Modification of the apolipoprotein B gene in HepG2 cells by gene targeting.
The HepG2 cell line has been used extensively to study the synthesis and secretion of apolipoprotein (apo) B. In this study, we tested whether gene-targeting techniques can be used to inactivate one of the apo B alleles in HepG2 cells by homologous recombination using a transfected gene-targeting vector. Our vector contained exons 1-7 of the apo B gene, in which exon 2 was interrupted by a promoterless neomycin resistance (neo(r)) gene. The recombination of this vector with the cognate gene would inactivate an apo B allele and enable the apo B promoter to activate the transcription of the neo(r) gene. To detect the rare homologous recombinant clone, we developed a novel solid phase RIA that uses the apo B-specific monoclonal antibody MB19 to analyze the apo B secreted by G418-resistant (G418r) clones. Antibody MB19 detects a two-allele genetic polymorphism in apo B by binding to the apo B allotypes MB19(1) and MB19(2) with high and low affinity, respectively. HepG2 cells normally secrete both the apo B MB19 allotypes. Using the MB19 immunoassay, we identified a G418r HepG2 clone that had lost the ability to secrete the MB19(1) allotype. The inactivation of an apo B allele of this clone was confirmed by the polymerase chain reaction amplification of an 865-bp fragment unique to the targeted apo B allele and by Southern blotting of genomic DNA. This study demonstrates that gene-targeting techniques can be used to modify the apo B gene in HepG2 cells and demonstrates the usefulness of a novel solid phase RIA system for detecting apo B gene targeting events in this cell line.
Farese
R V
RV
Gladstone Institute of Cardiovascular Disease, Department of Medicine, University of California, San Francisco 94141-9100.
Flynn
L M
LM
Young
S G
SG
D000483:Alleles; D000911:Antibodies, Monoclonal / Q000276:immunology; D001055:Apolipoproteins B / Q000235:genetics*; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D011863:Radioimmunoassay; D011995:Recombination, Genetic; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D001055:Apolipoproteins B