Isolation of NPC1-deficient Chinese hamster ovary cell mutants by gene trap mutagenesis

Chinese hamster ovary cell mutants defective in the NPC1 gene (NPC1-trap) w ere generated by retrovirus-mediated gene trap mutagenesis from a parental cell line JP17 expressing an ecotropic retrovirus receptor. Insertion of th e gene trap vector in the NPC1 gene and the absence of the gene product wer e verified by 5'RACE and immunological analyses, respectively, NPC1-trap ce lls showed intracellular accumulation of low-density lipoprotein (LDL)-deri ved cholesterol and had an increased level of unesterified cellular cholest erol. Cholesterol biosynthesis through the mevalonate pathway was upregulat ed in the mutant cells as assessed by [C-14]acetate incorporation into cell ular sterols, When JP17 cells were depleted of lipoproteins and then loaded with LDL, cell surface LDL receptors were promptly downregulated and the m ature form of the sterol regulatory element-binding protein-1 disappeared f rom the nucleus, These responses to LDL were obviously retarded in NPC1-tra p cells, suggesting an impaired response of the cholesterol-regulatory syst em to LDL, NPC1-trap cells will be a useful tool to study the regulation of cellular cholesterol homeostasis and the pathogenesis of Niemann-Pick dise ase type C.