Construction of lentiviral vector carrying Rab9 gene and its expression in mouse brain

doi:10.1007/s11684-009-0041-6

Front Med Chin

2009, Vol. 3

Issue (2) : 141-147 https://doi.org/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 Author(s): ZHANG Min,Email:zhang-min-3464@yahoo.com.cn

Issue Date: 05 June 2009

Cite this article:

Youguo HAO,Min ZHANG,Jinzhi XU, et al. Construction of lentiviral vector carrying Rab9 gene and its expression in mouse brain[J]. Front Med Chin, 2009, 3(2): 141-147.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-009-0041-6
https://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).

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/sj.gt.3301119
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

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