atas
DUSP9 phosphatase 9 Nucleus e
endothelin
converting enzyme Plasma
Ece2 ECEZ 2 Membrane peptidase
Ecml ECMl protein 1 Space orter
transcriptio
EGRl _-resonsel Nucleus n reulator
epithelial
membrane protein Plasma
Emp l EMF 1 l Membrane other
epithelial
membrane protein Plasma
Emp3 EMP3 3 Membrane other
Ephx epoxide hydrolase
EPHXZ 2, cytoplasmic Cytoplasm enzyme
coagulation factor transmemb activated
111 (thromboplastin, Plasma rane inant
F3 ’11 U.) tissue ) Membrane receptor human factor VII
Finkel-Biskis-
Reilly murine
sarcoma virus
(FBR-MuSV)
tously
Fau FAU expressed Cytoplasm other
Extracellular
an1 FBN1 fibrillin 1 Space other
Fbxol transcriptio
FBXO l 5 F-box protein 15 Other n regulator
four and a half transcriptio
FhlZ FHLZ LIM domains 2 Nucleus n tor
Flnc FLNC filamin C, gamma Cytoplasm other
FBJ murine
osteosarcoma Viral transcriptio
Fos FOS oncogene homolog Nucleus n regulator
Fundc FUNDC FUN 1 4 domain
2 containing 2 Cytoplasm other
gap junction
protein, beta 3, Plasma
Gjb3 GJB3 3 1kDa ne transporter
Gpa3 Plasma
GPA3 3 (transmembrane) Membrane other
GC-rich promoter
prp GPBP1L binding protein 1-
111 1 like 1 Other other
Plasma
Gpc3 GPC3 Membrane other
growth factor
receptor-bound
Grb 1 0 C) ._. O protein 10 Cytoplasm other
Gstm
GSTMS transferase mu 5 Cytoplasm enzyme
huntingtin-
associated protein
Hap 1 HAP 1 1 Cytoplasm other
HISTZH
Hist1 (includes histone cluster 2,
h2bc others) H2be Nucleus other
nga high mobility
HMGAZ ,urou AT-hook 2 Nucleus Cl’lZ me
high mobility
ngn group nucleosomal
ngn3 binding domain 3 s other
Horm HORMA HORMA domain
ad1 containing 1 Nucleus other
hydroxysteroid
Hsdl HSD17B ( l 7 -beta)
7b 1 4 14 dehydrogenase 14 asm enzyme
Hspb
HSPB1 protein 1 Cytoplasm other
Hspb
HSPB8 protein 8 asm kinase
Extracellular
Htra1 HTRAl peptidase 1 Space peptidase
Ifi204
des eron activated transcriptio
Ifi204 others) gene 204 Nucleus n regulator
interferon-induced
Ifi44 IFI44 0 rotein 44 C o .lasm other
interferon-induced
protein with
tetratricopeptide
Ifitl IFIT1B repeats 1B Cytoplasm other
interferon induced
If1tm3 IiEfi-fifiiillll transmembraneIFITMZ protein 2 Cytoplasm other
insulin-like growth
factor 2 Extracellular growth
Igf2 IGFZ (somatomedin A) Space factor
insulin-like growth
Igfbp factor binding Extracellular
IGFBP7 protein 7 Space transporter
transmemb
interleukin 1 Plasma rane
ll 1 rl l ILlRLl receptor-like l ne rece tor
Inhba HBA ibin,betaA S 0 ace factor
Inhbb HBB —-—inhibin,betaB Space factor
Irf7 regulatory factor 7 Nucleus n regulator
Isng 3 _-—likemodifier Space other
integrin, alpha 5
(fibronectin transmemb
receptor, alpha Plasma rane
Itga5 ITGAS polypeptide) VIembrane receptor
_-—junproto-oncogene \Iucleus n regulator
Junb U3 _—-oncogene us n regulator
lectin, galactoside- transmemb
Lgals LGALS3 binding, soluble, 3 rane
3bp binding n receptor
Lgals lectin, galactoside- Extracellular
LGALS9 , soluble, 9 Sace other
Lmna LMNA
Extracellular
Lox LOX l s loxidase Sace enz me
Lole LOXLZ yloxidase-like2 Space enzyme
Loxl3 LOXL3 _-—lysyloxidase-like 3 Space enzyme
low density
lipoprotein transmemb
receptor-related Plasma rane
Lrpl protein 1 Membrane receptor
Mage MAGEB
b1 6 l 6 family B, 16 Other other
Mcam MCAM _-—adhesionmolecule Membrane other
MGP _-—matrixGlaprotein Space other
matrix
metallopeptidase 2
(gelatinase A,
72kDa gelatinase,
72kDa type IV Extracellular
MMP2 collagenase) Space ase marimastat
matrix-remodelling
E><RA8 associated 8 Other other
Myl9 MYL9 9, regulatory Cytoplasm other
myosin light chain,
phosphorylatable,
Mylpf MYLPF fast skeletal muscle Cytoplasm other
NGFI-A binding
protein 2 (EGRl transcriptio
Nab2 Z binding protein 2) Nucleus n regulator
NADH
ogenase
(ubiquinone) 1 beta
Ndufb NDUFB subcomplex, 4,
4; l SkDa C o .lasm trans 0 orter
nucleophosmin
(nucleolar
phosphoprotein transcriptio
Npml NPMl B23, numatrin) Nucleus n regulator
ligand-
nuclear receptor ent
subfamily 0, group nuclear
Nr0bl NROBl B, member 1 Nucleus or
ligand-
nuclear or dependent
subfamily 4, group nuclear
Nr4al NR4Al A, member 1 Nucleus rece tor
Plasma
Nrp2 Z Membrane kinase
2'-5'-oligoadenylate
synthetase l,
Oasla OASl Da Cytoplasm enzyme
2'-5' denylate
Oale Oale synthetase-like 2 Other enzyme
prolyl 4-
hydroxylase, alpha
P4ha2 P4HA2 polypeptide 11 Cytoplasm enzyme
poly ibose)
polymerase family,
Parp3 PARP3 member 3 Nucleus enzyme
procollagen C-
Pcolc endopeptidase Extracellular
PCOLCE enhancer Space other
phosphate
Pcytl cytidylyltransferase
PCYT lB l, choline, beta Cytoplasm enzyme
Extracellular growth
Pdfc PDGFC rowth factor C S u ace factor
pleckstrin
homology-like
Phlda domain, family A,
PHLDAl member 1 Cytoplasm other
pleckstrin
homology-like
Phlda domain, family A,
PHLDAZ member 2 0‘5plasm other
phospholipase A2,
Pla2g PLAZGl group IB Extracellular
lb B (pancreas) Space enzyme niflumic acid
Pla2g PLA2G4 phospholipase A2,
A group IVA Cytoplasm enzyme quinacrine
WO 30706
(cytosolic,
dependent)
Porcn -_-PORCN (Drosophila) Cytoplasrn
periostin,
osteoblast specific Extracellular
Postn POSTN factor Space
Prrxl PRRXl_—-homeoboxl Nucleus n regulator
3 PRSSZ3 protease, serine, 23 Space ase
some
(prosorne,
rnacropain)
Psrnb subunit, beta type,
8 PSMB8 8 Cytoplasrn peptidase
acetaminophen/pe
ntazocine,
acetaminophen/cl
emastine/pseudoe
phedrine,
aspirin/butalbital/
caffeine
acetaminophen/ca
ffeine/dihydrocod
eine,
aspirin/hydrocodo
aspirin/oxycodone
acetaminophen/as
pirin/caffeine,
aspirin/praVastatin
acetaminophen/de
phenirarnin
e/pseudoephedrin
aspirin/meprobarn
ate,
n/caffeine/pr
opoxyphene,
aspirin/butalbital/
caffeine/codeine,
aspirin/caffeine/di
hydrocodeine,
chlorphenirarnine/
ibuprofen/pseudoe
phedrine,
licofelone,
menatetrenone,
icosapent,
suprofen,
prostaglandin— lornoxicarn,
endoperoxide tiaprofenic acid,
synthase 2 lurniracoxib,
aglandin G/H tenoxicarn,
synthase and naproxen/surnatri
Ptgs2 PTGSZ cyclooxygenase) Cytoplasrn ptan,
ibuprofen/phenyle
phrine,
acetaminophen/as
pirin/codeine,
naproxen/esomepr
azole,
famotidine/ibupro
fen,
ibuprofen/phenyle
phrine/chlorphenir
amine
enac/misopr
0st01,
acetaminophen/bu
talbital/caffeine,
hydroc0d0ne/ibup
r0fen,
acetaminophen/by
droc0d0ne,
acetaminophen/Ha
madol,
acetaminophen/c0
deine,
acetaminophen/0X
ycodone,
acetaminophen/pr
opoxyphene,
niflumic acid,
nitroaspirin,
ofen,
diclofenac,
etoricoxib,
naproxen,
meclofenamic
acid,
pomalidomide,
meloxicam,
celecoxib,
dipyrone,
nimesulide,
acetaminophen,
mefenamic acid,
sal,
ibuprofen,
GW4063 8 1X,
phenylbutazone ,
indomethacin,
sulfasalazine
piroXicam,
valdecoxib,
aspirin, carprofen,
zomepirac,
r0fecoxib,
n/methocarb
amol,
aspirin/caffeine/or
phenadrine ,
aspirin/carisoprod
aspirin/carisoprod
III%EIIIE[HI— 01/codeine,
acetaminophen/bu
talbital,
balsalazide,
aspirin/dipyridarn
ole,
inophen/bu
talbital/caffeine/c
odeine, racemic
flurbiprofen,
phenacetin,
sulindac,
nabumetone,
tolrnetin,
ketorolac,
oxaprozin,
mesalarnine,
salsalate,
fenoprofen,
salicylic acid,
inophen/ch
lorphenirarnine/hy
drocodone/phenyl
ephrine/caffeine,
bromfenac
Extracellular growth
Ptn PTN pleiotrophin Space factor
polymerase I and
transcript release transcriptio
Ptrf W factor Nucleus n regulator
etretinate,
adapalene,
isotretinoin,
ligand- tazarotene,
dependent acitretin, tretinoin,
retinoic acid r alitretinoin,
Rarg receptor, gamma Nucleus rece tor fenretinide
regulator of G-
protein signaling
Rgsl6 7‘ 1 6 Cytoplasrn other
45S pre-ribosornal
Rn45s E45s RNA Other other
RpllO
aL 1 0A L10a Other other
Rpl3l aL31 L31 Other other
Rpl37
a L3 7a Cytoplasrn other
Rple z rough Cytoplasrn other
translation
Rpsl4 aS14 S14 Cytoplasrn regulator
R. 820 Rs20 S20 C o n lasrn other
Rps26 aS26 S26 Cytoplasrn other
ribosomal protein translation
Rps9 E S9 asrn regulator
WO 30706
SlOOa
4 S100A4_-binding protein A4 Cytoplasm other
SlOOa
6 S100A6_-binding protein A6 Cytoplasm transporter
schwannomin
Sdnp interacting protein
1 SCHIPl l Cytoplasm other
Plasma
Sdc2 SDCZ syndecan 2 Membrane other
serpin peptidase
tor, clade E
(nexin,
nogen
Serpi SERPIN activator inhibitor Extracellular
nel ,_. type 1), member 1 Space co in alfa
serpin peptidase
inhibitor, clade E
(nexin,
nogen
Serpi SERPIN activator inhibitor Extracellular
ne2 mN type 1), member 2 Space other
serpin peptidase
inhibitor, clade F
(alpha-2
antiplasmin,
t epithelium
Serpi SERPIN derived factor), Extracellular
H member 1 Space other
Sh3gl
SH3GL2 GRBZ-like 2 Membrane enzyme
solute carrier
family 19
(thiamine
Slcl9 SLC19A transporter), Plasma
a2 member 2 Membrane transporter
solute carrier
family 25
(mitochondrial
carrier; adenine
nucleotide
Slc25 SLCZSA translocator),
a5 member 5 Cytoplasm trans orter clodronic acid
solute carrier
family 29
(equilibrative
nucleoside
SLC29A transporter), Plasma
member 1 Membrane transporter
solute carrier
SLC35F family 3 5, member
N F2 Other other
small r
ribonucleoprotein
SNRPN polypeptide N Nucleus other
SNX22 transporter
secreted protein,
acidic, cysteine- Extracellular
Sparc SPARC rich (osteonectin) Space other
Sppl SPPl Extracellular cytokine
phosphoprotein 1 Space
sulfotransferase
Su1t4a SULT4A family 4A, member
1 1 Cytoplasm enzyme
Tagln TAGLN transgelin Cytoplasm other
ription
elongation factor A transcriptio
Tcea3 TCEA3 (SH), 3 n regulator
transforming
growth factor, beta Extracellular growth
Tgfb3 TGFB3 3 Space factor
Thbsl THBSl _-thrombospondin1 Space other
_Extracellular Thbs2 THBSZ thrombospondin 2 Space other
transmembrane 4 L
Tm4s six family member Plasma
TM4SF1 1 Membrane other
transmembrane -
Tmbi TMBIM BAX inhibitor
1 motif containing 1 Cytoplasm other
m176 TMEMl
b 76B ein176B Other other
Tnc TNC _-tenascinC Space other
Tpd5 TPDSZL
211 1 _-like1 Cytoplasm other
Tme TPMZ _-(beta) Cytoplasm other
Usp 1 tin specific
8 USP18 ase 18 Cytoplasm peptidase
Vim VIM Cytoplasm other
WAP four-
Wfdc disu1f1de core Extracellular
2 WFDCZ domain 2 Space other
WNTl inducible
Wisp signaling pathway Extracellular growth
2 WISPZ protein 2 Space factor
Y box binding transcriptio
bel YBX 1 protein 1 n regulator
Table 13. The genes listed were expressed at levels 20-fold higher in rat ES cells than
the corresponding genes in mouse ES cells.
Entrez Gene Name on Type(S) Dmg(S)
adherens on
associated protein 1 other
adenosylmethionine
decarboxylase 1 Cytoplasm enzyme
ankyrin repeat
domain 2 (stretch transcription
responsive muscle) regulator
Cdc42 guanine
nucleotide exchange
factor (GEF) 9 Cytoplasm other
ATP synthase, H+
transporting,
mitochondrial F0
Atp5h complex, subunit d Cytoplasm enzyme
Btg3 BTG3 member 3 Nucleus other
olamide,
hydrochlorothiazi
de, olamide,
trichloromethiazid
e, chlorothiazide,
chlorthalidone,
benzthiazide,
carbonic anhydrase Extracellul sulfacetamide,
Car6 VI ar Space enzyme topiramate
calcium/calmodulin-
dependent protein
Camk4 CAMK4 kinase IV Nucleus kinase
Capnlz___—
chaperonin
containing TCPl,
Cct6b CCT6B subunit 6B (zeta 2) asm transporter
Cdx2 CDXZ homeobox 2 Nucleus regulator
Cldn5 ___-CLDNS claudin 5 Membrane other
C—type lectin
domain family 3,
Clec3a CLEC3A member A Other other
- de intracellular_- Clic6 CLIC6 channel 6 Membrane ion channel
dehydrogenase/redu
Dhrsx I DHRSX X-linked r enzyme
Dpyle DPYSLZ -like 2 Cytoplasm enzyme
dual specificity
phosphatase 26
Dusp26 DUSP26 (putative) Cytoplasm enzyme
Eci3 Eci3 delta isomerase 3 Other enzyme
eukaryotic
elongation factor-2
Eeka EEFZK kinase Cytoplasm kinase
Efnal ___-EFNAl ephrin-Al Membrane other
Epha4 ___-EPHA4 EPH receptor A4 Membrane kinase
fibronectin type III
and ankyrin repeat transcription
Fankl FANKl domains 1 Nucleus regulator
Fhit FHIT triad asm enzyme
Filipl FILIPl interacting protein 1 Cytoplasm other
Fmod ___-FMOD fibromodulin ar Space other
Foxel FOXEl (thyroid Nucleus regulator
—-2)
Fry FRY (Drosophila) ar Space other
gap junction protein,
Gjbs GJBS beta 5, 3 l . lkDa Membrane transporter
glutathione -
peroxidase 2
pr2 ointestinal) Cytoplasm enzyme
GRXCR glutaredoxin, -
ercr2 2 cysteine rich 2 Other other
HECT, C2 and WW
domain containing
E3 ubiquitin protein Extracellul
Hecw2 liase 2 ar Snace Cl’lZ The
enhancer-of—
split related with transcription
Hey2 YRPW motif 2 Nucleus regulator
inducible T-cell co-
Icos Icos stimulator Membrane
interferon induced
transmembrane Plasma transmembra
Ifitml IFITMl protein 1 Membrane ne rece tor
Illf‘8 Interleuk
in 3 6
beta
112 8ra Interleuk
in 2 8
receptor,
alpha
n-like growth
IGFBPL factor binding
I_nfl)ll 1 urotein—like 1 Other other
interaction protein
for cytohesin
Ipcefl IPCEFl exchange factors 1 Cytoplasm enzyme
Lctl Lctl lactase-like Cytoplasm other
lactate
thd LDHD dehydrogenase D Cytoplasm enzyme
lymphoid enhancer- transcription
Lefl LEFl binding factor 1 Nucleus regulator
left-right
determination factor Extracellul
Leftyl LEFTYl 1 ar Space growth factor
leukemia tory transmembra
Lifr LIFR factor receptor alpha Membrane ne receptor
G-protein
lysophosphatidic Plasma coupled
Lpar2 acid receptor 2 Membrane rece tor
myelin
oligodendrocyte ellul
Mog glycoprotein ar Space other
MORN repeat
Morn5 MORNS nin; 5 Other other
nuclear cap binding
protein subunit 2,
Pigz 20kDa Nucleus other
2014/017452
neuronal pentraXin Plasma transmembra
Nptxr
Ntm —--NTM neurotrimin Membrane other
nuclear transport --
Nutf2 NUTFZ factor 2 Nucleus transporter
Ocln OCLN occludin --Membrane enzyme
oxidized low density
lipoprotein (lectin- Plasma transmembra
Olr l like) receptor 1 Membrane ne receptor
poly(A) binding
protein, cytoplasmic translation
Pabpc4 PABPC4 4 (inducible form) Cytoplasm regulator
dyphylline,
nitroglycerin,
aminophylline,
dipyridamole,
tolbutamide,
tadalafil,
phosphodiesterase theophylline,
Pdel l a PDEl 1A 1 1A Cytoplasm enzyme pentoxifylline
den PDYN prodynorphin ar Space transporter
period circadian
Per3 PER3 clock 3 Nucleus other
Pllp —“as“PLLP plasmolipin ne transporter
protein atase
1, regulatory
Ppplrl4 PPP 1 R1 4 (inhibitor) subunit
O C 14C Cytoplasm other
preferentially
expressed antigen in
Pramel6 Pramel6 melanoma like 6 Other other
protein ne
phosphatase, non-
receptor type 18
Ptpnl 8 PTPNl 8 -derived) Nucleus phosphatase
pyrroline-S -
carboxylate
Pycr l reductase l Cytoplasm enzyme
RAB26, member
RAS oncogene Plasma
Rab26 family ne enzyme
receptor (G protein- -
coupled) activity Plasma
Ramp2 RAMPZ modifying protein 2 Membrane transporter pramlintide
RNA binding motif -
Rbm24 RBM24 protein 24 Other other
Rh-associated
E RHAG glycoprotein Membrane ase
ribosomal n - rringtonin
Rpl3 RPL3 L3 Cytoplasm other C
sal-like 3 -
Sall3 SALL3 (Drosophila) Nucleus other
transcription
Satb l SATBl SATB ox l -Nucleus regulator
ch2 SCGZ secretogranin II cytokine
solute carrier family
(oligopeptide
SLC15A orter), Plasma
Slc15a1 1 member 1 Membrane transporter
solute carrier family
(neuronal/epithelial
high ty
glutamate
transporter, system Plasma
Slc1a1 SLC1A1 Xag), member 1 Membrane transporter riluzole
solute carrier family
(sodium/potassium/c
alcium exchanger),
Slc24a5 member 5 Other other
solute carrier family
37 (glucose
phosphate
SLC37A transporter),
Slc37a2 2 member 2 Other transporter
syntrophin, beta 1
(dystrophin-
associated protein
A1, 59kDa, basic Plasma
40424 comonent 1 Membrane other
ST6 (alpha-N-
acetyl-neuraminyl-
2,3-beta—galactosyl-
1,3)-N-
acetylgalactosamini
St6galn de alpha-2,6-
ac3 transferase 3 asm enzyme
Tex12 TEX12 testis expressed 12 s other
Extracellul
Tex15 TEX15 testis expressed 15 ar Space other
transcription factor
AP-Z alpha
(activating er
binding protein 2 transcription
Tfap2a alpha) Nucleus regulator
transmembrane Plasma
Tmc1 TMC1 channel-like 1 Membrane other
Tmem1 TMEM1 transmembrane
30 protein 130 Other other
Tmem3 TMEM3 transmembrane
0b 0B protein 30B Other other
translocase of outer
mitochondrial
Tomm2 membrane 20
homolog (yeast) Cytoplasm transporter
TOX high mobility
group box family
Tox3 member 3 Other other
tetratricopeptide
th25 TTCZS repeat domain 25 Cytoplasm other
thymidine Extracellul
Tynm TYMP phosphorylase ar Space growth factor
-—__—
-_--_vesicle-associated
-_--_Wfdcl2 core domain 12 ar Space other
-—--WfdclSacore domain 15A Other other
dc6a core domain 6A Other other
Table 14. A subset of genes from Table 13 which are expressed at levels 20-fold higher
in rat ES cells than the corresponding genes in mouse ES cells.
ID Entrez Gene Name
Ajap1 Adheres Iunctions
Associate Protein
CldnS Claudin 5
Arhgef9 Cdc42 guanine
tide exchange
facter 9
Calcium/calmodulin-
dependent protein
kinase IV
Efna1 -Al
E oha4 EPH rece otor A4
Gjb5 gap junction protein beta
Igfbpll Insulin-like growth factor
bindin_ orotein-like 1
1128ra eukin 28 receptor,
aloha
Lefty1 left-right determination
factor 1
Lifr Leukemia inhibitory
factor rece otor aloha
Lpar2 Lysophosphatidic acid
receotor 2
rece otor
Protein tyrosine
phosphatase non-
receotor oe 18
Fibronectin type III and
ankyrin repeat domains
Forkhead box E1
(thyroid transcription
Hairy/enhancer-of—split
related with YRPW motif
id enhancer-
bindin; factor 1
2014/017452
Sa113 Sal-like 3 hila
SATB homeobox 1
An additional molecular signature employing the pluripotency markers/genes for
the rat ES cells has also been developed. Table 15 provides a gene list and their
sion ranks from the RNA profiling data. mRNA was isolated from rat ES cells
and the expression level of various pluripotency markers were compared relative to each
other. The "pluripotency genes" which are listed are genes that other groups have used
(mostly in mouse, but also in rat) as markers of ES cells. Mesoderm endoderm and
neural are similarly defined. By "rank" is refers to the expression in our experiment: the
higher the rank (1 is highest) the higher the expression. For example, Oct4's rank of 13
means that, of all the genes assayed, it was expressed higher than all but 12 genes.
Background in this experiment was any sion value below 30; 6107 genes had
expression values of 30 or higher.
Table 15. Rat ES cell molecular ure employing the pluripotency, mesodermal,
endodermal, neural and trohectoderm markers/genes.
Plurigotenc Plurigotenc Endoderma Endoderma
yRank I lRank
c—Myc 11195 Nestin 7761
Dnmt3L 11418 Pax6 13570
DppaZ Not tested 4571 30x2 681
DppaS Not tested
Ecatl 9714
Eras
Fbxols
6.13
Lefl
LIF receptor
Lin28
Nanog —
----
55w ----_----
Stet. Notttsttt ---_----
Notttsted ---_----
All publications and patent applications mentioned in the specification are
indicative of the level of those skilled in the art to which this invention pertains. All
publications and patent applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was specifically and
dually indicated to be incorporated by reference. Unless otherwise apparent from
the context of any embodiment, aspect, step or feature of the invention can be used in
combination with any other. Reference to a range es any integers within the range,
any ge within the range. Reference to multiple ranges includes composites of
such ranges.
Claims (50)
1. A method for making a targeted c modification in rat embryonic stem (ES) cells, comprising: (a) culturing a population of rat ES cells under conditions comprising a layer of feeder cells that are not modified to express leukemia inhibitory factor (LIF) and a medium comprising about 50 U/mL to about 150 U/mL LIF and a combination of inhibitors ting of a MEK inhibitor and a GSK3 inhibitor; and (b) modifying the rat ES cells to comprise the targeted genetic modification, wherein the rat ES cells are capable of transmitting the targeted genetic modification through the germline.
2. The method of claim 1, further comprising generating the population of rat ES cells in step (a), comprising: (i) culturing in vitro a first layer of feeder cells that are not modified to s leukemia inhibitory factor (LIF) and a morula or a blastocyst stage rat , wherein the zona pellucida of the morula or blastocyst stage rat embryo has been removed, and wherein the culture conditions maintain pluripotency of a rat ES cell and comprise a medium comprising about 50 U/mL to about 150 U/mL LIF and a combination of inhibitors consisting of a MEK inhibitor and a GSK3 inhibitor; (ii) transferring an outgrowth of an amorphous undifferentiated mass of rat ES cells obtained from step (i) to an in vitro culture well comprising a second layer of the feeder cells that are not modified to s LIF; and (iii) ing the outgrowth under conditions comprising the medium comprising about 50 U/mL to about 150 U/mL LIF and a ation of inhibitors consisting of a MEK inhibitor and a GSK3 inhibitor.
3. The method of claim 1 or 2, wherein the rat ES cells: (ii) lack expression of c-Myc; and/or (iii) have a normal karyotype; and/or (iv) form spherical, free-floating es in culture.
4. The method of any one of claims 1-3, further comprising: (c) introducing at least one of the modified rat ES cells from step (b) into a rat host embryo to produce an F0 embryo; (d) implanting the F0 embryo into a surrogate ; (e) gestating the F0 embryo in the surrogate mother to term; and (f) identifying an F0 rat having the targeted genetic modification.
5. The method of claim 4, wherein the modified rat ES cell introduced into the rat host embryo is from the same rat strain as the rat host embryo.
6. The method of claim 4, wherein the modified rat ES cell introduced into the rat host embryo is from a different rat strain than the rat host embryo.
7. A method of making a genetically modified rat, comprising: (a) providing a rat ES cell line comprising a population of rat ES cells obtained by culturing isolated rat ES cells on a layer of feeder cells that are not modified to express ia inhibitory factor (LIF) with a medium comprising about 50 U/mL to about 150 U/mL LIF and a combination of inhibitors consisting of MEK inhibitor PD0325901 and GSK3 inhibitor CHIR99021, and wherein the population of rat ES cells: lacks expression of c-Myc; forms cal, free-floating es in e; is diploid; and is germline competent; (b) obtaining a rat ES cell clone comprising a targeted cation, wherein the obtaining consists of: (i) modifying the population of rat ES cells to comprise the targeted genetic modification; and (ii) identifying in a single g step a rat ES cell clone comprising the targeted genetic modification; (c) introducing the rat ES cell clone into a rat host embryo, wherein the rat ES cell clone is from a different rat strain than the rat host embryo; and (d) gestating the rat host embryo comprising the rat ES cell clone in a surrogate , wherein the surrogate mother produces F0 progeny comprising the targeted c modification, n the targeted genetic modification is transmitted through the germline.
8. The method of claim 7, wherein the providing step (a) ses: (i) culturing in vitro a first layer of the feeder cells that are not modified to express leukemia inhibitory factor (LIF) and a morula or a blastocyst stage rat embryo, wherein the zona pellucida of the morula or blastocyst-stage rat embryo has been removed, and wherein the culture conditions maintain pluripotency of a rat ES cell and comprise a medium comprising about 50 U/mL to about 150 U/mL LIF and a combination of inhibitors consisting of MEK inhibitor 901 and GSK3 tor CHIR99021; and (ii) transferring an outgrowth of an amorphous undifferentiated mass of rat ES cells to an in vitro culture well comprising a second layer of the feeder cells that are not modified to express LIF; and (iii) culturing the outgrowth under conditions comprising the medium comprising about 50 U/mL to about 150 U/mL LIF and a combination of inhibitors consisting of MEK inhibitor PD0325901 and GSK3 inhibitor CHIR99021, thereby maintaining pluripotency of the rat ES cells.
9. The method of any one of claims 4-8, further comprising breeding a male F0 rat comprising the targeted genetic cation with a wild type female rat to e an F1 progeny that is heterozygous for the targeted genetic modification;, and optionally breeding a male rat of the F1 progeny with a female rat of the F1 progeny to obtain an F2 progeny that is homozygous for the targeted genetic cation.
10. The method of claim 9, wherein at least 3% of the F0 rats having the targeted genetic cation transmit the targeted genetic modification to the F1 progeny, optionally n at least 10% of the F0 rats having the ed genetic modification transmit the targeted genetic modification to the F1 progeny, and optionally n at least 60% of the F0 rats having the targeted genetic modification transmit the ed genetic modification to the F1 progeny.
11. The method of any one of claims 1-10, wherein: (I) the rat ES cells are derived from an ACI rat or derived from a Dark Agouti (DA) rat; and/or (II) the rat ES cells are d from a morula stage or a blastocyst stage embryo from a superovulated rat.
12. The method of any one of claims 1-11, wherein the targeted genetic modification comprises an insertion, a deletion, a knockout, a knockin, a point mutation, or a combination thereof.
13. The method of claim 12, wherein the targeted genetic modification comprises an insertion of a logous polynucleotide into the genome of the rat ES cells.
14. The method of any one of claims 1-13, wherein the ing step comprises modifying the rat ES cells to comprise two or more targeted modifications, wherein the rat ES cells can transmit the two or more targeted genetic modifications through the germline.
15. The method of any one of claims 1-14, wherein the rat ES cells are male (XY) rat ES cells or female (XX) rat ES cells.
16. The method of any one of claims 1-15, wherein the rat ES cells have one or more of the following characteristics: (a) the rat ES cells express at least one pluripotency marker selected from Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, and Utf1; (b) the rat ES cells do not express one or more pluripotency markers selected from c-Myc, Ecat1, and Rexo1; (c) the rat ES cells do not express one or more mesodermal s selected from Brachyury and Bmpr2; (d) the rat ES cells do not express one or more endodermal markers selected from Gata6, Sox17, and Sox7; (e) the rat ES cells do not express one or more neural markers selected from Nestin and Pax6; (f) the rat ES cells express one or more pluripotency markers selected from Oct-4, Sox2, and alkaline phosphatase; and (g) the rat ES cells express of one or more rat ESC-specific genes selected from Adherens ons Associated Protein 1 (Ajap1), n 5 (Cldn5), Cdc42 guanine tide exchange factor 9 (Arhgef9), Calcium/calmodulin-dependent protein kinase IV (Camk4), ephrin-A1 (Efna1), EPH receptor A4 (Epha4), gap junction protein beta 5 (Gjb5), Insulin-like growth factor binding n-like 1 (Igfbpl1), Interleukin 36 beta (Il1f8), Interleukin 28 receptor, alpha (Il28ra), ight determination factor 1 (Lefty1), Leukemia inhibitory factor receptor alpha (Lifr), osphatidic acid receptor 2 (Lpar2), Neuronal pentraxin receptor (Ntm), Protein tyrosine phosphatase non-receptor type 18 8), Caudal type homeobox 2 (Cdx2), Fibronectin type III and ankyrin repeat domains 1 (Fank1), Forkhead box E1 (thyroid transcription factor 2) ), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Forkhead box E1 (thyroid ription factor 2) (Foxe1), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Lymphoid enhancer-binding factor 1 (Lef1), Sal-like 3 (Drosophila) (Sall3), SATB homeobox 1 (Satb1), and miR-632.
17. The method of any one of claims 1-16, wherein the medium comprises about 75 U/mL to about 125 U/mL LIF, optionally n the medium comprises about 90 U/mL to about 110 U/mL LIF.
18. The method of claim 17, wherein the medium comprises about 100 U/mL LIF.
19. The method of any one of claims 1-18, wherein the feeder cells comprise a monolayer of mitotically inactivated mouse embryonic fibroblasts (MEFs).
20. The method of any one of claims 1-19, wherein the LIF in the medium is a mouse LIF or has at least 91% sequence identity to SEQ ID NO: 1.
21. The method of any one of claims 1-20, wherein the MEK inhibitor comprises PD0325901 and/or wherein the GSK3 inhibitor comprises CHIR99021.
22. The method of claim 21, wherein the MEK inhibitor is PD0325901 at a concentration of 0.8 µM to about 1.2 µM, and the GSK3 tor is CHIR99021 at a concentration of about 2.5 µM to about 3 µM or 3 µM to about 3.5 µM, ally, wherein the concentration of the MEK inhibitor is about 1 µM, and the concentration of the GSK3 inhibitor is about 3 µM.
23. The method of claim 22, wherein the concentration of LIF in the medium is about 100 U/mL, the MEK inhibitor is PD0325901 at a concentration of about 1 M, and the GSK3 inhibitor is 021 at a tration of about 3 M.
24. The method of any one of claims 1-23, wherein the medium further comprises DMEM/F12 basal medium and Neurobasal medium.
25. The method of claim 24, wherein the medium comprises: 1x 12 basal medium; 1x neurobasal medium; 1% penicillin/streptomycin; 4 mM L-glutamine; 0.1 mM 2-mercaptoethanol; 1x N2 ment; 1x B27 supplement; 100 U/mL LIF; 1 M 901; and 3 M CHIR99021.
26. The method of any one of claims 1-25, wherein the modifying step comprises one or more rounds of electroporation.
27. The method of any one of claims 1-26, wherein the targeted genetic modification is generated via a homologous ination event.
28. The method of any one of claims 1-27, wherein the targeted genetic modification is generated by employing a targeting vector comprising upstream and downstream homology arms flanking an insert polynucleotide, wherein the homology arms correspond to genomic regions within a targeted genomic locus.
29. The method of any one of claims 1-28, wherein the targeted genetic modification is generated using a nuclease agent that generates a single or double strand break at a targeted genomic locus.
30. The method of claim 29, wherein the nuclease agent is a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease (ZFN), a meganuclease, or a CRISPR/Cas system.
31. The method of any one of claims 1-30, wherein the modifying step comprises: (i) introducing into the rat ES cells a heterologous polynucleotide comprising a selection marker operably linked to a promoter active in the rat ES cells; and (ii) culturing in vitro the rat ES cells in alternating first and second culture media, n the first e medium comprises an effective amount of a selection agent for a first time period and the second e medium does not comprise the selection agent, and wherein the in vitro culture ions are sufficient to maintain pluripotency, y selecting the rat ES cells having the heterologous polynucleotide stably integrated into the genome.
32. The method of claim 31, wherein the first and second culture media are alternated every 24 hours, and n the selection marker imparts resistance to an antibiotic, optionally wherein the selection marker comprises one or more of neomycin phosphotransferase (neor), ycin B phosphotransferase (hygr), puromycin-N- acetyltransferase (puror), and cidin S deaminase (bsrr); and optionally wherein the antibiotic is G418.
33. The method of claim 31 or 32, wherein the selection marker has one or more of the ing characteristics: (a) the selection marker comprises a non-attenuated selection marker gene; (b) the selection marker has an sed activity compared to the wild type selection marker; and (c) the rat ES cells comprise multiple copies of the selection marker stably incorporated into the genome.
34. An in vitro e system for culturing and ining pluripotency of rat ES cells, comprising: (a) a feeder cell layer not modified to express leukemia inhibitory factor (LIF); (b) a medium comprising about 50 U/mL to about 150 U/mL LIF, and a combination of inhibitors consisting of a MEK inhibitor and a GSK3 inhibitor; and (c) a population of one or more rat ES cells comprising a targeted genetic modification, wherein the rat ES cells are capable of transmitting the targeted genetic modification through the germline.
35. The in vitro culture system of claim 34, wherein the rat ES cells: (i) lack expression of c-Myc; and/or (ii) have a normal karyotype; and/or (iii) form spherical, free-floating es in culture.
36. The in vitro culture system of claim 34 or 35, wherein the medium comprises about 75 U/mL to about 125 U/mL LIF, ally wherein the medium comprises about 90 U/mL to about 110 U/mL LIF.
37. The in vitro culture system of claim 36, wherein the medium comprises about 100 U/mL LIF.
38. The in vitro culture system of any one of claims 34-37, wherein the feeder cell layer comprises a monolayer of mitotically inactivated mouse embryonic fibroblasts (MEFs).
39. The in vitro culture system of any one of claims 34-38, wherein the LIF in the medium is a mouse LIF or has at least 91% sequence identity to SEQ ID NO: 1.
40. The in vitro culture system of any one of claims 34-39, wherein the MEK inhibitor ses PD0325901 and/or wherein the GSK3 inhibitor comprises CHIR99021.
41. The in vitro culture system of claim 40, wherein the MEK inhibitor is 901 at a concentration of 0.8 µM to about 1.2 µM, and the GSK3 inhibitor is 021 at a concentration of about 2.5 µM to about 3 µM or 3 µM to about 3.5 µM, optionally, wherein the concentration of the MEK inhibitor is about 1 µM, and the concentration of the GSK3 inhibitor is about 3 µM.
42. The in vitro culture system of claim 41, wherein the concentration of LIF in the medium is about 100 U/mL, the MEK inhibitor is PD0325901 at a concentration of about 1 M, and the GSK3 inhibitor is CHIR99021 at a concentration of about 3 M.
43. The in vitro culture system of any one of claims 34-42, n the medium further comprises DMEM/F12 basal medium and asal medium.
44. The in vitro culture system of claim 43, wherein the medium comprises: 1x DMEM/F12 basal ; 1x neurobasal medium; 1% penicillin/streptomycin; 4 mM L- glutamine; 0.1 mM 2-mercaptoethanol; 1x N2 supplement; 1x B27 supplement; 100 U/mL LIF; 1 µM PD0325901; and 3 µM CHIR99021.
45. The in vitro culture system of any one of claims 34-44, wherein the targeted genetic modification comprises an ion, a on, a knockout, a n, a point mutation, or a combination thereof.
46. The in vitro culture system of claim 45, wherein the targeted genetic modification comprises an insertion of a heterologous polynucleotide into the genome of the rat ES cells.
47. The in vitro culture system of claim 46, wherein the heterologous polynucleotide comprises a selection marker, and wherein the selection marker has one or more of the following characteristics: (a) the selection marker comprises a non-attenuated selection marker gene operably linked to a promoter; (b) the ion marker has increased activity compared to a wild type ion marker; and (c) the rat ES cells comprise multiple copies of the selection marker stably incorporated into the genome.
48. The in vitro culture system of any one of claims 34-47, wherein: (a) the rat ES cells are derived from an ACI rat or derived from a Dark Agouti (DA) rat; and/or (b) the rat ES cells are derived from a morula stage or a blastocyst stage embryo.
49. The in vitro culture system of any one of claims 34-48, n the rat ES cells are male (XY) rat ES cells or female (XX) rat ES cells.
50. The in vitro e system of any one of claims 34-49, wherein the rat ES cells have one or more of the following characteristics: (a) the rat ES cells express at least one pluripotency marker ed from Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, and Utf1; (b) the rat ES cells do not express one or more otency markers selected from c-Myc, Ecat1, and Rexo1; (c) the rat ES cells do not express one or more mesodermal markers selected from Brachyury and Bmpr2; (d) the rat ES cells do not express one or more endodermal markers selected from Gata6, Sox17, and Sox7; (e) the rat ES cells do not express one or more neural markers selected from Nestin and Pax6; (f) the rat ES cells express one or more pluripotency markers selected from Oct-4, Sox2, and alkaline phosphatase; and (g) the rat ES cells express of one or more rat ESC-specific genes selected from Adherens Junctions Associated Protein 1 ), Claudin 5 (Cldn5), Cdc42 guanine nucleotide exchange factor 9 (Arhgef9), Calcium/calmodulin-dependent n kinase IV (Camk4), ephrin-A1 (Efna1), EPH receptor A4 (Epha4), gap junction protein beta 5 (Gjb5), Insulin-like growth factor binding protein-like 1 (Igfbpl1), Interleukin 36 beta ), Interleukin 28 receptor, alpha (Il28ra), left-right ination factor 1 (Lefty1), Leukemia inhibitory factor receptor alpha (Lifr), Lysophosphatidic acid receptor 2 (Lpar2), Neuronal pentraxin receptor (Ntm), Protein tyrosine phosphatase non-receptor type 18 8), Caudal type homeobox 2 (Cdx2), Fibronectin type III and ankyrin repeat domains 1 (Fank1), Forkhead box E1 (thyroid transcription factor 2) (Foxe1), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Forkhead box E1 (thyroid transcription factor 2) ), Hairy/enhancer-of-split related with YRPW motif 2 , Lymphoid enhancer-binding factor 1 (Lef1), Sal-like 3 (Drosophila) (Sall3), SATB homeobox 1 (Satb1), and miR-632.
Applications Claiming Priority (3)
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US201361767093P | 2013-02-20 | 2013-02-20 | |
US61/767,093 | 2013-02-20 | ||
PCT/US2014/017452 WO2014130706A1 (en) | 2013-02-20 | 2014-02-20 | Genetic modification of rats |
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NZ711775A NZ711775A (en) | 2021-02-26 |
NZ711775B2 true NZ711775B2 (en) | 2021-05-27 |
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