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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front Med Chin    2009, Vol. 3 Issue (2) : 141-147     DOI: 10.1007/s11684-009-0041-6
RESEARCH ARTICLE |
Construction of lentiviral vector carrying Rab9 gene and its expression in mouse brain
Youguo HAO, Min ZHANG(), Jinzhi XU, Bitao BU, Jiajun WEI
Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
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Abstract  

Rab proteins and their effectors facilitate vesicular transport by tethering donor vesicles to their respective target membranes. Rab9 mediates late endosome-to-trans-Golgi-network trafficking. To explore the possibility of Rab9-related gene therapy for neurodegenerative diseases, we packed Lentivirus encoding Rab9. The expressing plasmid pCDH1-MCF1-Rab9-EF1-copGFP was constructed by using molecular biological techniques. The Lentivirus encoding Rab9 cDNA was packed by Lifectamine-2000 mediated co-transfection of the plasmid pPACKH1-GAG, pPACKH1-REV and pVSV-G into 293T cells. DNA sequencing proved the successful construction of pCDH1-MCF1-Rab9-EF1-copGFP. After 72 hours, the expression of GFP could be detected in BV-2 cells. Western blotting revealed that the Rab9 gene expression in BALB/c mice brain was up-regulated significantly 4 weeks after injection with Lentivirus encoding Rab9, which evidenced a satisfactory increasing effect of this virus. Administration of Lenti-Rab9 to postnatal day 3 Niemann-Pick disease type C (NPC) mice reduced motor defects and prevented the weight loss associated with female NPC mice, as well as modulating the death rate of Purkinje neurons. It is concluded that the packaging of Lentivirus encoding Rab9 was successful. Lentivirus encoding Rab9 can increase the expression of Rab9 gene effectively, which might offer a novel means for the treatment of neurodegenerative diseases.

Keywords Rab9      lentivirus      gene therapy      gene transfer     
Corresponding Authors: ZHANG Min,Email:zhang-min-3464@yahoo.com.cn   
Issue Date: 05 June 2009
URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-009-0041-6     OR     http://academic.hep.com.cn/fmd/EN/Y2009/V3/I2/141
Fig.1  I and HI enzyme digestion of pCDH1-MCF1-Rab9-EF1-copGFP plasmid. 1: pCDH1-MCF1-Rab9-EF1-copGFP; 2 and 3: pCDH1-Rab9/I+HI; 3: D2000 marker.
Fig.2  Sequencing result of the inserted element Rab9 of the recombinant. Plasmid pCDH1-MCF1-Rab9-EF1-copGFP is displayed partly.
Fig.3  Expression of green fluorescence protein (GFP) in microglia cells infected by the recombinant lentivirus. (a) No GFP expression in the control group; (b) abundant expression of GFP in microglia cells 72 h after infection (×20).
Fig.4  Expression of GFP in BALB/c mice brain 4 weeks after injection of recombinant lentivirus. (a) No expression of GFP in saline injected group; obvious expression of GFP in pons (b) and cerebellum (c) in virus group (×40).
Fig.5  The recombinant lentivirus enhanced expression of Rab9 in mice brain. The Rab9 level was increased [(30.2±6.5)%, =0.021, =6] significantly in the virus group, compared with that of the control group. β-actin served as the endogenous reference of loading amount.
Fig.6  Lenti-Rab9 improved motor activity and inhibited weight loss in female NPC mice. The motor activity and body weight were compared among four groups. (a) Comparison of body weight among four groups of female BALB/c mice (=0.0227, =6); (b) comparison of body weight among male BALB/c mice of four groups (=0.6617, =6); (c) comparison of motor activity among four groups (=0.0215, =6).
Fig.7  Lenti-Rab9 effectively reduced Purkinje neuron loss in NPC mice. Mice treated with either Lenti-Rab9 or control virus were analyzed for the presence of Purkinje neurons. (a) Hematoxylin-eosin staining (HE staining) showing missing Purkinje neurons (arrows) in control virus group; (b) HE staining showing surviving Purkinje neurons (arrows) in Lenti-Rab9-treated mice; (c) comparison of surviving Purkinje neurons among three groups (=0.0325, =6) (×40).
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