A new dual-vector approach to enhance recombinant adeno-associated virus-mediated gene expression through intermolecular cis activation
A new dual-vector approach to enhance recombinant adeno-associated virus-mediated gene expression through intermolecular cis activation
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DOI：
10.1038/75080
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发表时间：
2000-05-01
期刊：
影响因子：
82.9
通讯作者：
Engelhardt, JF
中科院分区：
文献类型：
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作者：
Duan, DS;Yue, YP;Engelhardt, JF
（AV. Luc）或具有最小启动子的构建体（AV.单独的SV（P）Luc）在感染后3天仅产生最小表达（图3a）。但是，与AV合并感染。SupEnh使来自AV的荧光素酶表达增加16倍和35倍。卢克和AV SV（P）Luc向量。这些数据支持我们最初的假设，顺式激活一个最小的SV 40启动子可以发生与第二个AAV载体含有增强子元件的共感染。然而，出乎意料的是，AV存在高水平的反式激活。Luc构建体，其不含异源启动子。这些发现支持了早期的研究，表明ITR包含一个神秘的启动子1。通过与阴性对照AV共同给药后缺乏反式激活进一步证明了该诱导的特异性。AmpOri病毒伴AV。卢克或AV。SV（P）Luc载体为了测量双载体方法的效率，我们比较了顺式激活后获得的荧光素酶表达与来自含有SV 40启动子和增强子的不同组合的单载体的荧光素酶表达。荧光素酶基因表达水平在AV。SV（P）Luc和AV. SupEnh达到单一载体AV的50%。SV（P/E）Luc和单载体AV的10%。成纤维细胞中的SV（P/SupEnh）Luc（图3a）。
(AV. Luc) or the construct with a minimal promoter (AV. SV (P) Luc) alone gave only minimal expression at 3 days after infection (Fig. 3a). However, co-infection with AV. SupEnh produced 16-fold and 35-fold increases in luciferase expression from the AV. Luc and AV. SV (P) Luc vectors, respectively. These data support our initial hypothesis that cis activation of a minimal SV40 promoter can occur by coinfection with a second AAV vector containing enhancer elements. Unexpectedly, however, there was high-level transactivation of the AV. Luc construct, which contains no heterologous promoter. These findings support earlier studies indicating that ITRs contain a cryptic promoter1. The specificity of this induction was further demonstrated by the lack of transactivation after co-administration of the negative control AV. AmpOri virus with AV. Luc or AV. SV (P) Luc vectors.To measure the efficiency of the dual-vector approach, we compared luciferase expression obtained after cis activation with that from single vectors containing different combinations of the SV40 promoter and enhancers. The level of luciferase gene expression after intermolecular concatamerization between AV. SV (P) Luc and AV. SupEnh reached 50% that of the single vector AV. SV (P/E) Luc and 10% that of the single vector AV. SV (P/SupEnh) Luc in fibroblasts (Fig. 3a).