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.

中文翻译：

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基于全基因组无序慢向量的短发夹 RNA 库的优化。

为了获得抑制人类 mRNA 总多样性的全基因组文库，优化了慢病毒载体构建体和短发夹 RNA (shRNA) 表达盒。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、copGFP 或H-2K(k) 作为选择标记获得基于慢病毒的shRNA 表达构建体。在筛选 shRNA 重新激活 HeLa 细胞中的肿瘤抑制因子 p53 时，证明了优化文库的效率。细胞携带报告构建体，确保荧光蛋白的 p53 响应合成，这允许通过流式细胞术选择具有重新激活的 p53 的细胞。