Transfer of genes into embryonal carcinoma cells by retrovirus infection: efficient expression from an internal promoter.
The expression of retroviral genomes after infection of embryonal carcinoma (EC) cells is usually restricted. We have constructed a retroviral vector which has the potential to express efficiently in EC cells an inserted sequence, the selectable neomycin (neo) gene, from an internal thymidine kinase promoter. A second retrovirus vector contained in addition a v-myc oncogene, which in NIH 3T3 cells could be expressed from the viral long terminal repeat (LTR) through a subgenomic mRNA. 10-100% of the EC cells infected with recombinant virus gave rise to neo-resistant colonies following drug selection. The neo gene was expressed in both the non-permissive EC and the permissive NIH 3T3 cells at similar levels, and transcription of neo-specific RNA was initiated at the proper site in the thymidine kinase promoter. In contrast, v-myc mRNA was produced only in the NIH 3T3 cells and not in EC cells, even after induction of differentiation. Thus, genes inserted into retrovirus constructs can easily be introduced into EC cells by virus infection, even without drug selection, and they can be efficiently expressed when transcribed from promoters other than the viral LTRs.
D000818:Animals; D002478:Cells, Cultured; D004279:DNA, Viral / Q000235:genetics; D004351:Drug Resistance; D054278:Embryonal Carcinoma Stem Cells; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D051379:Mice; D009355:Neomycin / Q000494:pharmacology; D014411:Neoplastic Stem Cells / Q000382:microbiology*; D009857:Oncogenes; D011401:Promoter Regions, Genetic; D012190:Retroviridae / Q000235:genetics*; D013234:Stem Cells / Q000382:microbiology*; D013937:Thymidine Kinase / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D004279:DNA, Viral; D013937:Thymidine Kinase; D009355:Neomycin