Investigation of coelectroporation as a method for introducing small mutations into embryonic stem cells.
We have investigated coelectroporation as a method for introducing minor genetic changes into specific genes in embryonic stem cells. A selectable marker (neo) and a targeting replacement vector designed to insert a 4-bp insertion into exon 3 of the mouse hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene were coelectroporated into embryonic stem cells and selected in G418 and 6-thioguanine (6-TG). HPRT-negative clones were obtained at a frequency of approximately 1 per 520 G418r clones. Southern analysis and the polymerase chain reaction were used to demonstrate that 3 of 36 of the 6-TG-resistant clones had the desired 4-bp insertion without any other disruption of the HPRT locus. Initial studies indicated that the other 33 6-TG-resistant clones probably resulted from the targeted integration of a concatemer containing both the targeting construct and the selectable neo gene.
Davis
A C
AC
Institute for Molecular Genetics, Baylor College of Medicine, Houston 77030.
Wims
M
M
Bradley
A
A
D015139:Blotting, Southern; D004252:DNA Mutational Analysis; D004560:Electricity; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D011995:Recombination, Genetic; D015183:Restriction Mapping; D013724:Teratoma; D014162:Transfection
D007041:Hypoxanthine Phosphoribosyltransferase