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 inhibitory 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 embryonic stem (ES) cell obtainable by culturing isolated rat ES cells on a feeder cell layer with a medium comprising N2 supplement, B27 supplement, a MEK inhibitor, a GSK3 inhibitor, and about 50 U/mL to about 150 U/mL leukemia inhibitory factor (LIF),
 wherein the feeder cell layer is not modified to express LIF, and   wherein the isolated rat ES cell:   (i) is capable of being modified to comprise a targeted genetic modification and transmitting the targeted genetic modification through the germline;   (ii) has a normal karyotype; and   (iii) lacks expression of c-Myc.   
     
     
         2 . The isolated rat ES cell of  claim 1 , wherein the isolated rat ES cell has been modified to comprise the targeted genetic modification and is capable of transmitting the targeted genetic modification through the germline. 
     
     
         3 . The isolated rat ES cell of  claim 1 , wherein the isolated rat ES cell is derived from an ACI rat or a Dark Agouti (DA) rat. 
     
     
         4 . The isolated rat ES cell of  claim 1 , wherein the isolated rat ES cell is a male (XY) rat ES cell. 
     
     
         5 . The isolated rat ES cell of  claim 1 , wherein the isolated rat ES cell is a female (XX) rat ES cell. 
     
     
         6 . The isolated rat ES cell of  claim 2 , wherein the germline transmission efficiency of the targeted genetic modification is at least 3%. 
     
     
         7 . The isolated rat ES cell of  claim 6 , wherein germline transmission efficiency of the targeted genetic modification is at least 60%. 
     
     
         8 . The isolated rat ES cell of  claim 2 , wherein the isolated rat ES cell exhibits a homologous recombination targeting efficiency of at least 2%. 
     
     
         9 . The isolated rat ES cell of  claim 2 , wherein the isolated rat ES cell is capable of transmitting the targeted genetic modification into progeny following one or more rounds of electroporation. 
     
     
         10 . The isolated rat ES cell of  claim 2 , wherein the isolated rat ES cell comprises two or more targeted genetic modifications and can transmit the two or more targeted genetic modifications through the germline. 
     
     
         11 . The isolated rat ES cell of  claim 2 , wherein the targeted genetic modification comprises an insertion, a deletion, a knockout, a knockin, a point mutation, or a combination thereof. 
     
     
         12 . The isolated rat ES cell of  claim 11 , wherein the targeted genetic modification comprises at least one insertion of a heterologous polynucleotide into the genome of the isolated rat ES cell. 
     
     
         13 . The isolated rat ES cell of  claim 1 , wherein the isolated rat ES cell, when cultured in vitro, loosely adheres to the feeder cell layer. 
     
     
         14 . The isolated rat ES cell of  claim 1 , wherein the isolated rat ES cell forms sphere-like colonies when plated on the feeder cell layer in vitro. 
     
     
         15 . The isolated rat ES cell of  claim 1 , wherein the isolated rat ES cell does not require paracrine LIF signaling for maintenance of pluripotency. 
     
     
         16 . The isolated rat ES cell of  claim 1 , wherein the isolated rat ES cell can be passaged up to at least 11 times in the medium without decreasing targeting efficiency in the isolated rat ES cell or germline transmission efficiency of the targeted genetic modification. 
     
     
         17 . The isolated rat ES cell of  claim 1 , wherein the isolated rat ES cell expresses at least one pluripotency marker selected from Dnmt3L, Eras, En-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, and Utf1. 
     
     
         18 . The isolated rat ES cell of  claim 1 , wherein the isolated rat ES cell does not express one or more pluripotency markers selected from Ecat1 and Rexo1. 
     
     
         19 . The isolated rat ES cell of  claim 1 , wherein the isolated rat ES cell exhibits one or more of the following characteristics:
 (a) the isolated rat ES cell does not express one or more mesodermal markers selected from Brachyury and Bmpr2;   (b) the isolated rat ES cell does not express one or more endodermal markers selected from Gata6, Sox17, and Sox7;   (c) the isolated rat ES cell does not express one or more neural markers selected from Nestin and Pax6; and   (d) the isolated rat ES cell expresses one or more pluripotency markers selected from Oct4, Sox2, and alkaline phosphatase.   
     
     
         20 . The isolated rat ES cell of  claim 1 , wherein the isolated rat ES cell is characterized by the expression of one or more rat ES-cell-specific genes selected from Adherens Junctions Associate Protein 1 (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), and miR-632. 
     
     
         21 . The isolated rat ES cell of  claim 1 , wherein the concentration of LIF in the medium is between about 75 U/mL to about 125 U/mL. 
     
     
         22 . The isolated rat ES cell of  claim 21 , wherein the concentration of LIF in the medium is between about 90 U/mL to about 110 U/mL. 
     
     
         23 . The isolated rat ES cell of  claim 22 , wherein the concentration of LIF in the medium is about 100 U/mL. 
     
     
         24 . The isolated rat ES cell of  claim 1 , wherein the MEK inhibitor is PD0325901 and/or the GSK3 inhibitor is CHIR99021. 
     
     
         25 . The isolated rat ES cell of  claim 1 , wherein the medium comprises a combination of inhibitors consisting of the MEK inhibitor and the GSK3 inhibitor. 
     
     
         26 . The isolated rat ES cell of  claim 1 , wherein the concentration of LIF in the medium is about 100 U/mL, the MEK inhibitor is PD0325901 at a concentration of about 1 μM, and the GSK3 inhibitor is CHIR99021 at a concentration of about 3 μM. 
     
     
         27 . The isolated rat ES cell of  claim 26 , wherein the medium comprises a combination of inhibitors consisting of PD0325901 and the CHIR99021. 
     
     
         28 . The isolated rat ES cell of  claim 1 , wherein the feeder cell layer comprises mitotically inactivated mouse embryonic fibroblasts, and the medium is a 2i medium comprising DMEM/F12 basal medium, Neurobasal medium, N2 supplement, B27 supplement, LIF, the MEK inhibitor, and the GSK inhibitor. 
     
     
         29 . The isolated rat ES cell of  claim 28 , wherein the concentration of the LIF is 100 U/ml, the MEK inhibitor is PD0325901 at a concentration of 1 μM, and the GSK inhibitor is CHIR99021 at a concentration of 3 μM. 
     
     
         30 . The isolated rat ES cell of  claim 29 , wherein the medium comprises a combination of inhibitors consisting of PD0325901 and CHIR99021.