ILKmRNA in colon cancer cell lines the expression and stable expression of RNA interference vectors.docVIP

 PAGE \* MERGEFORMAT 11 ILKmRNA in colon cancer cell lines the expression and stable expression of RNA interference vectors Abstract [Objective] To detect colon cancer cell lines the mRNA level of ILK, and build for the ILK gene-specific RNA interference (RNAi) expression vector. [Methods] RT-PCR detection of ILK gene in three kinds of colon cancer cell lines (SW480, LO-VO, and HT29) mRNA expression; available under the GenBank sequence of ILK gene mRNA to design a small hairpin structure of two DNA sequences , the chemical synthetic annealed to form double-stranded DNA fragments, to connect to through the Hind Ⅲ / Bgl Ⅱ digestion of linearized pSUPER-neo-EGFP/H1 (pSNE/H1) plasmid to form a recombinant vector pSNE-ILK, transformed into E. coli DH5α , extract plasmid Hind Ⅲ / EcoR Ⅰ pairs of restriction enzyme digestion and sequencing, stable transfection of colon cancer cells HT29. [Results] ① RT-PCR detection, ILK gene in three kinds of colon cancer cell lines were expressed, ILK / β-actin electrophoretic bands intensity ratio: SW480 to 0.63, HT29 is 0.65, LO-VO 0.33. ② successfully constructed for the ILK gene-specific RNA interference vector pSNE-ILK and control of the unrelated gene vector pSNE-Control, and stable transfection of colon cancer cell line HT-29. [Conclusion] successfully constructed for the ILK gene-specific RNAi vector and stably transfected HT29 cells, can effectively inhibit the ILK gene, for the next step to explore the process of ILK gene in colon cancer and colon cancer in the role of laying the foundation for gene therapy . Keywords: RNA interference vector RT-PCR integrin-linked kinase Expression of ILK mRNA in Colon Carcinoma Cell Lines and Construction of ILK-RNAi Stable Expressing Vector Abstract: [Purpose] To investigate the expression of ILK mRNA in the colon carcinoma cell line and to construct novel ILK-RNAi (RNA interference) vector. [Methods] The expression of ILK mRNA was detected by RT-PCR in thre