Faculty Research 1980 - 1989

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

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Abelson-Leukemia-Virus: ge, ph, Animal, Antibodies-Monoclonal, Base-Sequence, Cell-Transformation-Viral, Chromosome-Mapping, Clone-Cells: an, Genetic-Techniques, Heterozygote, Liver, Mice, Mice-Inbred-BALB-C: ge, Mice-Inbred-C57BL: ge, Moloney-Leukemia-Virus: ph, Mouse-Leukemia-Viruses: ge, Mutation, SUPPORT-NON-U-S-GOVT, SUPPORT-U-S-GOVT-P-H-S

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Proc Natl Acad Sci USA 1985 Oct; 82(19):6600-4.


1, CA13330, CA24220


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.

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