Figure 3.

Establishment of Slc52a3 KO ES cells. (A) Day 4.5 embryos (blastocysts) from heterozygous crossing were harvested and cultured in ES media with 2i/LIF (left panels). Five-day-culture of blastocysts led the outgrowth of the inner cell mass (middle panels). Outgrowing cell colonies in 2i/LIF condition on gelatin-coated dish were picked and cell lines were established (right panels). The total number of the established cell lines and the genotype ratio are shown shown in Table 1. (B) Genotyping results of the established ES cells. (C) Established cell lines stained with anti-pluripotent markers; Sox2 and Oct-3/4 and anti-Slc52a3 in each genotype. Slc52a3 −/− cell lines have limited expression of the target gene even though expression of the pluripotent markers seems to be normal. (D and E) Western blotting of pluripotent ES cell lysates. Relative expression levels were normalized by α-tubulin expression.