[Construction and identification of the eukaryotic expression vector expressing short hairpin RNA targeting human COL1A1].

OBJECTIVE: To construct the eukaryotic expression vector of short hairpin RNA (shRNA) targeting human collagen type 1 alpha 1 (COL1A1) and observe its inhibiting effect on the expression of target gene. METHODS: The complementary oligonucleotide sequences coding shRNA were designed and synthesized according to the sequence of human COL1A1 gene, and cloned into the linearized pSilencer(TM);2.1-U6 neo vector. The recombinant vector was confirmed by enzyme digestion analysis and DNA sequencing, and then the positive clones were transfected to human breast cancer cell MDA-MB-231 by Lipofectamine(TM);2000. The stable cell line was selected by G418. The expression of COL1A1 gene was detected by semi-quantitative RT-PCR and Western blot analysis. RESULTS: Double-enzyme digestion and DNA sequencing verified the correct sequences of the recombinant plasmid pshRNA-COL1A1. Compared with the control group, the expression level of COL1A1 mRNA and protein was inhibited markedly by pshRNA-COL1A1-1 or pshRNA-COL1A1-2 transfection, and the inhibitory rates were respectively (44.41±3.90)%, (63.05±3.13)% in RT-PCR and (45.50±2.71)%, (66.98±2.08)% in Western blot analysis. CONCLUSION: Specific shRNA interference plasmid vector targeting COL1A1 gene mRNA was constructed successfully.