Genetic modification of rats

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.

That which is claimed: 
     
         1 . An isolated 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  claim 1 , wherein the cell is derived from an ACI rat. 
     
     
         3 . The isolated rat ES cell of  claim 1 , wherein the cell is derived from a Dark Agouti (DA) rat.2. 
     
     
         4 . The isolated rat ES cell of  claim 1 , wherein the cell is euploid and capable of transmitting a targeted genetic modification through the germline. 
     
     
         5 . The isolated rat ES cell of  claim 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  claim 4 , wherein the rat ES cell has a germline transmission efficiency of the targeted genetic modification of at least 60%. 
     
     
         7 . The isolated rat ES cell of  claim 1 , wherein the rat ES cell exhibits a targeting efficiency of homologous recombination of at least 2%. 
     
     
         8 . The isolated rat ES cell of  claim 1 , wherein the rat ES cell is capable of transmitting a targeted genetic modification into progeny following a successive round of electroporation. 
     
     
         9 . The isolated rat ES cells of  claim 1 , wherein the rat ES cell comprises one or more, two or more, or three or more targeted genetic modification. 
     
     
         10 . The isolated rat ES cell of  claim 4 , wherein the targeted genetic modification comprises an insertion, a deletion, a knockout, a knockin, a point mutation, or a combination thereof. 
     
     
         11 . The isolated rat ES cell of  claim 9 , wherein the targeted genetic modification comprises at least one insertion of a heterologous polynucleotide into a genome of the cell. 
     
     
         12 . The isolated rat ES cell of  claim 11 , wherein the heterologous polynucleotide comprises a selection marker. 
     
     
         13 . The isolated rat ES cell of  claim 12 , 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 marker. 
     
     
         14 . The isolated rat ES cell of  claim 12 , wherein the selection marker has an increased activity compared to a wild type selection marker. 
     
     
         15 . The isolated rat ES cell of  claim 1 , 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  claim 1 , wherein the rat ES cell, when cultured in vitro, loosely adhere to the feeder cell layer. 
     
     
         17 . The isolated rat ES cell of  claim 1 , wherein the cell does not require paracrine LIF signaling for maintenance of pluripotency. 
     
     
         18 . The isolated rat ES cell of  claim 1 , wherein the cell is a male (XY) rat ES cell. 
     
     
         19 . The isolated rat ES cell of  claim 1 , wherein the cell is a female (XX) rat ES cell. 
     
     
         20 . The isolated rat ES cell of  claim 1 , 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 targeting efficiency or germline transmission efficiency of a targeted genetic modification. 
     
     
         21 . The isolated rat ES cell of  claim 1 , wherein 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, Utf1, or a combination thereof. 
     
     
         22 . The isolated rat ES cell of  claim 1 , wherein the rat ES cells do not express one or more pluripotency markers selected from c-Myc, Ecat1, Rexo1, or a combination thereof. 
     
     
         23 . The isolated rat ES cell of  claim 1 , wherein the rat ES cells do not express one or more mesodermal markers selected from Brachyury, Bmpr2, or a combination thereof. 
     
     
         24 . The isolated rat ES cell of  claim 1 , wherein the rat ES cells do not express one or more endodermal markers selected from Gata6, Sox17, Sox7, or combination thereof; 
     
     
         25 . The isolated rat ES cell of  claim 1 , 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  claim 1 , wherein the cell expresses a pluripotency marker comprising Oct-4, Sox2, alkaline phosphatase, or a combination thereof. 
     
     
         27 . The isolated rat ES cell of  claim 1 , wherein the rat ES cell is characterized by the expression of one or more of a rat ESC-specific gene selected from one or more of Adheres Junctions Associate Protein (Ajap1), Claudin 5 (Cldn5), Cdc42 guanine nucleotide 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 protein-like 1 (Igfbpl1), Interleukin 36 beta (Il1f8), Interleukin 28 receptor, alpha (Il28ra), left-right determination 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 (Ptpn18), 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) (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), miR-632, or a combination thereof. 
     
     
         28 . An isolated population 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  claim 28 , wherein the rat ES cells are derived from an ACI rat. 
     
     
         30 . The isolated population of rat ES cells of  claim 28 , wherein the rat ES cells are derived from a Dark Agouti (DA) rat. 
     
     
         31 . The isolated population of rat ES cells of  claim 28 , wherein the rat ES cells are capable of transmitting their genome through the germline. 
     
     
         32 . The isolated population of rat ES cells of  claim 28 , wherein the rat ES cells have a germline transmission efficiency of the targeted genetic modification of at least 3%. 
     
     
         33 . The isolated population of rat ES cells of  claim 28 , wherein the rat ES cells have a germline transmission efficiency of the targeted genetic modification of at least 60%. 
     
     
         34 . The isolated population of rat ES cells of  claim 28 , wherein the rat ES cells exhibit a targeting efficiency of homologous recombination of at least 2%. 
     
     
         35 . The isolated population of rat ES cells of  claim 28 , wherein the rat ES cells are capable of transmitting a targeted genetic modification into progeny following a successive round of electroporation. 
     
     
         36 . The isolated population of the rat ES cells of  claim 28 , 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  claim 36 , wherein the targeted genetic modification is at the rat Rosa26 locus. 
     
     
         38 . The isolated population of rat ES cells of  claim 36 , wherein the targeted genetic modification comprises an insertion, a deletion, a knockout, a knockin, a point mutation, or a combination thereof. 
     
     
         39 . 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  claim 39 , wherein the heterologous polynucleotide comprises a selection marker. 
     
     
         41 . The isolated population of rat ES cells of  claim 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 marker.   
     
     
         42 . The isolated population of rat ES cells of  claim 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  claim 28 , wherein the cells form a sphere-like colony when plated on a feeder cell layer in culture comprising a LIF polypeptide, a GSK3 inhibitor, and a MEK inhibitor. 
     
     
         44 . The isolated population of rat ES cells of  claim 28 , wherein the cells, when cultured in vitro, loosely adhere to the feeder cell layer. 
     
     
         45 . The isolated population of rat ES cells of  claim 28 , wherein the cells do not require paracrine LIF signaling for maintenance of pluripotency. 
     
     
         46 . The isolated population of rat ES cells of  claim 28 , wherein the rat ES cells are a male (XY) rat ES cells. 
     
     
         47 . The isolated population of rat ES cells of  claim 28 , wherein the rat ES cells are female (XX) rat ES cells. 
     
     
         48 . The isolated population of rat ES cells of  claim 28 , wherein 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. 
     
     
         49 . The isolated population of rat ES cells of  claim 28 , wherein 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, Utf1, or a combination thereof. 
     
     
         50 . The isolated population of rat ES cells of  claim 28 , wherein the rat ES cells do not express one or more pluripotency markers selected from c-Myc, Ecat1, Rexo1, or a combination thereof. 
     
     
         51 . The isolated population of rat ES cells of  claim 28 , 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  claim 28 , wherein the rat ES cells do not express one or more endodermal markers selected from Gata6, Sox17, Sox7, or combination thereof. 
     
     
         53 . The isolated population of rat ES cells of  claim 28 , 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  claim 28 , wherein the rat ES cells expresses a pluripotency marker comprising Oct-4, Sox2, alkaline phosphatase, or a combination thereof. 
     
     
         55 . The isolated population of rat ES cells of  claim 28 , 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 (Ajap1), Claudin 5 (Cldn5), Cdc42 guanine nucleotide 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 protein-like 1 (Igfbpl1), Interleukin 36 beta (Il1f8), Interleukin 28 receptor, alpha (Il28ra), left-right determination 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 (Ptpn18), 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) (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), miR-632, or a combination thereof. 
     
     
         56 . The isolated population of rat ES cells of  claim 28 , wherein the population comprises at least 10 4  cells. 
     
     
         57 . The isolated population of rat ES cells of  claim 28 , 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 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 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 paracrine 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 pluripotency marker selected from Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, Utf1, or a combination thereof;   j. the rat ES cells do not express one or more differentiation markers selected from c-Myc, Ecat1, Rexo1;   k. the rat ES cells do not express one or more mesodermal markers selected from Brachyury, Bmpr2, or a combination thereof;   l. the rat ES cells do not express one or more endodermal markers selected from Gata6, Sox 17, Sox7, or combination thereof; and/or   m. the rat ES cells do not express one or more neural markers selected from Nestin, Pax6, or combination thereof.   
     
     
         58 . The isolated population of rat ES cells of  claim 28 , 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 comprising a Leukemia Inhibitory Factor (LIF), GSK3 inhibitor, and a MEK inhibitor, wherein 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  claim 59 , wherein the rat ES cell, loosely adhere to the feeder cell layer. 
     
     
         61 . The in vitro culture of  claim 59 , wherein the rat ES cells are capable of transmitting their genome through the germline. 
     
     
         62 . The in vitro culture of  claim 59 , wherein the rat ES cells are derived from an ACI rat. 
     
     
         63 . The in vitro culture of  claim 59 , wherein the rat ES cells are derived from a Dark Agouti (DA) rat.2. 
     
     
         64 . The in vitro culture of  claim 59 , wherein the rat ES cells are capable of transmitting a targeted genetic modification through the germline. 
     
     
         65 . The in vitro culture of  claim 64 , wherein the rat ES cells comprise a germline transmission efficiency of the targeted genetic modification of at least 3%. 
     
     
         66 . The in vitro culture of  claim 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  claim 59 , wherein the rat ES cells exhibit a targeting efficiency of homologous recombination of at least 2%. 
     
     
         68 . The in vitro culture of  claim 59 , wherein the rat ES cell is capable of transmitting a targeted genetic modification into progeny following a successive round of electroporation. 
     
     
         69 . The in vitro culture of  claim 59 , wherein the rat ES cell comprises one or more, two or more, or three or more targeted genetic modification. 
     
     
         70 . The in vitro culture of  claim 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 culture of  claim 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  claim 71 , wherein the heterologous polynucleotide comprises a selection marker. 
     
     
         73 . The in vitro culture of  claim 72 , 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 marker. 
     
     
         74 . The in vitro culture of  claim 72 , wherein the selection marker has an increased activity compared to a wild type selection marker. 
     
     
         75 . The in vitro culture of  claim 59 , wherein the cell does not require paracrine LIF signaling for maintenance of pluripotency. 
     
     
         76 . The in vitro culture of claim of  claim 59 , wherein the cell is a male (XY) rat ES cell. 
     
     
         77 . The in vitro culture of claim of  claim 59 , wherein the cell is a female (XX) rat ES cell. 
     
     
         78 . The in vitro culture of claim of  claim 59 , 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 targeting efficiency or germline transmission efficiency of a targeted genetic modification. 
     
     
         79 . The in vitro culture of  claim 59 , wherein 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, Utf1, or a combination thereof. 
     
     
         80 . The in vitro culture of  claim 59 , wherein the rat ES cells do not express one or more pluripotency markers selected from c-Myc, Ecat1, Rexo1, or a combination thereof. 
     
     
         81 . The in vitro culture of  claim 59 , wherein the rat ES cells do not express one or more mesodermal markers selected from Brachyury, Bmpr2, or a combination thereof. 
     
     
         82 . The in vitro culture of claim of  claim 59 , wherein the rat ES cells do not express one or more endodermal markers selected from Gata6, Sox17, Sox7, or combination thereof. 
     
     
         83 . The in vitro culture of claim of  claim 59 , wherein the rat ES cells do not express one or more neural markers selected from Nestin, Pax6, or combination thereof. 
     
     
         84 . The in vitro culture of  claim 59 , wherein the cell expresses a pluripotency marker comprising Oct-4, Sox2, alkaline phosphatase, or a combination thereof. 
     
     
         85 . The in vitro culture of  claim 59 , 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 (Ajap1), Claudin 5 (Cldn5), Cdc42 guanine nucleotide 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 protein-like 1 (Igfbpl1), Interleukin 36 beta (Il1f8), Interleukin 28 receptor, alpha (Il28ra), left-right determination 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 (Ptpn18), 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) (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), miR-632, or a combination thereof. 
     
     
         86 . The in vitro culture of  claim 59 , wherein the concentration of LIF is 50 U/ml to 150 U/ml. 
     
     
         87 . The in vitro culture of  claim 59 , wherein the concentration of LIF is 100 U/ml. 
     
     
         88 . The in vitro culture of  claim 59 , wherein the LIF is from mouse or comprises at least 92% sequence identity to SEQ ID NO: 1. 
     
     
         89 . The in vitro culture of  claim 59 , wherein the rat ES cell is capable of maintaining a pluripotency without requiring a paracrine LIF signaling. 
     
     
         90 . The in vitro culture of  claim 59 , wherein the feeder cell layer is not genetically modified to express LIF. 
     
     
         91 . The in vitro culture of  claim 59 , wherein the feeder cell layer comprises a monolayer of mitotically inactivated mouse embryonic fibroblasts (MEFs). 
     
     
         92 . The in vitro culture of  claim 59 , wherein the MEK inhibitor comprises PD0325901. 
     
     
         93 . The in vitro culture of  claim 59 , wherein the GSK-3 inhibitor comprises CHIR99021. 
     
     
         94 . The in vitro culture of  claim 59 , wherein the population of rat ES cells is derived from a rat blastocyst-stage embryo or a rat morula-stage embryo. 
     
     
         95 . The in vitro culture of  claim 94 , wherein the blastocyst-stage or the morula-stage rat embryo further comprises an outgrowth of an amorphous undifferentiated mass of rat ES cells. 
     
     
         96 . The in vitro culture of  claim 94 , wherein the population of rat ES cells comprises an isolated outgrowth of an amorphous undifferentiated mass of rat ES cells. 
     
     
         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 wherein the culture conditions 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) 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 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  claim 97 , wherein the rat ES cell line is passaged at least 5 times. 
     
     
         99 . The method of  claim 97 , wherein the rat ES cell line is passaged at least 10 times. 
     
     
         100 . The method of  claim 97 , wherein the medium comprises about 50 U/ml to about 150 U/ml of mouse LIF. 
     
     
         101 . The method of  claim 97 , wherein the medium comprises about 100 U/ml of mouse LIF. 
     
     
         102 . The method of  claim 97 , wherein the feeder cell layer is not genetically modified to express LIF. 
     
     
         103 . The method of  claim 97 , wherein the feeder cell layer comprises a monolayer of mitotically inactivated mouse embryonic fibroblasts (MEFs). 
     
     
         104 . The method of  claim 97 , wherein the MEK inhibitor comprises PD0325901. 
     
     
         105 . The method of  claim 97 , wherein the GSK-3 inhibitor comprises CHIR99021. 
     
     
         106 . The method of  claim 97 , wherein (a) the rat ES cell line is derived from an ACI rat or derived from a Dark Agouti (DA) rat; (b) the rat ES cell line is derived from a morula-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 claim of  claim 97 , wherein the medium further comprises at least one of an FGF receptor inhibitor, a ROCK inhibitor, or an ALK inhibitor. 
     
     
         108 . The method of  claim 107 , wherein the FGF receptor inhibitor comprises PD184352, the ROCK inhibitor comprises Y-27632, or the ALK inhibitor comprises A-83-01. 
     
     
         109 . The method of  claim 97 , wherein at least one rat ES cell has a germline transmission efficiency of the targeted genetic modification is at least 3%. 
     
     
         110 . The method of  claim 69 , wherein the germline transmission efficiency of the targeted 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) 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 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  claim 111 , wherein the first and second culture media are alternated every 24 hours. 
     
     
         113 . The method of  claim 111 , wherein the selection marker imparts resistance to an antibiotic. 
     
     
         114 . The method of  claim 111 , wherein the antibiotic comprises G418. 
     
     
         115 . The method of  claim 111 , wherein the selection marker comprises neomycin phosphotransferase (neo r ), hygromycin B phosphotransferase (hyg r ), puromycin-N-acetyltransferase (puro r ), blasticidin S deaminase (bsr r ), xanthine/guanine phosphoribosyl transferase (gpt), and herpes simplex virus thymidine kinase (HSV-k), or a combination thereof. 
     
     
         116 . The method of  claim 111 , wherein (a) the selection marker has an increased activity compared to the wild type selection 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  claim 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 introducing into the genome of an isolated rat ES cell of  claim 1  a heterologous polynucleotide to form a genetically modified rat ES cell. 
     
     
         119 . A method of making a genetically modified rat comprising:
 (a) introducing into the genome of the isolated rat embryonic stem (ES) cell of  claim 1 , 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  claim 119 , further comprising breeding a male F0 rat with a wild type female rat to produce an F1 progeny that is heterozygous for the targeted genetic modification. 
     
     
         121 . The method of  claim 120 , further comprising breeding a male F0 rat with a wild type female rat to produce an F1 progeny that is heterozygous for the targeted genetic modification. 
     
     
         122 . The method of  claim 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 genetic modification. 
     
     
         123 . The method of  claim 119 , wherein at least 3% of the F0 rats having the genetic modification transmit the genetic modification to the F1 progeny. 
     
     
         124 . The method of  claim 119 , wherein at least 10% of the F0 rats having the genetic modification transmit the genetic modification to the F1 progeny. 
     
     
         125 . The method of  claim 119 , wherein at least 60% of the F0 rats having the genetic modification transmit the genetic modification to the F1 progeny. 
     
     
         126 . The method of  claim 119 , wherein the genetically modified rat ES cell is from the same rat strain as the rat host embryo. 
     
     
         127 . The method of  claim 119 , wherein 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  claim 1 , wherein the rat ES cells in the population comprise:
 (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 genetically 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 requiring 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 express at least one pluripotency marker selected from Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, Utf1, or a combination thereof;   (k) the rat ES cells do not express one or more differentiation markers selected from c-Myc, Ecat1, Rexo1.   (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 endodermal markers selected from Gata6, Sox17, Sox7, or combination thereof; and/or   (n) the rat ES cells do not express one or more neural markers selected from Nestin, Pax6, or combination thereof.