Accurate modification of a chromosomal plasmid by homologous recombination in human cells.
We have examined the consequences of modifying mammalian cellular DNA sequences by homologous recombination. A plasmid carrying a 248-base-pair deletion in the neomycin phosphotransferase (neo) gene was introduced into hamster and human cells. The integrated, defective neo gene was used as a target for modification by a second round of transfection with a plasmid carrying a different (283-base-pair) deletion in the neo gene. Recombinants resulting in an intact neo gene were selected by their G418 resistance phenotype. The best ratio of homologous to nonhomologous recombination events was about 1:80. Analyses of the functional neo genes in various independent cell lines establish that simple crossovers (single and double) generated the wild-type neo genes.
Song
K Y
KY
Center for Genetics, University of Illinois College of Medicine, Chicago 60612.
Schwartz
F
F
Maeda
N
N
Smithies
O
O
Kucherlapati
R
R
D000818:Animals; D002875:Chromosomes / Q000502:physiology; D006224:Cricetinae; D004262:DNA Restriction Enzymes; D005796:Genes; D006801:Humans; D006822:Hybrid Cells / Q000166:cytology; D019868:Kanamycin Kinase; D010770:Phosphotransferases / Q000235:genetics; D010957:Plasmids; D011995:Recombination, Genetic
D010770:Phosphotransferases; D019868:Kanamycin Kinase; D004262:DNA Restriction Enzymes