[Construction of a lentiviral vector for RNA interference of human CDH22 gene and its gene silencing effect in SW480 cells].
OBJECTIVE
To construct a lentiviral expression vector for RNA interference of human CDH22 gene, and assess its gene silencing effect in colorectal cancer cells to provide a basis for investigating the role of CDH22 gene in the signaling pathway involved in human colorectal carcinoma metastasis.


METHODS
Human CDH22 gene short hairpin RNA (shRNA) sequence was designed using a software available on-line. After synthesis and annealing, the double-stranded oligonucleotides (dsOligoe) were cloned into the pENTR(TM)/U6 plasmid followed by sequence analysis. A positive clone was subcloned into pLenti6/BLOCK-iT(TM)-DEST vector and transformed into stb13 competent cells, with also verification by sequencing. The recombinant lentivirus was harvested from 293FT cells contransfected with the positive recombined plasmid and lentiviral packing materials. SW480 cells were infected with the recombinant lentivirus and the cells with stable CDH22 knock-down were screened by blasticidin selection. CDH22 expression in the cells was determined by real-time reverse transcription-polymerase chain reaction.


RESULTS
A recombinant lentiviral vector expressing shRNAs against CDH22 gene was obtained and confirmed by DNA sequencing. Fifteen clones of SW480 cells infected with the recombinant lentivirus were selected, and clone 11 exhibited substantial knock-down of CDH22 mRNA expression.


CONCLUSIONS
The lentiviral shRNA expression vector targeting human CDH22 gene capable of stable CDH22 gene knock-down in SW480 cells has been successfully constructed, which provides a basis for further study of the relationship between human colorectal carcinoma and CDH22 gene.
Zhou
Jun
J
Department of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Li
Jian-ming
JM
Yang
Fa-da
FD
Liu
Yu-hong
YH
Ding
Yan-qing
YQ
D001483:Base Sequence; D015820:Cadherins / Q000096:biosynthesis / Q000235:genetics*; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008969:Molecular Sequence Data; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection
D015820:Cadherins; D034741:RNA, Small Interfering; C099417:CDH22 protein, human