Transformation of teratocarcinoma stem cells and fibroblasts with various vectors containing the Eco.gpt gene as a selection marker.
Eco.gpt, which codes for xanthine guanine phosphoribosyltransferase (XGPRT), when placed under the control of SV40 early genes regulating sequences (pSV2gpt) selects transformed teratocarcinoma cells with a low efficiency. The SV40 promoter may not function efficiently in teratocarcinoma stem cells, as suggested by the fact that such cells do not support SV40 T antigen expression. We have tested whether one could change the efficiency of gpt as a dominant selective marker in transformation by several operations. (1) Deletion of 121 base pairs (bp) upstream the bacterial coding sequence gpt (pQS14) did not make any difference. (2) Replacement of the SV40 regulating sequences by the HSV tk regulating sequences (pQS15) resulted in ten times fewer transformants with PCC4 teratocarcinoma cells as well as with L cells. No XGPRT activity was detectable in cultures 48 h after transfection. (3) Reintroduction of the PvuII-HindIII SV40 fragment (which contains an enhancing sequence together with the origin of replication and the early promoter of the virus) into the pQS15 vector, either in 5' or 3' from tk-gpt composite gene (pQS20 and pQS22) allows selection of ten to twenty times more transfected PCC4 or L cells colonies and restores transient XGPRT activity upon transfection. Whatever the vector used, the transformation frequency of PCC4 teratocarcinoma cells remains ten times lower than that of L cells. It appears that the presence of the SV40 PvuII-HindIII fragment in the vector increases cell transformation even with PCC4 cells and that the low frequency obtained with pSV2pgt is likely not due to the use of the SV40 early promoter.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002460:Cell Line; D004274:DNA, Recombinant; D054278:Embryonal Carcinoma Stem Cells; D005786:Gene Expression Regulation; D005814:Genes, Viral; D005819:Genetic Markers; D005822:Genetic Vectors; D007739:L Cells; D051379:Mice; D014411:Neoplastic Stem Cells / Q000502:physiology*; D009876:Operon; D010430:Pentosyltransferases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D013539:Simian virus 40 / Q000235:genetics*; D013234:Stem Cells / Q000502:physiology*; D013724:Teratoma / Q000473:pathology*; D014162:Transfection; D014170:Transformation, Genetic
D004274:DNA, Recombinant; D005819:Genetic Markers; D010430:Pentosyltransferases; C024827:xanthine phosphoribosyltransferase