[Construction of recombinant adeno-associated virus vectors carrying double gene of antisense multidrug resistance-associated protein and antisense multidrug resistance].
OBJECTIVE
To construct a recombinant adeno-associated virus vectors carrying double gene of antisense multidrug resistance-associated protein (MRP) and antisense multidrug resistance (MDR1) for use in studying the gene therapy to reverse the multidrug resistance (MDR) in hepatocellular carcinoma (HCC).


METHODS
The 500 bp fragment (mrp) of MRP cDNA 5' region and the 600 bp fragment (mdr1) of MDR1 cDNA 5' region were amplified through polymerase chain reaction (PCR), and then they were linked to a combined gene fragment (mrp+mdrl) by overlapping technique. The combined gene fragment(mrp+mdrl) was cloned reversely into the multiple cloning site (MCS) of the expression plasmid pAAV-IRES-hrGFP in AAV Helper-Free System to construct the recombinant expression plasmid pAAV-IRES-hrGFP-(mrp + mdr1)AS. The packaging cell line (HEK 293 cell) was co-transfected with the pAAV-IRES-hrGFP-(mrp+mdr1)AS together with the control plasmid pAAV-RC and pHelper in AAV Helper-Free System by means of lipofectamine. The recombinant adeno-associated virus vector : rAAV2-(mrp+mdr1)AS carrying the double gene of antisense multidrug resistance-associated protein (MRP)and antisense multidrug resistance (MDR1) was packaged. Then the viral titer was checked by GFP.


RESULTS
The recombinant adeno-associated virus vector : rAAV2-(mrp + mdr1)AS carrying antisense MRP and antisense MDR1 was constructed successfully, the strong green fluorescence was observed in HEK 293 cells under a fluorescence microscope. The viral titer was 2.5 X 10(6) efu/ml.


CONCLUSIONS
The rAAV2-(mrp+mdr1)AS thus constructed could introduce the antisense MRP and antisense MDR1 into the human drug-resistant hepatocellular cell line effectively, which might provide a sound basis for the mechanisms and reversal methods of the multidrug resistance in HCC.
Zhang
Ming-Man
MM
Department of General Surgery, West China Hospital, Sichuan University, Chengdu 610041, China.
Li
De-Hua
DH
Yan
Lü-Ian
LI
Gou
Xing-Hua
XH
Zhao
Yong-Heng
YH
Su
Zhi
Z
Huang
Ying-Chun
YC
Han
Lei
L
Zhao
Lan-Ying
LY
Hu
Hai-Yang
HY
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000235:genetics; D045744:Cell Line, Tumor; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy / Q000235:genetics*; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000096:biosynthesis / Q000235:genetics*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins