Rapid detection of homologous recombinants in nontransformed human cells.
Gene targeting is a technique by which a preselected site in the genome of a living cell can be modified by inserting, deleting, or exchanging DNA sequences. The application of this technology to cells with a limited life-span, such as nontransformed human somatic cells, requires the development of simplified and efficient procedures to allow the isolation of correctly modified cells from the much larger pool of random integrants. The current study describes the development of a widely applicable strategy for detecting homologous recombinants in human cells by using an ELISA-based screen. When this system is used accurately targeted clones can be detected with high efficiency as soon as 14 days following transfection. Data are presented demonstrating the utility of this detection system in isolating targeted recombinants at the beta 2-microglobulin locus in both human retinal pigmented epithelial cells and human keratinocytes.
Williams
S R
SR
Cell Genesys, Inc., Foster City, CA 94404.
Ousley
F C
FC
Vitez
L J
LJ
DuBridge
R B
RB
D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017931:DNA Primers; D018274:Electroporation; D005260:Female; D005822:Genetic Vectors; D015894:Genome, Human; D015698:Genomic Library; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D008969:Molecular Sequence Data; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D010920:Placenta / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011995:Recombination, Genetic; D016324:Sequence Tagged Sites; D014162:Transfection; D001613:beta 2-Microglobulin / Q000096:biosynthesis / Q000235:genetics*
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D001613:beta 2-Microglobulin