[Construction and identification of eukaryotic expression plasmid expressing siRNA targeting Rac1 and detection of its specific downregulation effect].
OBJECTIVE
To constructed eukaryotic expressing siRNA sections targeting human and rat Rac1 and to observe their effects on Rac1 gene expression in HeLa cell line.


METHODS
siRNA sequences were designed and synthesized targeting human and rat's Rac1 mRNA, and then directionally cloned into pSUPER plasmid. The recombinant vectors were confirmed by enzyme digestion analysis and DNA sequencing. The recombinant vectors were transfected into HeLa cell line and the transfection rate was evaluated with fluorescence microscope. The effects of the recombinants on Rac1 at mRNA levels were observed by RT-PCR.


RESULTS
Three siRNA expressing recombinants and corresponding negative control vector were constructed and transfected into HeLa cell successfully. Rac1 transcript was reduced significantly in three transfectants.


CONCLUSIONS
The construction of eukaryotic expression vectors expressing siRNA sections targeting Rac1 and identification successfully. This will be very helpful for further study on the function of Rac1 and it may be a useful tool in the treatment of the neovascular retinopathy.
Zhang
Mei-Xia
MX
Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu 610041, China.
Wu
Jing
J
Xin
Mei
M
Li
Juan-Juan
JJ
Zhang
Jun-Jun
JJ
Yan
Mi
M
D000818:Animals; D001483:Base Sequence; D005822:Genetic Vectors / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D015861:Retinal Neovascularization / Q000378:metabolism; D014162:Transfection; D020830:rac1 GTP-Binding Protein / Q000096:biosynthesis / Q000235:genetics*
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D020830:rac1 GTP-Binding Protein