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

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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.