Establishment of functional germ cell precursor cell lines

本研究的目的是从小鼠原始生殖细胞（PGCs）和精原细胞中获得细胞系，并将其分化为功能配子。为此，我首先研究了pgc衍生的多能性EG细胞在与胚胎性腺体细胞培养时是否能再分化为PGCs。培养后，我用pgc特异性vasa抗体对其进行染色，发现EG细胞分化为vasa阳性细胞。此外，EGF还能在没有性腺体细胞的EG细胞中诱导血管阳性细胞。我也尝试用过表达bcl-2的转基因小鼠在精原细胞中建立精原细胞系。将解离的转基因睾丸细胞培养在STO细胞的饲养层上，我发现通过免疫染色检测到的tra98阳性生殖细胞在培养45天后仍能检测到，而野生型睾丸细胞在培养中只能存活30天。此外，我发现转基因生殖细胞在培养10天后分化为tra369阳性的精细胞和/或精细胞。

The aim of this research is to derive cell lines from mouse primordial germ cells (PGCs) and spermatogonia and to differentiate them into functional gametes. For this purpose, Ifirst examined whether PGC-derived pluripotential EG cells could redifferentiate into PGCs when culturing them with somatic cells from embryonic gonads. After culture, I stained them with PGC-specific vasa antibody, and found that EG cells differentiated to vasa-positive cells. In addition, EGF could also induced vasa-positive cells from EG cells without gonadal somatic cells. I also attempted to establish spermatogonial cell lines form a transgenic mice overexpressing bcl-2 in spermatogonia. Dissociated transgenic testicular cells were cultured on a feeder layer of STO cells and I found that TRA98-positive germ cells determined by imnmunostaining could be detected up to 45 days after culture, while wild type testicular cells survived up to only 30 days in culture. In addition, I found that the transgenic germ cells differentiated intoTRA369-positive spermatocytes and/or spermatid after 10 days in culture.