Using genetically modified mice to study apolipoprotein B.

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.

载脂蛋白B，即载脂蛋白B48和载脂蛋白B100，是哺乳动物脂蛋白代谢中的关键蛋白质，是所有被认为有致动脉粥样硬化作用的各类脂蛋白的组成成分。我们实验室培育了一系列用于研究载脂蛋白B生物学的转基因小鼠。通过在小鼠胚胎干细胞中进行基因打靶，我们培育出了载脂蛋白B缺陷型小鼠。杂合子的血浆载脂蛋白B和胆固醇水平较低；纯合子在胚胎发育早期死亡，很可能是因为卵黄囊不分泌脂蛋白，从而干扰了脂质营养物质向发育中的胚胎的输送。我们还培育了携带一段80kb涵盖整个人类载脂蛋白B基因的基因组DNA片段的人类载脂蛋白B转基因小鼠；这些小鼠的低密度脂蛋白胆固醇血浆水平显著升高，并且对动脉粥样硬化的易感性增加。人类载脂蛋白B转基因小鼠也为了解不同组织中载脂蛋白B表达的调控提供了见解。尽管80kb的转基因包含近20kb的5′和3′侧翼序列，并且在肝脏中高水平表达，但在肠道中未检测到转基因表达。随后的转基因小鼠研究表明，肠道中载脂蛋白B基因的表达是由距离结构基因很远的DNA序列控制的。转基因小鼠也被证明对研究载脂蛋白B的结构 - 功能关系很有用。通过在转基因小鼠中表达人类载脂蛋白B的突变形式，我们研究了脂蛋白（a）形成所必需的载脂蛋白B分子的结构特征。我们已经证明，载脂蛋白B100的羧基末端半胱氨酸残基，即半胱氨酸 - 4326，是载脂蛋白B100与载脂蛋白（a）形成二硫键以形成脂蛋白（a）所必需的。最后，我们利用基因打靶技术培育出了只合成载脂蛋白B48的小鼠（仅载脂蛋白B48小鼠）和只合成载脂蛋白B100的小鼠（仅载脂蛋白B100小鼠）：这些小鼠有助于阐明这两种载脂蛋白B蛋白质独特的代谢作用。