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. Nontransfected 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.
Citation: Jin Ma. Construction and identification of small hairpin RNA expression plasmid targeting opticin gene in rat. Chinese Journal of Ocular Fundus Diseases, 2011, 27(1): 60-64. doi: Copy