Knockdown of human p53 gene expression in 293-T cells by retroviral vector-mediated short hairpin RNA
Knockdown of human p53 gene expression in 293-T cells by retroviral vector-mediated short hairpin RNA
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DOI：
10.1111/j.1745-7270.2005.00107.x
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发表时间：
2005-11-01
影响因子：
3.7
通讯作者：
Liang, CC
中科院分区：
文献类型：
--
作者：
Hao, DL;Liu, CM;Liang, CC
RNA干扰（RNAi）是多种生物体中进化上保守的基因沉默过程，已成为通过反向遗传学研究基因功能的有力工具。最近，许多研究小组报道了使用合成的寡核苷酸或siRNA编码质粒通过转染在哺乳动物细胞中诱导RNAi，但这在其应用中仍然受到限制，特别是当需要在体内产生长期基因沉默时。为了解决这个问题，逆转录病毒或慢病毒传递的 RNAi 被开发出来。在这里，我们描述了两种用于传递从 H1 启动子转录的短发夹 RNA (shRNA) 的逆转录病毒系统。结果表明，逆转录病毒载体介导的RNAi可以显着下调293-T细胞中人p53的表达。此外，我们的转导系统中的逆转录病毒载体介导的RNAi可以长时间稳定地灭活p53基因。与从 U6 启动子转录的 shRNA 相比，H1 驱动的 shRNA 还显着降低了 p53 的表达。 H1 和 U6 驱动的 shRNA 的 p53 下调效率几乎相同。结果表明，逆转录病毒载体传递的 RNAi 将成为功能基因组学和基因治疗的有用工具。
RNA interference (RNAi) is an evolutionarily conserved process of gene silencing in multiple organisms, which has become a powerful tool for investigating gene function by reverse genetics. Recently, many groups have reported to use synthesized oligonucleotides or siRNA encoding plasmids to induce RNAi in mammalian cells by transfection, but this is still limited in its application, especially when it is necessary to generate long-term gene silencing in vivo. To circumvent this problem, retrovirus- or lentivirus-delivered RNAi has been developed. Here, we described two retroviral systems for delivering short hairpin RNA (shRNA) transcribed from the H1 promoter. The results showed that retroviral vector-mediated RNAi can substantially downregulate the expression of human p53 in 293-T cells. Furthermore, the retroviral vector-mediated RNAi in our transduction system can stably inactivate the p53 gene for a long time. Compared to shRNAs transcribed from the U6 promoter, H1-driven shRNA also dramatically reduced the expression of p53. The p53 downregulation efficiencies of H1- and U6-driven shRNAs were almost identical. The results indicate that retroviral vector-delivered RNAi would be a useful tool in functional genomics and gene therapy.