- 作者: Wen-Ji Dong a, Xiao-Bing Wu b, De-Pei Liu a, Jia Li a, Guang Liu a, Zhen-Xiang Zu a, Na Zhao a, Yun-De Hou b, Chih-Chuan Liang a
- 作者服務機構: a National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Peking Union Medical College and Chinese Academy of Medical Sciences, b National Laboratory of Molecular Virology and Genetic Engineering, Institute of Virology, Chinese Academy of Preventive Medicine, Beijing, China
- 中文摘要: --
- 英文摘要: detect the expression of human β-globin gene, respec-tively. Genomic DNA was extracted from spleen nodulesof BM reconstituted mice 12 days after transplantation.Human β-globin gene was detected in 1 out of 6 nodulesusing PCR combined with Southern blot. Human β-glo-bin gene was also detected in the BM and thymus ofmouse Y6161, in the thymus and spleen of mouse Y6162and in the BM of mice Y6211 and Y6212. RT-PCRrevealed low levels of expression of human β-globingene in the BM of mouse Y6211. Our results suggestedthat the efficiency of AAV-mediated human β-globingene integration into hematopoietic stem/progenitorcells was very low. It is necessary to perform furtherresearch on AAV biology before applying gene therapythat requires integration of a foreign gene into host chro-mosomes.Adeno-associated virus (AAV)-2 was developed as a use-ful vector for human gene therapy. In this report, we ana-lyzed the integration and expression of AAV-mediated exvivo transferred human β-globin gene in bone marrow(BM) reconstituted mice. Recombinant AAV (rAAV) con-taining human β-globin gene was packaged by infectingindividual G418-resistant BHK-21 cell clones integratedwith the plasmid AV53HS432 β2.ONeo with recombi-nant herpes simplex virus, which can express rep andcap genes of wild-type AAV. The titer of rAAV was deter-mined using slot blot hybridization with a result of 10virus particles/ml (genome copy number). Low-densitymononuclear cells were isolated from fetal livers of em-bryos from pregnant C57BL/6 mice at 14-16 days of ges-tation and were infected with rAAV. The transducedhematopoietic cells were then reinfused into lethally irra-diated C57BL/6 recipient mice via tail vein injection. Toanalyze the provirus in short-term and long-term BMreconstituted mice, PCR/Southern blot and RT-PCR wereperformed to identify the integrity of the provirus and to
- 中文關鍵字: --
- 英文關鍵字: B-Thalassemia, Gene therapy, Ex vivo gene transfer, Adeno-associated virus vector