关键词: 基因, p53, 逆转录病毒载体, shRNA

Abstract: Objective To establish a retroviral vector of small hairpin RNA (shRNA) expression in which the sense and antisense sequences targeting wild type human p53 were linked together with a 9-nucleotide loop to detect the interfering role of p53 gene in mammalian cells. Methods The human H₁ promoter was inserted into the upstream of the cytomegalovirus (CMV) promoter by using Xhol I and EcoR I sites. The resistanting hygromycin gene was replaced with the human CD₄ gene. These oligonucleotides were annealed and ligated the downstream of the H₁ promoter. All oligonucleotides were synthesized with Qiagen. HEK293 was infected with the shRNA vector and the expression of p53 was detected 72 h after infection by flow cytometry. Results The retroviral vector was successfully constructed and the expression of P53 protein was definitely suppressed in the HEK293 72 h after infection. Conclusion The applying of shRNA expression vector is possible to provide a prompt and promising method for evaluating the gene function of mammalian cells.

Key words: genes, p53, retroviral vector, shRNA