Please wait a minute...
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
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
Download: PDF(242 KB)   HTML
Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks

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,   
Issue Date: 05 June 2009
URL:     OR
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).
1 Bu B, Klunemann H, Suzuki K, Li J, Bird T, Jin LW, Vincent Niemann-Pick disease type C yields possible clue for why cerebellar neurons do not form neurofibrillary tangles. Neurobiol Dis , 2002, 11(2): 285-297
doi: 10.1006/nbdi.2002.0551
2 Zhou L, Miller B L, McDaniel C H, Kelly L, Kim O J, Miller C A. Frontotemporal dementia: neuropil spheroids and presynaptic terminal degeneration. Ann Neurol , 1998, 44(3): 99-109
doi: 10.1002/ana.410440116
3 Loftus S K, Morris J A, Carstea E D, Gu J Z, Cummings C, Brown A, Ellison J, Ohno K, Rosenfeld M A, Tagle D A, Pentchev P G, Pavan W J. Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene. Science , 1997, 277(3): 232-235
doi: 10.1126/science.277.5323.232
4 Mukherjee S, Maxfield F R. Lipid and cholesterol trafficking in NPC. Biochim Biophys Acta , 2004, 1685(2): 28-37
5 Sturley S L, Patterson M C, Balch W, Liscum L. The pathophysiology and mechanisms of NPC disease. Biochim Biophys Acta , 2004, 1685(1-3): 83-87
6 Chang T Y, Reid P C, Sugii S, Ohgami N, Cruz J C, Chang C C. Niemann-Pick Type C Disease and Intracellular Cholesterol Trafficking. J Biol Chem , 2005, 280(1): 20917-20920
doi: 10.1074/jbc.R400040200
7 Underwood K W, Jacobs N L, Howley A, Liscum L. Evidence for a cholesterol transport pathway from lysosomes to endoplasmic reticulum that is independent of the plasma membrane. J Biol Chem , 1998, 273(7): 4266-4274
doi: 10.1074/jbc.273.7.4266
8 Wojtanik K M, Liscum L. The transport of low density lipoprotein-derived cholesterol to the plasma membrane is defective in NPC1 cells. J Biol Chem , 2003, 278(5): 14850-14856
doi: 10.1074/jbc.M300488200
9 Ganley I G, Pfeffer S R. Cholesterol accumulation sequesters Rab9 and disrupts late endosome function in NPC1-deficient cells. J Biol Chem , 2006, 281(26): 17890-17899
doi: 10.1074/jbc.M601679200
10 Narita K, Choudhury A, Dobrenis K, Sharma D K, Holicky E L, Marks D L, Walkley S U, Pagano R E. Protein transduction of Rab9 in Niemann-Pick C cells reduces cholesterol storage. FASEB J , 2005, 19(11): 1558-1560
11 Walter M, Davies J P, Ioannou Y A. Telomerase immortalization upregulates Rab9 expression and restores LDL cholesterol egress from Niemann-Pick C1 late endosomes. J Lipid Res , 2003, 44(11): 243-253
doi: 10.1194/jlr.M200230-JLR200
12 Englund U, Ericson C, Rosenblad C, Mandel RJ, Trono D, Wictorin K, Lundberg C. The use of a recombinant lentiviral vector for ex vivo gene transfer into the rat CNS. Neuroreport , 2000, 11(18): 3973-3977
doi: 10.1097/00001756-200012180-00014
13 Miyoshi H, Bl?mer U, Takahashi M, Gage FH, Verma I M. Development of a self-inactivating lentivirus vector. J Virol , 1998, 72(10): 8150-8157
14 Kafri T, van Praag H, Gage F H, Verma I M. Lentiviral vectors: regulated gene expression. Mol Ther , 2000, 1(6): 516-521
doi: 10.1006/mthe.2000.0083
15 Zhang M, Li J, Chakrabarty P, Bu B, Vincent I. Cyclin-dependent kinase inhibitors attenuate protein hyperphosphorylation, cytoskeletal lesion formation, and motor defects in Niemann-Pick type C mice. Am J Pathol , 2004, 165(10): 843-853
16 Voikar V, Rauvala H, Ikonen E. Cognitive deficit and development of motor impairment in a mouse model of Niemann-Pick type C disease. Behav Brain Res , 2002, 132(9): 1-10
doi: 10.1016/S0166-4328(01)00380-1
17 Riederer M A, Soldati T, Shapiro A D, Lin J, Pfeffer S R. Lysosome biogenesis requires Rab9 function and receptor recycling from endosomes to the trans-Golgi network. J Cell Biol , 1994, 125(12): 573-582
doi: 10.1083/jcb.125.3.573
18 Choudhury A, Dominguez M, Puri V, Sharma D K, Narita K, Wheatley C L, Marks D L, Pagano R E. Rab proteins mediate Golgi transport of caveolainternalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells. J Clin Invest , 2002, 109(5): 1541-1550
19 Costantini L C, Bakowska J C, Breakefield X O, Isacson O. Gene therapy in the CNS. Gene Ther , 2000, 7(2): 93-109
doi: 10.1038/
20 De Rijck J, Vandekerckhove L, Christ F. Lentiviral nuclear import: a complex interplay between virus and host. Bioessays , 2007, 29(5): 441-451
doi: 10.1002/bies.20561
21 Heiser W C. Delivery of DNA to cells in culture using particle bombardment. Methods Mol Biol , 2004, 245(23): 175-184
22 Naldini L, Bl?mer U, Gage F H, Trono D, Verma I M. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci U S A , 1996, 93(21): 11382-11388
doi: 10.1073/pnas.93.21.11382
23 Naldini L, Bl?mer U, Gallay P, Ory D, Mulligan R, Gage F H, Verma I M, Trono D. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science , 1996, 272(5259): 263-267
doi: 10.1126/science.272.5259.263
[1] Wei Lu,Qingzhang Zhou,Hao Yang,Hao Wang,Yexing Gu,Qi Shen,Jinglun Xue,Xiaoyan Dong,Jinzhong Chen. Gene therapy for hemophilia B mice with scAAV8-LP1-hFIX[J]. Front. Med., 2016, 10(2): 212-218.
[2] Ru Zhang,Qiang Wang,Lin Zhang,Saijuan Chen. Optimized human factor IX expression cassettes for hepatic-directed gene therapy of hemophilia B[J]. Front. Med., 2015, 9(1): 90-99.
[3] Chuanfeng Wu, Cynthia E. Dunbar. Stem cell gene therapy: the risks of insertional mutagenesis and approaches to minimize genotoxicity[J]. Front Med, 2011, 5(4): 356-371.
[4] Haiyan LU, Yongting WANG, Falei YUAN, Jianrong LIU, Lili ZENG, Guo-Yuan YANG. Overexpression of netrin-1 improves neurological outcomes in mice following transient middle cerebral artery occlusion[J]. Front Med, 2011, 5(1): 86-93.
[5] Jian XIN BM, Ze-Feng XIA MD, Kai-Xiong TAO MD, Kai-Lin CAI PhD, Gao-Xiong HAN MD, Xiao-Ming SHUAI MD, Ji-Liang WANG MD, Han-Song DU MD, Guo-Bin WANG PhD, Yan LUO MM, . Development of a magnetite-gene complex for gene transfection[J]. Front. Med., 2010, 4(2): 241-246.
[6] Xudong YU MM, Zengwu SHAO MD, Liming XIONG MD, Weiwei XU MM, Hezhong WANG MM, Huifa XU MM, . Adenovirus-mediated tissue inhibitor of metalloproteinase-3 gene transfection inhibits rabbit intervertebral disc degeneration in vivo[J]. Front. Med., 2009, 3(4): 415-420.
[7] Zhengjuan LIU, Jie BIAN, Yuchuan WANG, Yongli ZHAO, Dong YAN, Xiaoxia WANG. Construction and identification of lentiviral RNA interference vector of rat leptin receptor gene[J]. Front Med Chin, 2009, 3(1): 57-60.
[8] XIA Xi, WANG Beibei, CAO Li, CHEN Gang, WU Peng, LU Yunping, ZHOU Jianfeng, MA Ding. Investigation of gene therapy of denovirus in immune suppression[J]. Front. Med., 2008, 2(4): 386-390.
[9] XIONG Ying, GUO Wen, LI Ting, LI Ke. Influence of Survivin-targeted siRNA on the biological features of colorectal carcinoma cells[J]. Front. Med., 2007, 1(3): 304-307.
[10] TIAN Yongji, LI Guilin, GAO Jun, WANG Renzhi, KONG Yanguo, ZHANG Zhenxing, LI Shifang, TIAN Shiqiang, DOU Wanchen, ZHANG Bo. Construction of 6HRE-GFAP-Baxα system specific for glioma gene therapy[J]. Front. Med., 2007, 1(1): 49-53.
Full text