Patterns of integration of DNA microinjected into cultured mammalian cells: Evidence for homologous recombination between injected plasmid DNA molecules
The authors examined the fate of DNA microinjected into nuclei of cultured mammalian cells. The sequence composition and the physical form of the vector carrying the selectable gene affected the efficiency of DNA-mediated transformation. Introduction of sequences near the simian virus 40 origin of DNA replication or in the long terminal repeat of avian sarcoma provirus into a recombinant plasmid containing the herpes simplex virus thymidine kinase gene (pBR322/HSV-tk) enhanced the frequency of transformation of LMtk/sup -/ and RAT-2tk/sup -/ cells to the TK/sup +/ phenotype 20- to 40-fold. In cells receiving injections of only a few plasmid DNA molecules, the transformation frequency was 40-fold higher after injection of linear molecules than after injection of supercoiled molecules. By controlling the number of gene copies injected into a recipient cell, we could obtain transformants containing a single copy or as many as 50 to 100 copies of the selectable gene. By analyzing transformants obtained by coinjecting two vectors which were identical except that in one a portion of the vector was inverted, the authors were able to conclude that the head-to-tail concatemers were generated predominantly by homologous recombination. Surprisingly, these head-to-tail concatemers were found in transformants obtained by injecting either supercoiled or linear plasmid DNA.
- Research Organization:
- Dept. of Biology, Univ. of Utah, Salt Lake City, UT 84112
- OSTI ID:
- 5942028
- Journal Information:
- Mol. Cell. Biol.; (United States), Vol. 2:11
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
LIGASES
ENZYME ACTIVITY
PLASMIDS
CONFIGURATION
RECOMBINANT DNA
DNA-CLONING
MOLECULAR STRUCTURE
CELL CULTURES
CELL TRANSFORMATIONS
GENE RECOMBINATION
MAMMALS
MUTANTS
MUTATION FREQUENCY
PATTERN RECOGNITION
ANIMALS
CELL CONSTITUENTS
CLONING
DNA
ENZYMES
NUCLEIC ACIDS
ORGANIC COMPOUNDS
VERTEBRATES
550400* - Genetics