Retroviral insertional mutagenesis of a target allele in a heterozygous murine cell line.
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.
D000011:Abelson murine leukemia virus / Q000235:genetics* / Q000502:physiology; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002472:Cell Transformation, Viral; D002874:Chromosome Mapping; D002999:Clone Cells / Q000032:analysis; D005821:Genetic Techniques; D006579:Heterozygote; D009052:Leukemia Virus, Murine / Q000235:genetics*; D008099:Liver; D051379:Mice; D008807:Mice, Inbred BALB C / Q000235:genetics; D008810:Mice, Inbred C57BL / Q000235:genetics; D008979:Moloney murine leukemia virus / Q000502:physiology; D009154:Mutation; D001613:beta 2-Microglobulin / Q000235:genetics*
D000911:Antibodies, Monoclonal; D001613:beta 2-Microglobulin