Retroviral insertional mutagenesis of a target allele in a heterozygous murine cell line

W. Frankel, T. A. Potter, N. Rosenberg, J. Lenz, T. V. Rajan

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

A clonal murine cell line that is heterozygous at the β2-microglobulin locus (B2m) was obtained by Abelson murine leukemia virus (Ab-MuLV) transformation of liver cells from (C57BL/6 x BALB/c) F1 fetuses. To obtain proviral insertional mutants, we superinfected a subclone of these cells, which does not express the env surface protein of the Moloney leukemia virus (Mo-MuLV, the helper virus that was used to transmit the defective Ab-MuLV genome during transformation), with Mo-MuLV. Mutant clones that fail to express the C57BL/6 allele of B2m (B2mb) were then immunoselected by using a monoclonal antibody that specifically recognizes the B2mb gene product and not that of the B2ma allele. Of 22 independent clones obtained, one contains a proviral insertion that is near or in the first exon of the B2mb gene. Surprisingly, the insertion is of the Ab-MuLV genome and not of replication-competent Mo-MuLV. This indicates that superinfection with Mo-MuLV 'rescued' the defective Ab-MuLV genome, which then inserted into the B2mb gene. We conclude that when an allele-specific selection procedure exists, proviral insertion is a potential method for obtaining mutations in heterozygous mammmalian cells. This approach may thereby provide a method for molecular cloning of such selectable genes, using a retroviral hybridization probe.

Original languageEnglish (US)
Pages (from-to)6600-6604
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume82
Issue number19
DOIs
StatePublished - Dec 1 1985
Externally publishedYes

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