Targeted introduction of a diphtheria toxin-resistant point mutation into the chromosomal EF-2 locus by in vivo homologous recombination.
We have introduced a specific point mutation into the gene for chromosomal elongation factor 2 (EF-2) in Chinese hamster ovary cells (CHO-K1) by in vivo homologous recombination. To obtain a selectable construct for gene-targeting, we modified a diphtheria toxin-resistant mutant EF-2 gene (Gly717 to Arg) by deleting its promoter and first exon so that homologous recombinants could be distinguished from randomly integrated transformants, and also by inserting a second positive selection marker, the neomycin-resistance gene, into the 3'-flanking region to increase the selective accuracy. More than 80% of the clones surviving after selection for resistance to both the toxin and neomycin were the desired homologous recombinants in which the wild-type, toxin-sensitive EF-2 gene was replaced by the modified gene giving resistance to both the toxin and neomycin. This result shows that the specific point mutation was co-introduced with a second selective marker into an endogenous chromosomal gene and that the modified gene was expressed.
Kido
M
M
Institute for Molecular and Cellular Biology, Osaka, Japan.
Miwatani
H
H
Kohno
K
K
Uchida
T
T
Okada
Y
Y
D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004167:Diphtheria Toxin / Q000494:pharmacology*; D004351:Drug Resistance / Q000235:genetics; D000922:Immunotoxins; D009154:Mutation / Q000235:genetics; D009355:Neomycin / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics*; D012641:Selection, Genetic; D012689:Sequence Homology, Nucleic Acid
D004167:Diphtheria Toxin; D000922:Immunotoxins; D004247:DNA; D009355:Neomycin