High-throughput site-directed mutagenesis in ES cells.
Introduction of nonselectable mutations into the genome of embryonic stem cells by homologous recombination allows to investigate the function of genes at the molecular level and has been achieved, however, at very low efficiencies by the Hit and Run, Tag and Exchange, and Double Replacement strategies. Comparing those strategies at a single locus with vectors derived from a single fragment of the desmin gene led to the improvement of two strategies by employing a new selection cassette and modified selection procedures. Modified strategies resulted in the introduction of nonselectable point-mutations in 53% of the Hit and Run derived embryonic stem cell clones and in 0.7% of the Tag and Exchange clones. Efficiency of intrachromosomal recombination at Hit alleles outscored replacement-type recombination at the tagged alleles making the modified Hit and Run strategy the method of choice for the efficient introduction of nonselectable point mutations into the genome of embryonic stem cells.
Höllrigl
A
A
Institute of Medical Biochemistry, University of Vienna, Vienna Bio Center, Dr. Bohrgasse 9/3, A1030 Vienna, Austria.
Hergovich
A
A
Görzer
I
I
Bader
A
A
Ellersdorfer
G
G
Habegger
K
K
Hammer
E
E
Enzinger
S
S
Capetanaki
Y
Y
Weitzer
G
G
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D002478:Cells, Cultured; D018899:CpG Islands; D018076:DNA, Complementary / Q000378:metabolism; D003893:Desmin / Q000235:genetics*; D004622:Embryo, Mammalian / Q000166:cytology*; D015774:Ganciclovir / Q000494:pharmacology; D005821:Genetic Techniques; D005822:Genetic Vectors; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D017354:Point Mutation; D011995:Recombination, Genetic; D013234:Stem Cells / Q000378:metabolism*; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection
D000998:Antiviral Agents; D018076:DNA, Complementary; D003893:Desmin; D015774:Ganciclovir