Establishment of Embryonic Stem Cells and Generation of Genetically Modified Rats

Authentic embryonic stem (ES) cells are derived from the inner cell mass (ICM) of preimplantation blastocysts in rodents. ES cells have been routinely derived since 1981 (Evans & Kaufman, 1981; Martin, 1981). They are capable of generating germline chimeras following injection into blastocysts. A very large number of knockin/knockout mice have been produced so far, leading to significant progress in both basic research and clinical investigation. However, recent reports indicate that the phenotypes of knockout mice sometimes do not correspond to human diseases (Rogers et al., 2008). Thus, the ES cells of other species, especially rats, have been desired for the generation of new animal models for human diseases. In 2008, we successfully established rat ES cells with a chimeric contribution (Ueda et al., 2008). Soon after our report, authentic rat ES cells that could complete a germline transmission were established (Buehr et al., 2008; Li et al., 2008). These reports suggest that a removal of serum from a culture medium is necessary for maintaining the pluripotency of rat ES cells (Kawamata & Ochiya, 2010a). However, despite the assertion in these reports, we recently established high-quality rat ES cells by using a combination of 20% serum and signaling inhibitors. Furthermore, this culture condition enabled the ES cells to receive gene manipulation, leading to obtaining genetically modified rats via germline transmission. We also discovered an indispensable technique during a blastocyst injection process for the generation of germline chimeras (Kawamata & Ochiya, 2010b). This new technology should provide valuable animal models for the study of human diseases by the induction of gene-targeting manipulations in the rat ES cells. In this chapter, we discuss the techniques for the establishment of rat ES cells compared to mouse ES cells and the creation of genetically modified rats.