Wang D M, Taylor S, Levy-Wilson B
Palo Alto Medical Foundation Research Institute, CA 94301, USA.
J Lipid Res. 1996 Oct;37(10):2117-24.
The human apolipoprotein B (apoB) gene resides in a 47.5 kb DNasel-sensitive chromosomal domain in hepatic and intestinal cells, flanked by the 5' distal matrix association region (MAR) and the 3' proximal MAR. A third MAR, the 5' proximal MAR, is found only in transcriptionally active hepatic (HepG2) cells. Hepatic expression of the apoB gene requires a tissue-specific promoter (-898 to +121) and an enhancer from the second intron of the gene (+360 to +1064). A vector containing this portion of the gene linked to the beta-galactosidase reporter is sufficient for low level expression in the livers of transgenic mice. Expression in transgenic mice was increased when the promoter-enhancer beta-gal vector was flanked by MARs. The results were similar whether the 5' distal, the 5' proximal or the 3' proximal MARs were placed at both ends of the construct, or whether the construct was flanked by the 5' distal and the 3' MAR, suggesting that the apoB MARs play a role in gene expression in vivo. When the MAR-containing constructs were transiently transfected into HepG2 cells, the resulting beta-gal activities were similar to that of the construct lacking MARs, thus demonstrating that the MARs do not exhibit any enhancer activity. Recent experiments (Kalos, M., and R. E. K. Fournier. 1995. Mol. Cell. Biol. 15: 198-207) examining stable integration of some of our constructs into human and rat hepatoma transfectants suggest that in single and double copy transfectants, the apoB MARs behave as boundary "insulators", protecting the integrated transgenes against position effects regardless of their site of integration. However, multicopy transfectants are transcriptionally inactive and when the MARs are absent, expression of the transgenes drops to background levels. Our results to date with single and low-copy number transgenes do not support an insulator function for the apoB MARs, although they appear to be required to increase the levels of expression.
人类载脂蛋白B（apoB）基因位于肝脏和肠道细胞中一个对核酸酶敏感的47.5 kb染色体结构域内，两侧分别是5'远端基质附着区域（MAR）和3'近端MAR。第三个MAR，即5'近端MAR，仅在转录活跃的肝脏（HepG2）细胞中发现。apoB基因的肝脏表达需要一个组织特异性启动子（-898至+121）和来自该基因第二个内含子的增强子（+360至+1064）。一个包含该基因这部分与β-半乳糖苷酶报告基因相连的载体足以在转基因小鼠肝脏中实现低水平表达。当启动子-增强子β-半乳糖苷载体两侧带有MAR时，转基因小鼠中的表达会增加。无论将5'远端、5'近端还是3'近端MAR置于构建体两端，或者构建体两侧是5'远端和3' MAR，结果都相似，这表明apoB MAR在体内基因表达中起作用。当将含MAR的构建体瞬时转染到HepG2细胞中时，产生的β-半乳糖苷酶活性与缺乏MAR的构建体相似，从而证明MAR不表现出任何增强子活性。最近的实验（Kalos, M., and R. E. K. Fournier. 1995. Mol. Cell. Biol. 15: 198 - 207）研究了我们的一些构建体在人和大鼠肝癌转染细胞中的稳定整合情况，结果表明在单拷贝和双拷贝转染细胞中，apoB MAR表现为边界“绝缘子”，无论整合位点如何，都能保护整合的转基因免受位置效应的影响。然而，多拷贝转染细胞转录不活跃，且当没有MAR时，转基因的表达会降至背景水平。我们目前关于单拷贝和低拷贝数转基因的结果不支持apoB MAR具有绝缘子功能，尽管它们似乎是提高表达水平所必需的。
