Identification of synthetic endothelial cell-specific promoters by use of a high-throughput screen
Identification of synthetic endothelial cell-specific promoters by use of a high-throughput screen
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DOI：
10.1128/jvi.78.12.6209-6221.2004
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发表时间：
2004-06-01
影响因子：
5.4
通讯作者：
Sutton, RE
中科院分区：
文献类型：
--
作者：
Dai, C;McAninch, RE;Sutton, RE
转录靶向是许多基因转移应用的理想特性。由于内皮细胞排列在大多数血管上，它们是引入治疗性基因产品的有吸引力的候选者。作为一项概念验证研究，我们试图通过使用基于自我灭活（SIN）人类免疫缺陷病毒1型（HIV-1）载体的高通量筛选来鉴定一种合成的内皮细胞特异性启动子。在基于SIN hiv -1的载体中，选择转录因子识别的、位于内皮细胞特异性mRNA转录物5'的双工寡核苷酸，随机连接并克隆到最小ICAM-2启动子上游，驱动增强的绿色荧光蛋白（eGFP）。从bbb10(6)载体重组文库中制备水疱性口炎病毒G蛋白假型颗粒，并将其用于内皮细胞系的转导。对最高的eGFP表达子进行重复排序，回收合成的启动子并通过荧光素酶报告基因重新检测。几种启动子对不同种类的内皮细胞具有特异性和活性，具有高选择性指数和在模拟缺氧条件下的诱导性。然后将其中一种特别的病毒引入到基于SIN hiv -1的载体中，以确认其内皮细胞活性和特异性。本研究表明，利用SIN载体可以高通量地鉴定高活性的组织特异性启动子，在转录靶向和基因转移方面具有潜在的应用前景。
Transcriptional targeting is a desirable property for many gene transfer applications. Because endothelial cells line most blood vessels, they are attractive candidates for the introduction of therapeutic gene products. As a proof-of-concept study, we attempted to identify a synthetic, endothelial cell-specific promoter by use of a high-throughput screen involving self-inactivating (SIN) human immunodeficiency virus type 1 (HIV-1)based vectors. Select duplex oligodeoxynucleotides recognized by transcription factors and located 5' of endothelial cell-specific mRNA transcripts were randomly ligated and cloned upstream of a minimal ICAM-2 promoter driving enhanced green fluorescent protein (eGFP) in a SIN HIV-1-based vector. Vesicular stomatitis virus G protein-pseudotyped particles were prepared from a library of >10(6) vector recombinants and used to transduce an endothelial cell line. The highest eGFP expressers were repeatedly sorted, and the synthetic promoters were recovered and retested by a luciferase reporter. Several promoters were active and specific to endothelial cells of varied species, with high selectivity indexes and inducibility under hypoxia-mimetic conditions. One in particular was then introduced back into a SIN HIV-1-based vector to confirm its endothelial cell activity and specificity. This study suggests that SIN vectors may be used in a high-throughput manner to identify tissue-specific promoters of high activity, with potential applications for both transcriptional targeting and gene transfer.