Using genetically modified mice to study apolipoprotein B.

Abstract

The B apolipoproteins, apo-B48 and apo-B100, are key proteins in mammalian lipoprotein metabolism and are components of all classes of lipoproteins considered to be atherogenic. Our laboratory has generated an array of genetically modified mice for studying apo-B biology. Using gene targeting in mouse embryonic stem cells, we have generated apo-B-deficient mice. Heterozygotes had low plasma levels of apo-B and cholesterol; homozygotes died early in embryonic development, most likely because the absence of lipoprotein secretion by the yolk sac interfered with the delivery of lipid nutrients to the developing embryo. We have also generated human apo-B transgenic mice with an 80-kb genomic DNA fragment spanning the entire human apo-B gene; those mice had markedly increased plasma levels of low density lipoprotein cholesterol and exhibited increased susceptibility to atherosclerosis. The human apo-B transgenic mice have also yielded insights regarding the regulation of apo-B expression in different tissues. Although the 80-kb transgene contained nearly 20 kb of 5' and 3' flanking sequences and was expressed at high levels in the liver, no transgene expression was detectable in the intestine. Subsequent transgenic mouse studies have demonstrated that the expression of the apo-B gene in the intestine is controlled by DNA sequences that are very distant from the structural gene. Transgenic mice have also proved useful for studying apo-B structure/function relationships. By expressing mutant forms of human apo B in transgenic mice, we have examined the structural features of the apo-B molecule that are required for lipoprotein (a) formation. We have demonstrated that the carboxyl terminal cystine residue of apo-B100, cysteine-4326, is required for apo-B100's disulfide linkage with apo(a) to form lipoprotein (a). Finally, we have used gene targeting techniques to generate mice that synthesize exclusively apo-B48 (apo B48-only mice) and mice that synthesize exclusively apo-B100 (apo-B100 only mice): These mice have helped to clarify the unique metabolic roles of the two apo-B proteins.