Establishment of rat embryonic stem cell lines that can participate in germline chimerae at high efficiency

Laboratory rats (Rattus norvegicus) have been used more extensively than mice in the research fields of neuroscience, pharmacology, and toxicology. Functional germline-competent embryonic stem (ES) cell lines or induced pluripotent stem cell lines, which are essential in reverse genetics approach, have been recently established in the rat (Buehr et al., 2008; Li et al., 2008; Liao et al., 2009). Sooner or later, homologous recombinationbased gene targeting in such stem cell lines will allow for the production and analysis of individual rats with targeted mutation as routinely done in mice. Here we report rat ES cell lines that can participate in chimerae and transmit the characteristics through germline at high efficiency. Blastocysts at E4.5 were harvested from Wistar (WI) females copulated with a homogenous CAG/Venus transgenic male rat, and zona-free embryos were plated on mitomycin-treated mouse embryonic fibroblasts in N2B27 medium containing 3i (FGF receptor inhibitor SU5402, 2mM; MEK inhibitor PD0325901, 1 mM; GSK3 inhibitor CHIR99021, 3 mM) and rat LIF (1,000 U/ ml ESGRO), as described by Buehr et al. (2008) with a few modifications (replacement of 0.8 mM MEK activation inhibitor PD1843521 with 1 mM MEK inhibitor PD0325901, and supplementation of 1,000 U/ml rat LIF). Both lines of ES cells established (rESWIv3i-1 and rESWIv3i-5) have been cryopreserved at a relatively early passage number (<10). Subculture of post-thaw ES cells resulted in progressive self-renewal and colony formation. At passage 17, both of the ES cell lines were alkaline phosphatase-positive. PCR analysis to detect rat Sry gene showed that both rESWIv3i-1 and rESWIv3i-5 lines were female. To examine pluripotency in vivo, the ES cells (line rESWIv3i-1, 2.5 10 cells) were injected subcutaneously into a nude rat. Five weeks after the injection, a tumor was observed and its histological section was evaluated after hematoxylin--eosin staining. The tumor was found to be a teratoma with various tissues including gut-like epithelium or hepatic cells (endoderm), bone, cartilage or muscle (mesoderm), and neural tissues (ectoderm). To generate ES cell-derived chimerae, host blastocysts derived from WI females or WI Dark-Agouti F1 females were microinjected with 10 ES cells each at passage 6 or 8 and allowed to develop to fetus (E15.5) or full-term pups in pseudopregnant WI recipients. All of the E15.5 fetuses (100%) and majority of the newborn pups (81.8--100%) were chimeric and expressed the Venus gene (Table 1). The characteristics of the ES cells was successfully transmitted to their next generations in both lines. The ability of the ES cells to participate in chimerae was still high (78.6--100%) at advanced passage numbers (17--18). Overall efficiency of producing chimerae in our study (50.3%, 94 chimerae/187 injected embryos) was higher than 8.2% (20/ 245) as reported in Buehr et al. (2008) and 11.0% (26/237) in Li et al. (2008). This higher efficiency of chimera ratproduction inour study isprobably due to the rat strain combination used for donor (ES cells) and host (blastocysts) and/ormodificationof culturemedium, such as supplementation of MEK inhibitor and rat LIF. In conclusion, ES cell lines have been established from CAG/venuscarrying transgenic rat blastocysts, with evidences by the progressive proliferation while remaining undifferentiated, the contribution to teratoma formation, the participation into chimerae, and/or the transmission through germline.