Generation of a mouse mutant by oligonucleotide-mediated gene modification in ES cells.
Oligonucleotide-mediated gene targeting is emerging as a powerful tool for the introduction of subtle gene modifications in mouse embryonic stem (ES) cells and the generation of mutant mice. However, its efficacy is strongly suppressed by DNA mismatch repair (MMR). Here we report a simple and rapid procedure for the generation of mouse mutants using transient down regulation of the central MMR protein MSH2 by RNA interference. We demonstrate that under this condition, unmodified single-stranded DNA oligonucleotides can be used to substitute single or several nucleotides. In particular, simultaneous substitution of four adjacent nucleotides was highly efficient, providing the opportunity to substitute virtually any given codon. We have used this method to create a codon substitution (N750F) in the Rb gene of mouse ES cells and show that the oligonucleotide-modified Rb allele can be transmitted through the germ line of mice.
Aarts
Marieke
M
Division of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Dekker
Marleen
M
de Vries
Sandra
S
van der Wal
Anja
A
te Riele
Hein
H
D019943:Amino Acid Substitution; D000818:Animals; D002478:Cells, Cultured; D003062:Codon; D053843:DNA Mismatch Repair; D004268:DNA-Binding Proteins / Q000235:genetics; D015536:Down-Regulation; D053595:Embryonic Stem Cells / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D016161:Genes, Retinoblastoma; D051379:Mice; D018345:Mice, Knockout; D008817:Mice, Mutant Strains; D051718:MutS Homolog 2 Protein / Q000037:antagonists & inhibitors / Q000235:genetics; D000074081:MutS Homolog 3 Protein; D016296:Mutagenesis; D009841:Oligonucleotides; D017354:Point Mutation; D011506:Proteins / Q000235:genetics; D034622:RNA Interference
D003062:Codon; D004268:DNA-Binding Proteins; C089762:Msh3 protein, mouse; C102997:Msh6 protein, mouse; D000074081:MutS Homolog 3 Protein; D009841:Oligonucleotides; D011506:Proteins; C497173:Msh2 protein, mouse; D051718:MutS Homolog 2 Protein