Genetic modification of rats

A method of making a genetically modified rat whose genome comprises a targeted genetic modification that is transmitted through the germline, comprising:

• (a) providing a rat ES cell line comprising a population of rat ES cells obtained by culturing isolated rat ES cells on a feeder cell layer that is not modified to express leukemia inhibitory factor (LIF) with a medium comprising N2 supplement, B27 supplement, about 50 U/mL to about 150 U/mL LIF, and a combination of inhibitors consisting of MEK inhibitor PD0325901 and GSK3 inhibitor CHIR99021, wherein the population of rat ES cells:
• lacks expression of c-Myc;
• forms spherical, free-floating colonies in culture;
• is diploid;
• and
• is germline competent;
• (b) obtaining a rat ES cell clone comprising the targeted genetic modification, wherein the obtaining consists of:
• (i) modifying the population of rat ES cells to comprise the targeted genetic modification;
• and
• (ii) culturing the rat ES cells from step (b)(i) under the conditions set forth in step (a) and identifying in a single cloning step a rat ES cell clone comprising the targeted genetic modification and capable of transmitting the targeted genetic modification through the germline;
• (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;
• (d) gestating the rat host embryo comprising the rat ES cell clone in a surrogate mother, wherein the surrogate mother produces an F0 progeny genetically modified rat comprising the targeted genetic modification;
• and
• (e) breeding the F0 progeny genetically modified rat with another rat to produce an F1 progeny genetically modified rat comprising the targeted genetic modification, wherein the targeted genetic modification is transmitted through the germline.