Construction Of SiRNA Lentiviral Lectors Specific To Human CCL20 Gene And The Preliminary Research Of Its Infection To The Human Keratinocyte Cell Line

Objectives1. To design the small interference RNA (siRNA) specific to human CCL20 gene by RNA interfering technique, construct its recombinant lentiviral expression vectors, and identify these vectors by DNA sequencing.2. The packaging cells 293FT were transduced by recombinant lentiviral expression vectors which having been successfully constructed. The relevant supernatant was collected and concentrated and titrated.3. The human keratinocyte cell line HaCaT cells were infected by lentivral particles and were observed preliminarily.Methods1. According to the Tuschl's principle, the siRNA was designed and converted into cDNA of shRNA (small hairpin RNA) of siRNA specific to human CCL20 gene. The cDNA was synthesized and inserted into plasmid pHSER-dsRNA-GFP-SIN which was linearized by restriction endonucleases Spe I and Sal I. The recombinant plasmid was transformed into competent E. coli. DH5αcells. The positive recombinant colony was selected by ampicillin medium agar and identified by DNA sequencing.2. The packaging cells 293FT were transduced by jetPEI and the mixture of the recombinant lentiviral expression vector pHSER-CCL20-siRNA-SIN,the packaging plasmid Lentipack and the envelop plasmid Lentienv in a certain proportion. A few days later, the lentiviral particles in the 293FT media supernatant were collected and concentrated by centrifuging.3. Hela cells were infected by the concentrated lentiviral particles. The GFP was expressed in the infected cells. The lentiviral titer was analyzed by flow cytometry and and calculated by using the formula.4. HaCaT cells were infected by concentrated lentiviral particles and observed...