Immortalization of murine male germ cells at a discrete stage of differentiation by a novel directed promoter-based selection strategy

We developed a novel promoter-based selection strategy that could be used t o produce cell lines representing sequential stages of spermatogenesis, The method is based on immortalization and subsequent targeted selection by us ing differentiation-specific promoter regions. As an example for this appro ach, a new murine germ cell line (CC-4spc) was established using a vector c onstruct that contains the SV40 large T antigen and the neomycin phosphotra nsferase II gene under the control of the SV40 early promoter and a spermat ocyte-specific promoter for human phosphoglycerate kinase 2, respectively. The GC-4spc was characterized as a cell line at the stage between preleptot ene and early pachytene spermatocytes. Transcription of three germ cell-spe cific expressed genes, Pgk2, proacrosin, and the A-myb proto-oncogene, were detected in the CC-4spc cell line using reverse transcription-polymerase c hain reaction. Furthermore, TSPY (human testis-specific protein, Y-encoded) and PGK2 (human phosphoglycerate kinase 2) promoter regions showed differe nt transcriptional activities in the GC-4spc cell line compared with the sp ermatogonia-derived cell line GC-1spg. Thus, our strategy could be used for immortalization of cells at specific stages of differentiation, allowing p roduction of a series of cultured cell lines representing sequential stages of differentiation in given cell lineages.