Introduction of specific point mutations into RNA polymerase II by gene targeting in mouse embryonic stem cells: evidence for a DNA mismatch repair mechanism.
We have introduced two specific point mutations, located 20 base pairs apart, into the endogenous murine gene that encodes the largest subunit of RNA polymerase II (RPII215). The first mutation conferred resistance to the mushroom toxin alpha-amanitin (amar), and the second mutation generated a restriction fragment length polymorphism without altering the protein sequence. Targeted amar clones were generated at a frequency of 1 in 30 totipotent embryonic stem cells that expressed stably integrated DNA vectors after electroporation. Thirty to 40% of these clones had acquired both mutations, whereas, surprisingly, the remaining clones had acquired the specific amar point mutation but lacked the restriction fragment length polymorphism. We suggest that the latter clones were generated by independent DNA mismatch repair rather than by double crossover or gene conversion. These results demonstrate that it is possible to introduce specific point mutations into an endogenous gene in embryonic stem cells. Thus it should be possible to introduce single base substitutions into other cellular genes, including nonselectable genes, by optimizing the efficiency of gene transfer and/or the sensitivity of screening for targeted clones.
Steeg
C M
CM
Division of Molecular and Developmental Biology, Mount Sinai Hospital Research Institute, Toronto, Ontario, Canada.
Ellis
J
J
Bernstein
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D002999:Clone Cells; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004260:DNA Repair; D004622:Embryo, Mammalian; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D012150:Polymorphism, Restriction Fragment Length; D012319:RNA Polymerase II / Q000235:genetics*; D015183:Restriction Mapping
D046911:Macromolecular Substances; D004247:DNA; D012319:RNA Polymerase II