Cotransformation and gene targeting in mouse embryonic stem cells.
We have investigated cotransformation in mammalian cells and its potential for identifying cells that have been modified by gene targeting. Selectable genes on separate DNA fragments were simultaneously introduced into cells by coelectroporation. When the introduced fragments were scored for random integration, 75% of the transformed cells integrated both fragments within the genome of the same cell. When one of the cointroduced fragments was scored for integration at a specific locus by gene targeting, only 4% of the targeted cells cointegrated the second fragment. Apparently, cells that have been modified by gene targeting with one DNA fragment rarely incorporate a second DNA fragment. Despite this limitation, we were able to use the cotransformation protocol to identify targeted cells by screening populations of colonies that had been transformed with a cointroduced selectable gene. When hypoxanthine phosphoribosyltransferase (hprt) targeting DNA was coelectroporated with a selectable neomycin phosphotransferase (neo) gene into embryonic stem (ES) cells, hprt-targeted colonies were isolated from the population of neo transformants at a frequency of 1 per 70 G418-resistant colonies. In parallel experiments with the same targeting construct, hprt-targeted cells were found at a frequency of 1 per 5,500 nonselected colonies. Thus, an 80-fold enrichment for targeted cells was observed within the population of colonies transformed with the cointroduced DNA compared with the population of nonselected colonies. This enrichment for targeted cells after cotransformation should be useful in the isolation of colonies that contain targeted but nonselectable gene alterations.
Reid
L H
LH
Department of Pathology, University of North Carolina, Chapel Hill 27599.
Shesely
E G
EG
Kim
H S
HS
Smithies
O
O
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004558:Electric Stimulation; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000502:physiology; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D019868:Kanamycin Kinase; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010770:Phosphotransferases / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection; D014170:Transformation, Genetic
D015345:Oligonucleotide Probes; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase; D010770:Phosphotransferases; D019868:Kanamycin Kinase