Construction and identification of the lentiviral vector of repressor element-1/neuron-restrictive silencer element double-stranded RNA.
- Author:
Hong-tu LI
1
;
Xiao-yu LIU
;
Xi-ning PANG
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Bone Marrow Cells; Cells, Cultured; Genetic Vectors; Lentivirus; genetics; Mesenchymal Stromal Cells; Plasmids; genetics; RNA, Double-Stranded; genetics; Rats; Repressor Proteins; genetics; Silencer Elements, Transcriptional; genetics; Transfection
- From: Acta Academiae Medicinae Sinicae 2009;31(6):760-764
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct a lentiviral vector of repressor element-1/neuron-restrictive silencer element (RE-1/NRSE) double-stranded RNA (dsRNA).
METHODSThe RE-1/NRSE cDNA containing both sense and antisense oligo DNA fragments of the targeting sequence was synthesized and cloned into the pGC-LV vector. The obtained lentiviral vector containing RE-1/NRSE dsRNA was confirmed by PCR and sequencing. A total of 293T cells were cotransfected with lentiviral vector of L-smNRSE/RE-1, pHelper 1.0, and pHelper 2.0. The titer of virus was measured based on the expression level of green fluorescent protein. The transfection efficiency of green fluorescent protein into rat mesenchymal stem cells was calculated.
RESULTSPCR and DNA sequencing demonstrated that the constructed lentivirus vector of L-smNRSE/RE-1 produced RE-1/NRSE dsRNA.The titer of the concentrated virus was 4x108 TU/m1. The virus was stably transfected into rat mesenchymal stem cells, and the infection efficiency reached 100% when the multiplicity of infection was 80.
CONCLUSIONThe lentivirus vector of RE-1/NRSE dsRNA is successfully constructed.
