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 to produce cell lines representing sequential stages of spermatogenesis. The method is based on immortalization and subsequent targeted selection by using differentiation-specific promoter regions. As an example for this approach, a new murine germ cell line (GC-4spc) was established using a vector construct that contains the SV40 large T antigen and the neomycin phosphotransferase II gene under the control of the SV40 early promoter and a spermatocyte-specific promoter for human phosphoglycerate kinase 2, respectively. The GC-4spc was characterized as a cell line at the stage between preleptotene and early pachytene spermatocytes. Transcription of three germ cell-specific expressed genes, Pgk2, proacrosin, and the A-myb proto-oncogene, were detected in the GC-4spc cell line using reverse transcription-polymerase chain reaction. Furthermore, TSPY (human testis-specific protein, Y-encoded) and PGK2 (human phosphoglycerate kinase 2) promoter regions showed different transcriptional activities in the GC-4spc cell line compared with the spermatogonia-derived cell line GC-1spg. Thus, our strategy could be used for immortalization of cells at specific stages of differentiation, allowing production of a series of cultured cell lines representing sequential stages of differentiation in given cell lineages.
Tascou
S
S
Institute of Human Genetics, University of Göttingen, 37073 Göttingen, Germany Institute of Human Genetics, Medical High School of Hannover, Hannover, Germany.
Nayernia
K
K
Samani
A
A
Schmidtke
J
J
Vogel
T
T
Engel
W
W
Burfeind
P
P
D000818:Animals; D019677:Artificial Gene Fusion; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D002469:Cell Separation; D002478:Cells, Cultured; D003584:Cytological Techniques; D015870:Gene Expression / Q000187:drug effects; D005854:Germ Cells / Q000502:physiology*; D007124:Immunoenzyme Techniques; D008297:Male; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics*; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger / Q000096:biosynthesis; D012641:Selection, Genetic; D013737:Testis / Q000166:cytology; D014158:Transcription, Genetic
C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger