Gene targeting in Chinese hamster ovary cells is conservative.
Two fundamentally different pathways for homologous recombination have been identified in mammalian cells. For most chromosomal recombination events, two copies of a homologous sequence recombine to yield two copies in the products; such events are said to be conservative because the number of copies is preserved. By contrast, virtually all extrachromosomal recombination events are nonconservative; two copies recombine to give a product containing a single intact copy (the other copy is destroyed in the mechanism). Since gene targeting involves an introduced (extrachromosomal) plasmid and a chromosomal target, it was not clear which pathway would apply. We used a marked vector to determine whether targeted integrants were products of recombination events that involved two copies (the conservative pathway) or three copies (the nonconservative pathway) of the homologous sequence. Among 51 gene targeting events, we identified 17 homologous integrants and analyzed their structures. All match the predictions for a conservative pathway. We conclude that the principal pathway for gene targeting in mammalian cells is conservative.
Pennington
S L
SL
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030.
Wilson
J H
JH
D000228:Adenine Phosphoribosyltransferase / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D016466:CHO Cells / Q000502:physiology*; D003001:Cloning, Molecular; D006224:Cricetinae; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D015183:Restriction Mapping; D014162:Transfection
D009841:Oligonucleotides; D000228:Adenine Phosphoribosyltransferase