title: Transfer of nonselectable genes into mouse teratocarcinoma cells and transcription of the transferred human beta-globin gene.
abstract: Teratocarcinoma (TCC) stem cells can function as vehicles for the introduction of specific recombinant genes into mice. Because most genes do not code for a selectable marker, we investigated the transformation efficiency of vectors with a linked selectable gene. In one series, TCC cells first selected for thymidine kinase deficiency were treated with DNA from the plasmid vector PtkH beta 1 containing the human genomic beta-globin gene and the thymidine kinase gene of herpes simplex virus. A high transformation frequency was obtained after selection in hypoxanthine-aminopterin-thymidine medium. Hybridization tests revealed that the majority of transformants had intact copies of the human gene among three to six total copies per cell. These were associated with cellular DNA sequences as judged from the presence of additional new restriction fragments and from stability of the sequences in tumors produced by injecting the cells subcutaneously. Total polyadenylate-containing RNA from cell cultures of two out of four transformants examined showed hybridization to the human gene probe: one RNA species resembled mature human beta-globin mRNA transcripts; the others were of larger size. In differentiating tumors, various tissues, including hematopoietic cells of TCC provenance could be found. In a second model set of experiments, wild-type TCC cells were used to test a dominant-selection scheme with pSV-gpt vectors. Numerous transformants were isolated, and their transfected DNA was apparently stably integrated. Thus, any gene of choice can be transferred into TCC stem cells even without mutagenesis of the cells, and selected cell clones can be characterized. Cells of interest may then be introduced into early embryos to produce new mouse strains with predetermined genetic changes.
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant; D005799:Genes, Dominant; D005914:Globins / Q000235:genetics*; D006801:Humans; D051379:Mice; D009374:Neoplasms, Experimental; D012641:Selection, Genetic; D013724:Teratoma; D013937:Thymidine Kinase / Q000235:genetics; D014158:Transcription, Genetic; D014170:Transformation, Genetic
chemical_list: D004274:DNA, Recombinant; D005914:Globins; D013937:Thymidine Kinase; D004262:DNA Restriction Enzymes