Construction and package of recombinant adeno-associated virus encoding shRNA-Snail
10.3969/j.issn.1001-3733.2017.06.026
- VernacularTitle:shRNA-Snail载体的构建及其病毒的包装
- Author:
Zemeng LI
1
;
Genjian ZHENG
;
Lan ZHOU
;
Wen LUO
;
Man YUN
;
Pengcheng LI
Author Information
1. 海南医学院第一附属医院
- Keywords:
Adeno-associated virus;
shRNA-Snail;
Oral squamous cell carcinoma
- From:
Journal of Practical Stomatology
2017;33(6):833-837
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct an adeno-associated vector expressing Snail "tough decoy" (TUD).Methods:The nucleotide sequence of Snail gene were obtained from GenBank,then 2 cDNAs were designed and synthesized coding expression of small hairpin RNAs for Snail genes.The pAAV-U6-shRNA-Snail-TUD-EGFP expression vector was constructed by molecular biological techniques.Then pAAV-U6-shRNA-Snail-TUD-EGFP was co-transfected with Helper Free adeno-associated virus system pAAV-RC and pAAV-Helper into AAV-293 cell line to form rAAY-U6-shRNA-Snail-TUD(shRNA-Snail).The silencing effect of shRNA-Snail in Tca8113 and Cal-27 cells was detected by RT-PCR.The titer of the virus was measured.The expression level of green fluorescent protein in AAV-293 cells was monitored by the fluorescent microscopy.Results:The result of gene sequencing showed that shRNA-Snail was successful constructed in pAAV-U6-shRNA-Snail-TUD-EGFP vector.The titer of the recombined virus was 4.4 × 1010/ml.The Snail mRNA and protein expression level was significantly reduced in Tca8113 and Cal-27 cells by rAAY-U6-shRNA-Snail-TUD-EGFP transfection.Conclusion:rAAY-U6-shRNA-Snail-TUD-EGFP may inhibite Snail gene expression in cells.
