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  • Title: [Construction of a lentiviral vector for RNA interference of PRL-3 gene and its stable expression in SW480 cells].
    Author: Liu YH, Li JM, Zhou J, Ding YQ.
    Journal: Nan Fang Yi Ke Da Xue Xue Bao; 2008 Apr; 28(4):509-12. PubMed ID: 18495578.
    Abstract:
    OBJECTIVE: To construct a lentiviral vector for RNA interference (RNAi) of PRL-3 gene and establish a human colon carcinoma cell line with PRL-3 gene knock-down. METHODS: The plasmids were constructed expressing two different short hairpin RNAs (shRNA) targeting PRL-3 gene under control by the U6 promoter by lentiviral vector. An entry clone was generated in the pENTR/U6 vector. After identification with sequencing, a recombinant lentiviral vector was obtained using the pENTR/U6 construct and pLenti6/BLOCK-iT TM-DEST vector. The recombinant lentivirus was harvested from 293FT cells co-transfected with optimized ViraPower Packaging Mix and the pLenti6/BLOCK-iT -DEST expression construct. SW480 cells were infected with the recombinant lentivirus and the cells with stable PRL-3 gene knock-down were screened by blasticidin selection. PRL-3 expression was detected using real-time reverse transcription-polymerase chain reaction and Western blotting. RESULTS: A recombinant lentiviral vector expressing shRNAs targeting PRL-3 gene was successfully established and confirmed by DNA sequencing. The recombinant lentivirus were harvested from 293FT cells with a viral titer of 6 x 10(5) pfu/L. Twelve clones of SW480 cells infected with the recombinant lentivirus were selected, and the Clone 1 exhibited significant knock-down of PRL-3 protein expression (72.9%). CONCLUSION: The successful establishment of human colon carcinoma cell line with PRL-3 gene knock-down provide a basis for investigation of the role of PRL-3 gene in the metastasis of human colorectal carcinoma.
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