Construction and identification of RNAi leniviral vector targeting neuroD
10.3724/SP.J.1008.2013.01257
- Author:
Yang WANG
1
Author Information
1. Department of Pathology, Changhai Hospital, Second Military Medical University
- Publication Type:Journal Article
- Keywords:
Lentivirus;
NeuroD gene;
Pancreatic neoplasms;
RNA interference
- From:
Academic Journal of Second Military Medical University
2013;34(11):1257-1261
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct and identify RNAi lentiviral expression vectors targeting human neurogenic differentiation gene (NeuroD). Methods The oligo DNA sequences of 4 pairs of shRNA, named as NeuroDl, NeuroD2, NeuroD3, and NeuroD4, were designed according to NeuroD gene sequence (GenBank:NM_002500). The single strand of oligo DNA was annealed to form double strand DNA, and then was cloned into the empty plasmid pcDNA™ 6. 2-GW/EmGFP-miR. Four interference plasmids were constructed and transformed into competent cells DH5a. Interference carrier was transiently transfected into target cells and the interference effect against target genes was detected by real-time PCR. The interference plasmid pcDNA™ 6. 2-GW/EmGFP-miR-NeuroDl was linked to lentiviral destination vector pLenti6. 3/V5-DEST to form the lentiviral expression vector pLenti6. 3-EGFP-NeuroDl-miR. Constructed lentiviral vector carrier and packaging plasmids (Packaging Mix) were cotransfected into 293T cells, and followed by packaging virus, collecting the virus stock solution, ultra-centrifugating, condensing, and detecting the titer. PCR method was used to identify the recombinant vector; enhanced green fluorescent protein (EGFP) expression was used to determine the titer and the infection rate of the recombinant lentivirus under fluorescent microscope. Results Four interference plasmids for target gene were successfully constructed. The sequences of expression vector pcDNA™ 6. 2-GW/EmGFP-miR-NeuroDl/2/3/4 were proven correct using sequencing method. miR-NeuroDl sequence showed the best silencing effect after transfected into 293T cells (P<0. 01). Restriction endonuclease and PCR analysis confirmed that the pcDNA™ 6. 2-GW/EmGFP-miR-NeuroDl was successfully inserted into the lentivirus vector. The titer of the recombinant lentivirus harboring NeuroDl shRNA was 1. 18 X 108 ifu/mL. Conclusion The recombinant lentivirus pLenti6. 3-EGFP-NeuroDl-miR has been constructed successfully, which lays a found ation for future study of NeuroD function.
