Construction And Identification Of PIAS-1Gene Eukaryotic Expression Plasmid

Objective In this experiment, shRNA as a tool,pLKO.1as a viral-vector, build lentiviral vectors for human PIAS-1target genes, to intend to silence the PIAS-1in the cancer cell lines. We can study the expression of PIAS-1in the cervical cancer and the PIAS-1how it affects tumor.Methods For the gene sequence of PIAS-1in GenBank, according the siRNA design principles and the characteristics of the pLKO.1vector, design the upstream and downstream primers and synthesis by the company. Then anneale to form shRNA Oligos. Use double restriction enzyme enzyme digestion and purify the pLKO.1. Connect the shRNA Oligos and the digested plasmid with the ligase. Then the recombinant plasmid is transformed into the competent cells of E.coli DH5a. Positive clones are amplified. Extract the PIAS-1-pLKO.1-puro the recombinant plasmid. Identify the recombinant plasmid by double digestion and PCR. Save bacteria containing the recombinant plasmid. Bacteria containing the recombinant plasmid is sent to the sequencing company sequenced.Results In accordance with the extraction kit of instructions,we extract a small amount of plasmid plasmids pLKO.1. Target band appeares in the gel electrophoresis. Digest of the purified plasmid, two bands are apparent in the gel electrophoresis. Based on PIAS-1gene in the GenBank design upstream and downstream primers and are synthesized by the company. Then use the recombinant plasmid as a template, PCR verify recombinant plasmid. A DNA fragment7000bp is obtained by PCR and its sequence is in conformity with the sequence reported in GenBank. The PIAS-1gene is inserted into the lentiviral vector exactly.Conclusions Human PIAS-1gene is cloned correctly and the recombinant retroviral vector is constructed successfully, and all these lay a foundation for research correlation between expression of PIAS-1gene and chemosensitivity of cervical cancer cell line and itsfunctions in tumors.