Cointegration of DNA molecules introduced into mammalian cells by electroporation.
Electroporation was used to introduce a mixture of two plasmid-cloned genes into Chinese hamster ovary (CHO) cells, and the location of the two genes was subsequently determined by fluorescence in situ hybridization (FISH). The 25 kb Chinese hamster gene for dihydrofolate reductase (dhfr) in the form of a cosmid-derived 40 kb BglI fragment and the SV40 promoter-driven E. coli gene for guanine phosphoribosyltransferase (gpt) were co-electroporated and gpt + transfectants selected. Clones that had also integrated a single copy of the dhfr gene were studied by 2-color fluorescence in situ hybridization (FISH) to localize the integration site(s) of the exogenous DNA in metaphase chromosomes. All 9 clones examined showed co-localization of the two transgenes. The chromosomal site of integration was different in each clone. Co-integration was confirmed by co-amplification experiments. We conclude that, even when provided at low concentrations, separate soluble DNA molecules become linked upon gene transfer by electroporation, either by intracellular ligation prior to integration, or by co-integration at a common site in a given recipient cell.
Chen
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Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
Chasin
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D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002874:Chromosome Mapping; D006224:Cricetinae; D004247:DNA / Q000235:genetics*; D018274:Electroporation; D005822:Genetic Vectors / Q000235:genetics; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D017404:In Situ Hybridization, Fluorescence; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014162:Transfection / Q000379:methods*
D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase; D007041:Hypoxanthine Phosphoribosyltransferase