OBJECTIVE: To construct the recombinant adenoviral RNA interference (RNAi) vector in order to inhibit the expression of multidrug resistance MDR1 gene, and probe whether gene therapy for multidrug resistance of epilepsy is feasibility. METHODS: Three target sequences for short hairpin RNA (shRNA) expression were selected and designed according to MDR1 gene sequence of rat. Annealed oligos were inserted into the downstream of treated pSIREN-shuttle U6 promoter to construct RNAi plasmid pSIREN-shuttle-MDR1. Next, MDR1 shRNA sequence was cloned to pAdeno-X, a transfer vector of adenovirus, to produce the pAdeno-MDR1, which was then packed and amplified in HEK293 cells. Further the recombinant adenovirus was purified by CsCl and used to infect the rat astrocytes with P170-glucoprotein (P-gp) over-expression which have been induced by coriaria lactone (CL). RESULTS: It was confirmed by restriction digestion, PCR and sequencing that MDR1 shRNA expression structure was correctly cloned to pSIREN-shuttle and pAdeno-X vector respectively. Virus titer was 6 x 10(9) pfu/mL. The interference efficiency of pAdeno-MDR1 to the expression drop of multidrug resistance gene in astrocyte model neared to 100%. CONCLUSION: RNAi adenovirus vector of rat MDR1 gene has been constructed and found its high interference efficiency. It is the essential building required for the remedy of refractory epilepsy and the research on mechanism of multidrug resistance.
OBJECTIVE: To construct the recombinant adenoviral RNA interference (RNAi) vector in order to inhibit the expression of multidrug resistance MDR1 gene, and probe whether gene therapy for multidrug resistance of epilepsy is feasibility. METHODS: Three target sequences for short hairpin RNA (shRNA) expression were selected and designed according to MDR1 gene sequence of rat. Annealed oligos were inserted into the downstream of treated pSIREN-shuttle U6 promoter to construct RNAi plasmid pSIREN-shuttle-MDR1. Next, MDR1 shRNA sequence was cloned to pAdeno-X, a transfer vector of adenovirus, to produce the pAdeno-MDR1, which was then packed and amplified in HEK293 cells. Further the recombinant adenovirus was purified by CsCl and used to infect the rat astrocytes with P170-glucoprotein (P-gp) over-expression which have been induced by coriaria lactone (CL). RESULTS: It was confirmed by restriction digestion, PCR and sequencing that MDR1 shRNA expression structure was correctly cloned to pSIREN-shuttle and pAdeno-X vector respectively. Virus titer was 6 x 10(9) pfu/mL. The interference efficiency of pAdeno-MDR1 to the expression drop of multidrug resistance gene in astrocyte model neared to 100%. CONCLUSION: RNAi adenovirus vector of ratMDR1 gene has been constructed and found its high interference efficiency. It is the essential building required for the remedy of refractory epilepsy and the research on mechanism of multidrug resistance.