[Construction and identification of the lentiviral vector of repressor element-1/neuron-restrictive silencer element double-stranded RNA].
OBJECTIVE
To construct a lentiviral vector of repressor element-1/neuron-restrictive silencer element (RE-1/NRSE) double-stranded RNA (dsRNA).


METHODS
The 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.


RESULTS
PCR 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.


CONCLUSIONS
The lentivirus vector of RE-1/NRSE dsRNA is successfully constructed.
Li
Hong-tu
HT
Department of Developmental Biology, China Medical University, Shenyang 110001, China.
Liu
Xiao-yu
XY
Pang
Xi-ning
XN
D000818:Animals; D001854:Bone Marrow Cells; D002478:Cells, Cultured; D005822:Genetic Vectors; D016086:Lentivirus / Q000235:genetics*; D059630:Mesenchymal Stem Cells; D010957:Plasmids / Q000235:genetics; D012330:RNA, Double-Stranded / Q000235:genetics*; D051381:Rats; D012097:Repressor Proteins / Q000235:genetics*; D037941:Silencer Elements, Transcriptional / Q000235:genetics; D014162:Transfection; D000099229:RE1-Silencing Transcription Factor
D012330:RNA, Double-Stranded; D012097:Repressor Proteins; D000099229:RE1-Silencing Transcription Factor