[Construction of a lentiviral vector for RNA interference of PRL-3 gene and its stable expression in SW480 cells].
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%).


CONCLUSIONS
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.
Liu
Yu-hong
YH
Department of Pathology and Guangdong Provincial Key Laboratory of Molecular Tumor Pathology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. liuyuhongyantai@163.com
Li
Jian-ming
JM
Zhou
Jun
J
Ding
Yan-qing
YQ
D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D009363:Neoplasm Proteins; C484730:PTP4A3 protein, human; D017027:Protein Tyrosine Phosphatases