Nielsen L B, McCormick S P, Young S G
Department of Medicine, Nakskov Hospital, Denmark.
Scand J Clin Lab Invest Suppl. 1999;229:33-9. doi: 10.1080/00365519950185931.
Apolipoprotein B (apo-B) plays a crucial role in the assembly of lipoproteins in the liver and the intestine. Here, we review how transgenic mouse expression studies with large genomic clones have been used to define distant cis-acting regulatory DNA sequences that control the expression of the apo-B gene. In early studies, apo-B transgenic mice were generated with approximately 80-kb P1 bacteriophage clones spanning either the human or the mouse apo-B genes. Both the human and mouse clones directed high levels of transgene expression in the liver, but transgene expression was absent in the intestine. The absence of transgene expression in the intestine was surprising because both P1 clones contained more than 11 kb of flanking sequences both 5' and 3' to the gene. Subsequently, we isolated and characterized 145-kb and 207-kb bacterial artificial chromosome (BAC) clones that spanned the human apo-B gene. Each of these BAC's contained extensive 5 and 3' flanking sequences and each directed spatially and physiologically appropriate apo-B gene expression in the intestines of transgenic mice. To define the location of the sequences that control intestinal expression of the apo-B gene, we generated transgenic mice by co-microinjecting the approximately 80-kb P1 bacteriophage clone (which did not confer intestinal expression of apo-B) with either the 5' sequences or the 3' sequences from the 145-kb BAC. Analysis of the apo-B expression pattern in those mice revealed that the DNA sequences controlling intestinal expression were located 5' to the apo-B gene. Next, we used recA-assisted restriction endonuclease (RARE) cleavage to truncate specific segments of the 5' and 3' flanking sequences from the 145-kb BAC. A series of the truncated BAC's containing different lengths of 5' and 3' sequences was used to generate more than 40 additional lines of human apo-B transgenic mice. Analysis of human apo-B gene expression in those mice demonstrated that the sequences controlling the expression of the apo-B gene in the intestine are located more than 50 kb 5' to the apo-B gene. Our studies demonstrate that the RARE cleavage/transgenic expression strategy is a powerful approach for examining gene regulation by distant gene-regulatory elements.
载脂蛋白B（apo-B）在肝脏和肠道中脂蛋白的组装过程中起着关键作用。在此，我们回顾了如何利用携带大片段基因组克隆的转基因小鼠表达研究来确定控制apo-B基因表达的远距离顺式作用调控DNA序列。在早期研究中，利用跨越人类或小鼠apo-B基因的约80 kb P1噬菌体克隆构建了apo-B转基因小鼠。人类和小鼠的克隆在肝脏中均能引导高水平的转基因表达，但在肠道中却没有转基因表达。肠道中缺乏转基因表达令人惊讶，因为这两个P1克隆在基因的5'端和3'端均包含超过11 kb的侧翼序列。随后，我们分离并鉴定了跨越人类apo-B基因的145 kb和207 kb细菌人工染色体（BAC）克隆。这些BAC中的每一个都包含广泛的5'和3'侧翼序列，并且在转基因小鼠的肠道中均能引导空间和生理上适当的apo-B基因表达。为了确定控制apo-B基因肠道表达的序列位置，我们通过将约80 kb的P1噬菌体克隆（其不能赋予apo-B肠道表达）与来自145 kb BAC的5'序列或3'序列共显微注射来构建转基因小鼠。对这些小鼠中apo-B表达模式的分析表明，控制肠道表达的DNA序列位于apo-B基因的5'端。接下来，我们使用recA辅助的限制性内切酶（RARE）切割来截断145 kb BAC的5'和3'侧翼序列的特定片段。一系列包含不同长度5'和3'序列的截短BAC被用于构建另外40多株人类apo-B转基因小鼠品系。对这些小鼠中人类apo-B基因表达的分析表明，控制apo-B基因在肠道中表达的序列位于apo-B基因5'端超过50 kb处。我们的研究表明，RARE切割/转基因表达策略是研究远距离基因调控元件对基因调控的一种强大方法。
