Positive genetic selection for gene disruption in mammalian cells by homologous recombination.
AUTOR(ES)
Sedivy, J M
RESUMO
Efficient modification of genes in mammalian cells by homologous recombination has not been possible because of the high frequency of nonhomologous recombination. An efficient method for targeted gene disruption has been developed. Cells with substitution of exogenous sequences into a chromosomal locus were enriched, by a factor of 100, using a positive genetic selection that specifically selects for homologous recombination at the targeted site. The selection is based on the conditional expression of a dominant selectable marker by virtue of in-frame gene fusion with the target gene. The dominant selectable marker was derived by modification of the Escherichia coli neo gene so that it retains significant activity in mammalian cells after in-frame fusion with heterologous coding sequences. In the example presented here, homologous recombinants were efficiently recovered from a pool in which the targeted gene was disrupted in 1 per 10,000 cells incorporating exogenous DNA.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=286437Documentos Relacionados
- Recombination walking: genetic selection of clones from pooled libraries of yeast artificial chromosomes by homologous recombination.
- A crtB homolog essential for photochromogenicity in Mycobacterium marinum: isolation, characterization, and gene disruption via homologous recombination.
- High-frequency disruption of the N-myc gene in embryonic stem and pre-B cell lines by homologous recombination.
- Improved genetic selection for screening bacteriophage libraries by homologous recombination in vivo.
- Gene repeat expansion and contraction by spontaneous intrachromosomal homologous recombination in mammalian cells