PMID- 12065900
OWN - NLM
STAT- MEDLINE
DCOM- 20030303
LR  - 20190607
IS  - 1021-7770 (Print)
IS  - 1021-7770 (Linking)
VI  - 9
IP  - 3
DP  - 2002 May-Jun
TI  - Analysis of adeno-associated virus-mediated ex vivo transferred human beta-globin 
      gene in bone marrow engrafted mice.
PG  - 253-60
AB  - Adeno-associated virus (AAV)-2 was developed as a useful vector for human gene 
      therapy. In this report, we analyzed the integration and expression of 
      AAV-mediated ex vivo transferred human beta-globin gene in bone marrow (BM) 
      reconstituted mice. Recombinant AAV (rAAV) containing human beta-globin gene was 
      packaged by infecting individual G418-resistant BHK-21 cell clones integrated 
      with the plasmid AV53HS432Deltabeta2.0Neo with recombinant herpes simplex virus, 
      which can express rep and cap genes of wild-type AAV. The titer of rAAV was 
      determined using slot blot hybridization with a result of 10(13) virus 
      particles/ml (genome copy number). Low-density mononuclear cells were isolated 
      from fetal livers of embryos from pregnant C57BL/6 mice at 14-16 days of 
      gestation and were infected with rAAV. The transduced hematopoietic cells were 
      then reinfused into lethally irradiated C57BL/6 recipient mice via tail vein 
      injection. To analyze the provirus in short-term and long-term BM reconstituted 
      mice, PCR/Southern blot and RT-PCR were performed to identify the integrity of 
      the provirus and to detect the expression of human beta-globin gene, 
      respectively. Genomic DNA was extracted from spleen nodules of BM reconstituted 
      mice 12 days after transplantation. Human beta-globin gene was detected in 1 out 
      of 6 nodules using PCR combined with Southern blot. Human beta-globin gene was 
      also detected in the BM and thymus of mouse Y6161, in the thymus and spleen of 
      mouse Y6162 and in the BM of mice Y6211 and Y6212. RT-PCR revealed low levels of 
      expression of human beta-globin gene in the BM of mouse Y6211. Our results 
      suggested that the efficiency of AAV-mediated human beta-globin gene integration 
      into hematopoietic stem/progenitor cells was very low. It is necessary to perform 
      further research on AAV biology before applying gene therapy that requires 
      integration of a foreign gene into host chromosomes.
CI  - Copyright 2002 National Science Council, ROC and S. Karger AG, Basel
FAU - Dong, Wen-Ji
AU  - Dong WJ
AD  - National Laboratory of Medical Molecular Biology, Institute of Basic Medical 
      Sciences, Peking Union Medical College and Chinese Academy of Medical Sciences, 
      Beijing, China.
FAU - Wu, Xiao-Bing
AU  - Wu XB
FAU - Liu, De-Pei
AU  - Liu DP
FAU - Li, Jia Li
AU  - Li JL
FAU - Liu, Guang
AU  - Liu G
FAU - Zu, Zhen-Xiang
AU  - Zu ZX
FAU - Zhao, Na
AU  - Zhao N
FAU - Hou, Yun-De
AU  - Hou YD
FAU - Liang, Chih-Chuan
AU  - Liang CC
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - J Biomed Sci
JT  - Journal of biomedical science
JID - 9421567
RN  - 9004-22-2 (Globins)
SB  - IM
MH  - Animals
MH  - *Bone Marrow Transplantation
MH  - Cell Line
MH  - Dependovirus/*genetics/metabolism
MH  - Embryo, Mammalian
MH  - Female
MH  - *Gene Transfer Techniques
MH  - Genetic Therapy
MH  - *Genetic Vectors
MH  - Globins/*genetics
MH  - Humans
MH  - Mice
MH  - Mice, Inbred C57BL
MH  - Pregnancy
EDAT- 2002/06/18 10:00
MHDA- 2003/03/04 04:00
CRDT- 2002/06/18 10:00
PHST- 2002/06/18 10:00 [pubmed]
PHST- 2003/03/04 04:00 [medline]
PHST- 2002/06/18 10:00 [entrez]
AID - 59426 [pii]
AID - 10.1007/BF02256072 [doi]
PST - ppublish
SO  - J Biomed Sci. 2002 May-Jun;9(3):253-60. doi: 10.1007/BF02256072.