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首页> 外文期刊>Gene Therapy and Molecular Biology >Hepatocyte-specific gene expression by arecombinant adeno-associated virus vector carryingthe apolipoprotein E enhancer and ? 1-antitrypsinpromoter
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Hepatocyte-specific gene expression by arecombinant adeno-associated virus vector carryingthe apolipoprotein E enhancer and ? 1-antitrypsinpromoter

机译：携带载脂蛋白E增强子和α的重组腺相关病毒载体表达肝细胞特异性基因1-抗胰蛋白酶启动子

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An adeno-associated virus vector was constructed to express exogenous genes to the liver. The original plasmid construct carried two expression units; a neomycin resistant gene and human ? 1- antitrypsin cDNA under the control of hepatocyte specific transcription elements. Cells were transfected with the constructed plasmid DNA with another packaging plasmid, and recombinant adeno-associated viruses (rAAV) were then recovered after adenovirus infection. Alternatively, rAAV were recovered by transduction of DNAs of the packaging plasmid and adenovirus into pre- selected cells carrying constructed proviral DNA. When the transducing abilities were evaluated based on G418 resistant colony formation on HeLa cells, the latter method was found to give almost 10-fold more rAAV. We then isolated G418 resistant colonies and established several independent clones for the HeLa and Hepa1A cells infected with the rAAV. All of the eight clones derived from Hepa1A cells produced significant amounts of the human ? 1-antitrypsin protein. In contrast, none of the five clones derived from HeLa cells produced a detectable level of ? 1- antitrypsin. Our results suggest that liver-specific promoter and enhancer maintain the tissue specificity in the rAAV construct, and that the rAAV vector system would be useful in hepatocyte directed gene therapy.
机译：构建腺相关病毒载体以向肝脏表达外源基因。原始的质粒构建体带有两个表达单元;即两个表达单元。一个新霉素抗性基因和人类？ 1-抗胰蛋白酶cDNA在肝细胞特异性转录元件的控制下。用构建的质粒DNA和另一个包装质粒转染细胞，然后在腺病毒感染后回收重组腺相关病毒（rAAV）。备选地，通过将​​包装质粒和腺病毒的DNA转导到携带构建的原病毒DNA的预选细胞中来回收rAAV。当基于HeLa细胞上对G418的抗性集落形成来评估转导能力时，发现后者的方法可产生近10倍的rAAV。然后，我们分离了G418抗性菌落，并为感染了rAAV的HeLa和Hepa1A细胞建立了几个独立的克隆。来自Hepa1A细胞的八个克隆全部产生了大量的人肝癌细胞。 1-抗胰蛋白酶蛋白。相反，来自HeLa细胞的5个克隆均未产生可检测水平的α。 1-抗胰蛋白酶。我们的结果表明，肝特异性启动子和增强子可维持rAAV构建体中的组织特异性，并且rAAV载体系统可用于肝细胞定向基因治疗。

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