Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules.

Update item information
Publication Type Journal Article
School or College College of Science; School of Medicine
Department Oncological Sciences; Biology; Human Genetics
Program Institute of Human Genetics; Howard Hughes Medical Institute (HHMI)
Creator Capecchi, Mario R.
Other Author Folger, Kim R.; Wong, Eric A.
Title Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules.
Date 1982-11
Description We 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- and RAT-2tk- cells to the TK+ 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. Multiple copies of the transforming gene were not scattered throughout the host genome but were integrated as a concatemer at one or a very few sites in the host chromosome. Independent transformants contained the donated genes in different chromosomes. The orientation of the gene copies within the concatemer was not random; rather, the copies were organized as tandem head-to-tail arrays. By analyzing transformants obtained by coinjecting two vectors which were identical except that in one a portion of the vector was inverted, we 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. Even though we demonstrated that cultured mammalian cells contain the enzymes for ligating two DNA molecules very efficiently irrespective of the sequences or topology at their ends, we found that even linear plasmid DNA was recruited into the concatemer by homologous recombination.
Type Text
Publisher American Society for Microbiology
Volume 2
Issue 11
First Page 1372
Last Page 1387
Subject Base Sequence; Cell Line; Genes, Viral; Genetic Vectors; Mice; Microinjections
Subject MESH Models, Genetic; Nucleic Acid Conformation; Plasmids; Recombination, Genetic; Transformation, Genetic
Language eng
Bibliographic Citation Mol Cell Biol. 1982 Nov;2(11):1372-87: Folger KR, Capecchi MR. Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules. Retrieved on September 15.2006 from http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=6298598.
Rights Management Copyright C 1982, American Society for Microbiology. All Rights Reserved.
Format Medium application/pdf
Identifier ir-main,421
ARK ark:/87278/s6cz3r7z
Setname ir_uspace
Date Created 2012-06-13
Date Modified 2012-06-13
ID 702282
Reference URL https://collections.lib.utah.edu/ark:/87278/s6cz3r7z
Back to Search Results