[Construction and activity evaluation of pSUPER RNAi system that inhibits Smad3].
OBJECTIVE
To construct the expressing vector of siRNA which can inhibit the Smad3 activity.


METHODS
Sixty-four bases of 2 pair oligos for hairp in RNA expression which targeted Smad3 gene were chemically synthesized and annealed. pSUPER vector was linearized with BgL II and Hin d III treated with alkaline phosphatase (CIP). Anneled oligos were inserted into the downstream of the treated pSUPER's pol III H1 promoter to construct RNAi plasmid (pSUPER Smad3). Oligos with a scrambled sequence were used as a negative control. pSUPER Smad3 was transfected into human renal tubular epithelial cells (HKC).


RESULTS
Recombinant pSUPER Smad3 vector was identified by the digestion with Eco R I and Hin d III, and confirmed by the sequencing analysis with T3 primer. Sixty-four bases had been inserted into the expected site. Furthermore, the insertion sequence was exactly corrected. The activity evaluation indicated that mRNA and protein of Smad3 but not Smad2 were inhibited by pSUPER Smad3 in HKC.


CONCLUSIONS
The pSUPER Smad3 system has been constructed successfully, and has high inhibition and specificity in vitro.
Zhang
Dong-shan
DS
Department of Nephrology; Second Xiangya Hospital, Central South University, Changsha 410011, China. zhkidney@yahoo.com.cn
Liu
Fu-you
FY
Peng
You-ming
YM
Xiong
Guan-zhong
GZ
Chai
Xiang-ping
XP
D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D007684:Kidney Tubules / Q000166:cytology; D010957:Plasmids; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering; D051900:Smad3 Protein / Q000037:antagonists & inhibitors / Q000235:genetics*; D014162:Transfection
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051900:Smad3 Protein