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PMID: 8524285 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

High-frequency illegitimate integration of transfected DNA at preintegrated target sites in a mammalian genome.

Molecular and cellular biology ·Vol. 16 ·No. 1 ·1996-01-00 ·Pages 10-8

Merrihew RV, Marburger K, Pennington SL, Roth DB, Wilson JH

Abstract

To examine the mechanisms of recombination governing the illegitimate integration of transfected DNA into a mammalian genome, we developed a cell system that selects for integration events in defined genomic regions. Cell lines with chromosomal copies of the 3' portion of the adenine phosphoribosyltransferase (APRT) gene (targets) were established. The 5' portion of the APRT gene, which has no homology to the integrated 3' portion, was then electroporated into the target cell lines, and selection for APRT gene function was applied. The reconstruction of the APRT gene was detected at frequencies ranging from less than 10(-7) to 10(-6) per electroporated cell. Twenty-seven junction sequences between the integrated 5' APRT and its chromosomal target were analyzed. They were found to be randomly distributed in a 2-kb region immediately in front of the 3' portion of the APRT gene. The junctions fell into two main classes: those with short homologies (microhomologies) and those with inserted DNA of uncertain origin. Three long inserts were shown to preexist elsewhere in the genome. Reconstructed cell lines were analyzed for rearrangements at the target site by Southern blotting; a variety of simple and complex rearrangements were detected. Similar analysis of individual clones of the parental cell lines revealed analogous types of rearrangement, indicating that the target sites are unstable. Given the high frequency of integration events at these sites, we speculate that transfected DNA may preferentially integrate at unstable mammalian loci. The results are discussed in relation to possible mechanisms of DNA integration.

MeSH Terms
Adenine Phosphoribosyltransferase/genetics Animals Base Sequence CHO Cells Cricetinae DNA/genetics DNA Primers/genetics DNA Transposable Elements Gene Rearrangement Genome Models, Genetic Molecular Sequence Data Polymerase Chain Reaction Recombination, Genetic Transfection
Chemicals
DNA Primers DNA Transposable Elements DNA Adenine Phosphoribosyltransferase
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Merrihew R V
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030, USA.
Marburger K
Pennington S L
Roth D B
Wilson J H
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.16.1.10
Published
1996-01-00
Pages
10-8
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC230973
Subset
IM
Grants
NIGMS NIH HHS · GM38219 · United States
External Links
PubMed source DOI: 10.1128/MCB.16.1.10 PMC Full Text View journal details
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