Chao Chen and Lawrence A. Chasin
Cointegration of DNA Molecules Introduced into Mammalian Cells by Electroporation f
Somatic Cell and Molecular Genetics. 1999. 24: 249-256.
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) was provided as a cosmid-derived 40 kb BglI fragment and the E. coli gene for guanine phosphoribosyltransferase (gpt), driven by the SV40 early promoter, was in the form of the linearized plasmid pSV2gpt. Colonies selected for expression of the gpt gene were screened for the co-transfer of the unselected dhfr genes by quantitative Southern blotting. Nine clones that had integrated a single copy of the dhfr gene were studied further: all 9 exhibited a DHFR-positive growth phenotype (e.g., purine independence). Two-color fluorescence in situ hybridization (FISH) of metaphase chromosomes, using probes labeled with biotin and digoxigenin, showed co-localization of the two genes in all 9 clones. The chromosomal site of integration was different in each clone. Five clones were subjected to gradually increasing concentrations of methotrexate to select for amplification of the dhfr gene. In each case the se selected populations had co-amplified the gpt gene, as shown by FISH and by the polymerase chain reaction (PCR). 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.
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