A far-downstream hepatocyte-specific control region directs expression of the linked human apolipoprotein E and C-I genes in transgenic mice.
A far-downstream hepatocyte-specific control region directs expression of the linked human apolipoprotein E and C-I genes in transgenic mice.
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DOI：
10.1016/s0021-9258(18)53085-4
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发表时间：
1993-04
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影响因子：
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通讯作者：
Scott Sirnonet;Nathan Bucay;S. J. Lauer;John;Taylor
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文献类型：
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作者：
Scott Sirnonet;Nathan Bucay;S. J. Lauer;John;Taylor
人载脂蛋白（apo）E和apoC-I基因在19号染色体上以相同的转录方向相隔5个碱基，它们在肝脏中高水平表达，在选定的其他组织中表达水平较低。对转基因小鼠中一系列重叠的人apoE和apoC-Ⅰ基因组片段的分析表明，这些转基因在肝脏中的表达需要一个共同的顺式作用调控结构域。该肝控制区（HCR）位于apoE启动子下游约18个碱基和apoC-1启动子下游约9个碱基的764个碱基对区域。用含有该区域的构建体制备的所有转基因动物在肝脏中具有相对高水平的转基因表达，而缺乏该区域的构建体在肝脏中不显示表达。原位杂交研究表明，HCR指导apoE和apoC-Ⅰ转基因在肝细胞中的表达。当将来自apoE/C-I基因座的HCR连接到通常不在肝脏中表达的人apoA-IV基因片段的近端时，所得的apoA-IV/HCR融合构建体在肝脏中以高水平表达，表明HCR可以指导异源启动子/基因构建体的高水平肝脏表达。载脂蛋白E转基因在肝脏和肾脏，也许其他组织中的表达，需要在载脂蛋白E基因启动子，位于161和141 bp之间的转录起始位点的非特异性近端增强子元件的存在。然而，近端apoE基因启动子，包括该增强子元件，不包含能够在不存在HCR的情况下指导肝细胞表达的序列。因此，远下游的HCR似乎包含确定高水平肝脏特异性基因表达所需的所有序列。
The human apolipoprotein (apo) E and apoC-I genes are located 5 kilobases apart in the same transcriptional orientation on chromosome 19, and they are expressed at high levels in the liver with lower levels of expression in selected other tissues. Analysis of a series of overlapping human apoE and apoC-I genomic fragments in transgenic mice revealed that the expression of these transgenes in the liver requires a common cis-acting regulatory domain. This hepatic control region (HCR) was localized to a 764-base pair region that is located about 18 kilobases downstream of the apoE promoter and about 9 kilobases downstream of the apoC-I promoter. All the transgenic animals that had been prepared with a construct that contained this region had relatively high levels of transgene expression in the liver, whereas constructs that lacked this region showed no expression in the liver. In situ hybridization studies showed that the HCR directed apoE and apoC-I transgene expression in hepatocytes. When the HCR from the apoE/C-I gene locus was ligated proximal to a human apoA-IV gene fragment, which is not normally expressed in the liver, the resulting apoA-IV/HCR fusion construct was expressed at high levels in the liver, indicating that the HCR could direct high level liver expression of a heterologous promoter/gene construct. Expression of the apoE transgene in the liver and kidney, and perhaps other tissues, required the presence of a nonspecific proximal enhancer element in the apolipoprotein E gene promoter, located between 161 and 141 bp relative to the transcription initiation site. However, the proximal apoE gene promoter, including this enhancer element, contained no sequences capable of directing hepatocyte expression in the absence of the HCR. Thus, the far-downstream HCR appears to contain all of the sequences necessary for determining high level liver-specific gene expression.