Open AccessRetroviral insertional mutagenesis of a target allele in a heterozygous murine cell line.
Author(s) -
Wayne N. Frankel,
Terry A. Potter,
Naomi Rosenberg,
Jack Lenz,
T. V. Rajan
Publication year - 1985
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.82.19.6600
Subject(s) - murine leukemia virus , biology , microbiology and biotechnology , insertional mutagenesis , gene , mutant , helper virus , locus (genetics) , allele , genetics , virology , insertion , genome
A clonal murine cell line that is heterozygous at the beta 2-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 mammalian cells. This approach may thereby provide a method for molecular cloning of such selectable genes, using a retroviral hybridization probe.