Introduction of a point mutation into the mouse genome by homologous recombination in embryonic stem cells using a replacement type vector with a selectable marker.
The introduction of small mutations instead of null alleles into the mouse genome has broad applications to the study of protein structure-function relationships and the creation of animal models of human genetic diseases. To test a simple mutational strategy we designed a targeting vector for the mouse proopiomelanocortin (POMC) gene containing a single nucleotide insertion that converts the initial tyrosine codon of beta-endorphin 1-31 to a premature translational termination codon and introduces a unique Hpal endonuclease restriction site. The targeting vector also contains a neo cassette immediately 3' to the last POMC exon and a herpes simplex virus thymidine kinase cassette to allow positive and negative selection. Homologous recombination occurred at a frequency of 1/30 clones of electroporated embryonic stem cells selected in G418 and gancyclovir. 10/11 clones identified initially by a polymerase chain reaction (PCR) strategy had the predicted structure without evidence of concatemer formation by Southern blot analysis. We used a combination of Hpa I digestion of PCR amplified fragments and direct nucleotide sequencing to further confirm that the point mutation was retained in 9/10 clones. The POMC gene was transcriptionally silent in embryonic stem cells and the targeted allele was not activated by the downstream phosphoglycerate kinase-1 promoter that transcribed the neo gene. Under the electroporation conditions used, we have demonstrated that a point mutation can be introduced with high efficiency and precision into the POMC gene using a replacement type vector containing a retained selectable marker without affecting expression of the allele in the embryonic stem cells. A similar strategy may be useful for a wide range of genes.
Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland 97201-3098.
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M
Rubinstein
M A
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Japón
M J
MJ
Low
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D004247:DNA / Q000302:isolation & purification; D004622:Embryo, Mammalian; D005091:Exons; D015774:Ganciclovir / Q000494:pharmacology; D005819:Genetic Markers; D005822:Genetic Vectors; D016678:Genome; D005839:Gentamicins / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011333:Pro-Opiomelanocortin / Q000235:genetics*; D014176:Protein Biosynthesis; D011995:Recombination, Genetic; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics; D013234:Stem Cells / Q000187:drug effects / Q000378:metabolism*; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine; D001615:beta-Endorphin / Q000235:genetics*
D005819:Genetic Markers; D005839:Gentamicins; D009838:Oligodeoxyribonucleotides; D014443:Tyrosine; D001615:beta-Endorphin; D011333:Pro-Opiomelanocortin; D004247:DNA; C010680:antibiotic G 418; D013937:Thymidine Kinase; D015774:Ganciclovir