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

用膜相关钢因子、LIF和bFGF培养小鼠原始生殖细胞（PGCs）可发育成多能细胞系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成生殖细胞还不清楚。本研究项目的目的是建立一个直接分化为功能配子的PGC系。我们从各种近交系小鼠中建立了EG细胞，发现DBA/2小鼠的一些细胞系形成了类似于原代培养的PGCs的散点型菌落。这些细胞系似乎保持了运动能力，这是PGCs的一个特征，我们接下来通过将重组性腺移植到成年睾丸中，外源PGCs分化成配子，来测试它们发育成配子的能力。将DBA/2小鼠EG细胞系与13.5 dpc的雄性生殖脊体细胞在基质中重新聚集体，然后将聚集体移植到生殖细胞缺陷的W/W^睾丸中。2个月后恢复睾丸，用生殖细胞特异性TRA98单克隆抗体进行组织学检查。EG细胞移植的睾丸在精管内可见TRA98阳性细胞，而单独移植体细胞的睾丸未见TRA98阳性细胞。这表明EG细胞在睾丸中向生殖细胞分化。作为一种替代方法，我们计划从转基因小鼠中制备PGC细胞系，在PGC中过表达c-Kit。我们获得了几种具有种系特异性oct-3基因启动子/c-kit嵌合基因的转基因系，其中一些在睾丸中表达转基因。原代培养实验的初步结果表明，转基因小鼠的PGCs比正常小鼠生长得更快或更长。我们将从它们身上建立PGC系，并检验它们向配子分化的能力。

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.