慢病毒敲减质粒pLKO.1-hSRF的构建及鉴定

doi:10.3969/j.issn.1004-616x.2018.03.014

Abstract

Abstract: OBJECTIVE: To construct a plasmid which interfered with expression of the human SRF gene,and to study the role of SRF in oral squamous cell carcinoma. METHODS: A SRF gene-specific knockdown fragment was designed by using the Thermo Fisher's RNAi design tool,and then by double digestion of the pLKO.1 vector,gel extraction,and connection of specific fragment and the linearized vector by T4 ligase,then the pLKO.1-hSRF plasmid was obtained. The recombinant plasmids were identified by sequencing and restriction enzyme digestion. 293T cells were used to generate lentivirus by co-transfecting pLKO.1-hSRF with helper plasmids pVSV-G and pHR. The virus were collected and used to infect the oral squamous carcinoma cells,SAS. Stable cells were selected by puromycin and verified by Western blot and real-time quantitative PCR. RESULTS: The lentiviral knockdown plasmid pLKO.1-hSRF was identified by sequencing and restriction enzyme digestion. The expression of SRF protein and mRNA level of SAS cells infected with the lentiviral plasmid were significantly decreased compared with the control group. CONCLUSION: The lentiviral knockdown plasmid pLKO.1-hSRF was successfully constructed and the SAS cell line of low expression of SRF was obtained.

Key words: lentiviral vector, plasmid construction, serum response factor, oral squamous cell carcinoma

CLC Number:

R735.35

CAI Yihuang, DENG Xiaoling, HUANG Wenxia, HUANG Jie, JI Qing, XU Mingyan. Construction and identification of lentiviral knockdown plasmid pLKO.1-hSRF[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2018, 30(3): 234-238.

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Construction and identification of lentiviral knockdown plasmid pLKO.1-hSRF

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