Derivation of primordial germ cell lines and their application to transgenic technology

小鼠原始生殖细胞（PGCs）在膜结合Steel因子、LIF和bFGF的共同作用下，发育为多能性细胞系EG细胞。虽然它对嵌合小鼠的所有组织包括生殖细胞都有贡献，但它是否能直接发育为生殖细胞尚不清楚。本研究的目的是建立一个直接向功能配子转化的PGC系。我们从不同的近交系小鼠中建立了EG细胞，并发现DBA/2小鼠的一些细胞系形成了类似原代培养PGCs的散在型集落。这些细胞系似乎维持了PGCs的运动性，我们接下来通过将重建的性腺移植到成年睾丸中来测试它们发育成配子的能力，其中外源PGCs分化为配子。将DBA/2小鼠的EG细胞系与13.5dpc雄性生殖嵴的体细胞在基质胶中重组，然后将重组体移植到生殖细胞缺陷的W/W小鼠睾丸。2个月后，回收睾丸，并使用生殖细胞特异性TRA98单克隆抗体进行组织学检查。移植EG细胞的睾丸在曲细精管内可见TRA98阳性细胞，而单独移植体细胞的睾丸则无此现象。作为一种替代方法，我们计划从PGC中过表达c-Kit的转基因小鼠中制备PGC系。我们获得了几个带有生殖系特异性oct-3基因启动子/c-kit嵌合基因的转基因株系，其中一些在睾丸中表达了转基因。来自原代培养实验的初步结果表明，来自转基因小鼠的PGCs比正常小鼠的PGCs生长得更快或更长。我们将从它们建立PGC系，并检查它们分化为配子的能力。

Murine primordial germ cells (PGCs) cultured with membrane associated Steel factor, LIF and bFGF develop to apluripotent cell line, EG cell. Although it contributes to all tissues including germ cell in chimaeric mice, it has not been known whether it can directly develop to germ cells. The aim of this research project is to establish a PGC line that directly differetiates to functional gamete. We have established EG cells from various inbred mice and have found that some cell lines from DBA/2 mouse formed scatter type colonies resembling those of primary cultured PGCs. These cell lines seemed to sustain motility that id a character of PGCs, we next tested their ability to develop to gamates by transplantation of reconstituted gonads to adult testis in which exogenous PGCs differentiate to gametes. A EG cell line derived from DBA/2 mice was reaggregate with somatic cells of 13.5 dpc male genital ridges in matrigel, then the aggregates were transplanted to germ cell-deficient W/W^Vtestes. After 2 months, the testes were recovered and were histologically examined using germ cell-specific TRA98 monoclonal antibody. The EG cell-transplanted testis showed some TRA98 positive cells inside seminiferous tubules, but testes transplanted somatic cells alone did not. This result suggests that the EG cell differentiates to germ cells in testis.As an alternative approach, we planed to make PGC lines from transgenic mice that overexpress c-Kit in PGCs. We got several transgenic lines with germ line-specific oct-3 gene promotor/c-kit chimaeric gene and some of which express the transgene in testis. preliminary results from primary culture experiments suggest that PGCs from the transgenic mice grow more rapidly or longer than those of normal mice. We will establish PGC lines from them and examine their ability to differentiate to gametes.