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[Construction of recombinant lentiviral vectors for mouse NMNAT1 gene expression and its interference RNA]
Hong Zhao1, Zi-chao Yang, Jing-yu Zhang
1Department of Neurology, The Fourth Hospital, Harbin Medical University, Harbin 150001, China.
Objective:
To construct two recombinant lentiviral vectors carrying mouse NMNAT1 gene and RNAi targeting NMNAT1.
Methods:
According to GenBank, the full-length cDNA sequence of mouse NMNAT1, an interfering sequence targeting NMNAT1 and a negative sequence were designed, synthesized and inserted into plasmid pLenti6 lentiviral vector. The viral stock was prepared by cotransfection of plasmids and the packaging plasmid mix to 293T cells. The virus titer was tested by qPCR methods. After infection of Hela cells with these lentiviruses, the expression of NMNAT1 was detected by qPCR and Western blot.
Results:
All the recombinant plasmids were confirmed by sequencing. The titer of virus was over 2 X10(8) TU/mL. Hela cells infected with lentiviral vector carrying full length NMNAT1 gene successfully expressed high-level NMNAT1. The expression of NMNAT1 reduced to less than 30% after delivery of lentiviral vector carrying RNAi sequence.
Conclusion:
The lentiviral vectors carrying full length NMNAT1 gene and RNAi sequence targeting NMNAT1 have been successfully constructed.
