Abstract
Rat embryonic stem (ES) cell lines can be established in culture medium containing inhibitors for glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase (MEK). We confirmed reproducibility of the 2i culture system in establishing rat ES cell lines (Hirabayashi et al. 2010 Mol. Reprod. Dev. 77, 94) and the likelihood of successful germline transmission (genuine) of rat ES cell lines established in leukemia inhibitory factor (LIF)- and forskolin (FK)-supplemented 2i medium (Hirabayashi et al. 2013 Transgenic Res. 22, 411–416). This study was designed to investigate whether LIF and/or FK supplemented to the 2i medium support establishment of germline-competent rat ES cell lines. E4.5 blastocysts were recovered from BLK rat females, and zona-free embryos were plated on mitomycin-treated mouse embryonic fibroblasts in N2B27 medium containing 1 mM MEK inhibitor PD0325901 and 3 mM GSK3 inhibitor CHIR99021, with rat 1000 U mL–1 LIF and/or 10 μM FK. Outgrowth rate of the blastocysts after 1 wk culture and establishment efficiency of ES cell lines after third passage were analyzed by Fisher's exact probability test. Arcsin-transformed percentage data on full-term development of ES cell-injected blastocysts, chimeric rat production, and germline-competent chimeras were analyzed by Fisher's least significant difference test after one-way ANOVA. Because of the higher outgrowth rates of blastocysts, supplementation of rat LIF, FK, or both contributed to the higher (P < 0.05) establishment efficiency of ES cell lines in BLK rat strain (76% to 92% v. 50% in LIF/FK-free 2i medium). Neither efficiency of producing chimeric rats (14% to 39% of blastocysts injected) nor germline transmission competency of the chimeric rats (67% to 100% of cell lines analyzed) was influenced by the pre-treatment of ES cell lines. When the LIF/FK-supplemented 2i medium was used, rat strain for blastocyst donor such as F344 or WI was a possible factor negatively influencing the establishment efficiency of ES cell lines. Once ES cell lines were established, however, all of them (9/9 in overall) were found to be germline-competent by progeny test of chimeric rats. In conclusion, both LIF and FK are not essential, but can play a beneficial role, for the establishment of genuine rat ES cell lines. This work was supported by a grant-in-aid for basic research from Japan Society for the Promotion of Science (No. 25290037; to M.H.).
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