Efficient correction of mismatched bases in plasmid heteroduplexes injected into cultured mammalian cell nuclei.

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Publication Type Journal Article
School or College College of Science; School of Medicine
Department Internal Medicine; Oncological Sciences; Human Genetics; Biology
Program Institute of Human Genetics; Howard Hughes Medical Institute (HHMI)
Creator Thomas, Kirk R.; Capecchi, Mario R.
Other Author Folger, Kim R.
Title Efficient correction of mismatched bases in plasmid heteroduplexes injected into cultured mammalian cell nuclei.
Date 1985-01
Description Heteroduplexes were prepared from two plasmids, pRH4-14/TK and pRH5-8/TK, containing different amber mutations in the neomycin resistance gene (Neor). The Neor gene was engineered to be expressed in both bacterial and mammalian cells. A functional Neor gene conferred kanamycin resistance to bacteria and resistance to the drug G418 to mammalian cells. In addition, the plasmids contained restriction site polymorphisms which did not confer a selectable phenotype but were used to follow the pattern of correction of mismatched bases in the heteroduplexes. In a direct comparison of the efficiency of transforming mouse LMtk- cells to G418r, the injection of heteroduplexes of pRH4-14/TK-pRH5-8/TK was 10-fold more efficient than the coinjection of pRH4-14/TK and pRH5-8/TK linear plasmid DNA. In fact, injection of 5 to 10 molecules of heteroduplex DNA per cell was as efficient in transforming LMtk- cells to G418r as the injection of 5 to 10 molecules of linear plasmid DNA per cell containing a wild-type Neor gene. To determine the pattern of mismatch repair of the injected heteroduplexes, plasmids were "rescued" from the G418r cell lines. From this analysis we conclude that the generation of wild-type Neor genes from heteroduplex DNA proceeds directly by correction of the mismatched bases, rather than by alternative mechanisms such as recombination between the injected heteroduplexes. Our finding that a cell can efficiently correct mismatched bases when confronted with preformed heteroduplexes suggests that this experimental protocol could be used to study a wide range of DNA repair mechanisms in cultured mammalian cells.
Type Text
Publisher American Society for Microbiology
Volume 5
Issue 1
First Page 70
Last Page 74
Subject Cell Nucleus; Cells, Cultured; Microinjections
Subject MESH Plasmids; Recombination, Genetic; DNA Repair
Language eng
Bibliographic Citation Mol Cell Biol. 1985 Jan;5(1):70-4: Folger KR, Thomas KR, Capecchi MR. Efficient correction of mismatched bases in plasmid heteroduplexes injected into cultured mammalian cell nuclei. Retrieved on September 15.2006 from http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=3982420.
Rights Management Copyright 1985 by American Society for Microbiology
Format Medium application/pdf
Identifier ir-main,414
ARK ark:/87278/s66q2fmt
Setname ir_uspace
Date Created 2012-06-13
Date Modified 2012-06-13
ID 704906
Reference URL https://collections.lib.utah.edu/ark:/87278/s66q2fmt