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

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 cDNA. 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 h, 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 cDNA, 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 modulated the death rate of Purkinje neurons. It is concluded that the packaging of lentivirus encoding Rab9 cDNA was successful. lentivirus encoding Rab9 can increase the expression of Rab9 cDNA gene effectively, which might offer a novel means for the treatment of neurodegenerative diseases.