Homologous integration in mammalian cells without target gene selection.
Homologous integrations into a nonselectable target locus have been highly enriched for following DNA transfections into mammalian cells. The target gene, the SV40 early region in COS1 cells, provides transcription signals to activate a defective selectable marker, the gpt gene. We find that nearly half of the selected clones have integrated the gpt gene at the homologous sequence in the COS1 genome. This is an estimated 100-fold enrichment for homologous events compared with transfections in which the gpt gene is transcriptionally active. As shown for yeast integration events, a double-strand break at a position of homology between the transfected DNA and the genomic target is necessary to achieve a high frequency of homologous integrations. Furthermore, the arrangement of sequences at the integration site includes a repair of the double-strand gap, which was present on the transfected DNA, suggesting that similarities exist between yeast and mammalian integrations. The experimental design, in which a defective marker is activated following a homologous integration, may have general applications for gene targeting in mammalian cells.
Jasin
M
M
Department of Biochemistry, Stanford University Medical Center, California 94305.
Berg
P
P
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002478:Cells, Cultured; D004260:DNA Repair; D004274:DNA, Recombinant; D004742:Enhancer Elements, Genetic; D005819:Genetic Markers; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection
D004274:DNA, Recombinant; D005819:Genetic Markers