Optimization of a genome-wide disordered lentivector-based short hairpin RNA library
Optimization of a genome-wide disordered lentivector-based short hairpin RNA library
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DOI：
10.1134/s002689330603006x
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发表时间：
2006-05-01
影响因子：
1.2
通讯作者：
Frolova, E. I.
中科院分区：
文献类型：
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作者：
Guryanova, O. A.;Makhanov, M.;Frolova, E. I.
为了获得抑制人mRNA的总多样性的全基因组文库，对慢病毒载体构建体和短发夹RNA（shRNA）表达盒进行了优化。载体的优化使制剂中的病毒滴度提高了15-20倍。具有21-bp茎的简单shRNA结构被证明是最有效的。通过使用puro（R）、copGFP或H-2K（k）作为选择标记获得基于慢病毒载体的shRNA表达构建体。当在HeLa细胞中筛选重新激活肿瘤抑制基因p53的shRNA时，证明了优化文库的效率。细胞携带一个报告构建体，确保荧光蛋白的p53响应性合成，这允许通过流式细胞术选择具有重新激活的p53的细胞。
To obtain a whole genome library that suppresses the total diversity of human mRNAs, lentiviral vector constructs and a short hairpin RNA (shRNA) expression cassette were optimized. The optimization of the vector increased the virus titer in preparations by 15-20 times. A simple shRNA structure with a 21-bp stem proved to be the most effective. Lentivector-based shRNA expression constructs were obtained by using puro(R), copGFP or H-2K(k) as a selectable marker. The efficiency of the optimized library was demonstrated when screening for shRNAs reactivating the tumor suppressor p53 in HeLa cells. Cells carried a reporter construct ensuring p53-responsive synthesis of a fluorescent protein, which allowed selection of cells with reactivated p53 by flow cytometry.