title: [Construction of recombinant adeno-associated virus vectors carrying double gene of antisense multidrug resistance-associated protein and antisense multidrug resistance].
abstract: 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.
authors.affiliation: Department of General Surgery, West China Hospital, Sichuan University, Chengdu 610041, China.
authors.forename: Ming-Man
authors.initials: MM
authors.lastname: Zhang
authors.forename: De-Hua
authors.initials: DH
authors.lastname: Li
authors.forename: Lü-Ian
authors.initials: LI
authors.lastname: Yan
authors.forename: Xing-Hua
authors.initials: XH
authors.lastname: Gou
authors.forename: Yong-Heng
authors.initials: YH
authors.lastname: Zhao
authors.forename: Zhi
authors.initials: Z
authors.lastname: Su
authors.forename: Ying-Chun
authors.initials: YC
authors.lastname: Huang
authors.forename: Lei
authors.initials: L
authors.lastname: Han
authors.forename: Lan-Ying
authors.initials: LY
authors.lastname: Zhao
authors.forename: Hai-Yang
authors.initials: HY
authors.lastname: Hu
mesh_terms: 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
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins