Open AccessNovel use of synthetic oligonucleotide insertion mutants for the study of homologous recombination in mammalian cells.
Author(s) -
Guy Shapira,
Janet L. Stachelek,
Anthea Letsou,
L. K. Soodak,
R. Michael Liskay
Publication year - 1983
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.80.15.4827
Subject(s) - plasmid , thymidine kinase , homologous recombination , recombination , biology , mutant , microbiology and biotechnology , transformation (genetics) , gene , cre lox recombination , insertion , dna , genetics , genetic recombination , oligonucleotide , flp frt recombination , non allelic homologous recombination , homologous chromosome , mutation , herpes simplex virus , virus , transgene , genetically modified mouse
Thymidine kinase-deficient mouse L cells have been transformed with plasmid DNAs carrying 8-base-pair Xho I linker insertion mutations in the coding region of the herpes simplex virus type 1 thymidine kinase gene. When the mutant plasmids are introduced individually into LTK- cells, transformation efficiencies are greatly reduced relative to the wild type. However, when two mutant plasmids are cotransferred into the same LTK- recipients, significantly higher frequencies of transformation are observed (30-300 times). Here we demonstrate the usefulness of linker insertions for the study of homologous recombination in detecting the existence of normal thymidine kinase gene sequences (i.e., sequences lacking the insertions after recombination are substantiated by DNA . DNA hybridization). In addition, the frequencies of recombination in the various "crosses" are consistent with the known positions of the mutations.