Construction and identification of small hairpin RNA expression plasmid targeting opticin gene in rat
10.3760/cma.j.issn.1005-1015.2011.01.014
- VernacularTitle:大鼠opticin表达基因小发夹RNA真核表达质粒的构建及鉴定
- Author:
Jin MA
;
Tiepei ZHU
;
Qianru ZHANG
- Publication Type:Journal Article
- Keywords:
RNA,Small Interfering;
Gene transfer techniques;
OPTC;
Animal experimentation
- From:
Chinese Journal of Ocular Fundus Diseases
2011;27(1):60-64
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct small hairpin RNA (shRNA) expression plasmid targeting rat opticin gene. Methods Four pairs of opticin oligonucleotides were synthesized and inserted into the plasmid vector, resulting into four plasmids: shRNA-1, shRNA-2, shRNA-3 and shRNA-4. Then the four constructed shRNA expression vectors and empty vector were transfected into rat ciliary non-pigment epithelium (NPE) cells by lipofectmaine 2000. Non-transfected NPE cells were set as control group. The expression of opticin mRNA and protein were measured by Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot respectively. Results The opticin mRNA expression of the shRNA-1,shRNA-2, shRNA-3, shRNA-4 group were decreased compared with the control group (F = 10. 239, P = 0. 000);the inhibitory rate were 85.7% ,62. 87% ,54.87% and 48.77% respectively. The opticin protein expression of the shRNA-1,shRNA-2, shRNA-3, shRNA-4 group were also decreased compared with the control group (F=17.870, P= 0.000);the inhibitory rate were 78.7%, 34.6%, 31.1% and 16.8% respectively.Conclusions The shRNA-1 expression plasmid has most potent inhibitory effect on opticin expression in rat ciliary NPE cells.
