Construction and Identification of Transforming Growth Factor-β1 shrna Expressing Vector

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1 β1shrna TGF -β1 RNA shrna TGF-β1 mrna Ambion Target Finder TGF-β1shRNA BglⅡ HindⅢ psuper gfp-neo shrna psuper gfp-neo DH5a ELISA TGF-β1shRNA ELISA shrna-a shrna-b shrna-c TGF-β1 shrna-b P < TGF-β1shRNA TGF-β1 RNA -β1 R A Construction and Identification of Transforming Growth Factor-β1 shrna Expressing Vector ZHANG Biao GUO Ling-chuan DONG Liang ZHOU Yun WU Chun HU Xin PAN Zhan-teng ZHONG Liang-jun WANG Shou-li Dept of Pathology Medical College Soochow University Jiangsu Suzhou China Abstract Objective To construct transforming growth factor-β1 TGF-β1 short hairpin RNA shrna -expressing vector and provide the basis for further experiments in cancer biology. Methods TGF-β1-specific shrnas were designed with the Target Finder of Ambion according to mrna of TGFβ1 and were ligated into the mammalian expression vector psuper gfp-neo at the BglII and HindIII cloning sit. The recombinant shrnapsuper gfp-neo was used to transform Escherichia coli DH5a which was selected and verified by sequencing. Then select the optimal site for TGFB1 gene silencing with enzyme-linked immunosorbent assay ELISA and Immunofluorescence. Results ELISA and immunofluorescence staining showed reduced expression of TGF-b1 protein in RD cells transfected with shrna-a shrna-b and shrna-c. The reduction was most pronounced P < in cells transfected with shr- NA-b. Conclusion These data suggest that the constructed TGF-β1shRNA-expressing vector produces efficient TGF-β1 knockdown in the tumor cell line and may be a method of cancer biological research. Key words RNA interference transforming growth factor-β1 expressing vector construction KY B

2 976 SUZHOU UVIVERSITY JOURNAL OF MEDICAL SCIENCE transforming growth factor TGF-β1 Santa Cruz TGF -β FITC - IgG 1 1 h RNA RNA interference RNAi ELISA RD / 96 RNA d 6 d ELISA TGF-β1 96 RD TGF-β1 HRP TGFβ1 A TβR Smad TGFβ1 2 shrna 1. 5 SPSS χ 2 TGF-β1 t P < shrna 2. 1 TGF-β1-shRNA psuper OligoEngine H1RNA Ⅲ psuper BglⅡ HindⅢ H1RNA RNA 19bp shrna RNA Ambion Target Finder TGF-β1shRNA 9-nt 1 19nt 19nt BLAST 3 Target Finder BglⅡ HindⅢ shrna psuper DH5a TGF-β1 shrna 1. 2 DMEM Lipofectamine TM TGF-β1shRNA 1 1 Lip- shrna RD /ml 6 24 h DMEM 2 ml 250 μl 1 TGF-β1shRNA RD TGF-β1 TGF-β1shRNA TGF-β1shRNA TGF-β1 4 4% 15 min PBS 37 2 TGFβ1shRNA 1 5% 30 min 4 1 shrna

3 TGF-β1 TGF-β1shRNA-b sirna TGF-β1 sirna P < TGF-β1shRNA RD H1 RNA RD TGF-β1 % sirna TGF-β1shRNA-un ± TGF-β1shRNA-a ± * TGF-β1shRNA-b 27 * ± 2. 2 * TGF-β1shRNA-c ± * * P < TGF-β1shRNA TGF-β1 6 RNA 7 ELISA 3 shrna-a shrnab shrna-c RD TGF-β1 -sirna shrna-b P < TGF-β1 shrna ELISA TGF-β1shRNA TGF- 3 3 ELISA TGF-β1shRNA TGF-β1 sirna 5 psuper psuper BglⅡ HindⅢ RNA 19nt 9-nt H1RNA shrna β1shrna TGF-β1 TGF-β1shRNA-b ELISA TGF-β1 shrna-b P < TGF-β1-shRNA TGF-β ~ shrna-b RNAi RNAi shrna TGF-β1 8 RNAi 3 TGF-β1 4 dsr- NA sirna RNAi 1001

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