[Construction of recombinant adenovirus vector for RNA interference with multidrug resistance MDR1 gene].
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%.


CONCLUSIONS
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.
Chen
Lei
L
Department of Neurology, West China Hospital, Sichuan University, Chengdu 610041, China.
Feng
Pei-min
PM
Yang
Tian-hua
TH
Tian
Lin-yu
LY
Cheng
Xin-wang
XW
Zhou
Dong
D
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics*; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001253:Astrocytes; D002478:Cells, Cultured; D004827:Epilepsy / Q000628:therapy; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000096:biosynthesis*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D034741:RNA, Small Interfering