Constructing miR-100-3p inhibitor lentiviral silencing vector and its stable expression of mouse embryonic fibroblast cells
10.13699/j.cnki.1001-6821.2017.10.016
- VernacularTitle:人miR-100-3p抑制序列慢病毒载体及其稳定表达的小鼠胚胎成纤维细胞的建立
- Author:
Qi-Ping ZHU
1
;
Qiang GUO
;
Rui-Qing DENG
;
Wei DONG
;
Hai-Feng REN
;
Fang NI
;
Si-Ying WANG
Author Information
1. 安徽医科大学基础医学院病理生理学教研室
- Keywords:
lentivirus;
mouse embryonic fibroblast cells;
cancer
- From:
The Chinese Journal of Clinical Pharmacology
2017;33(10):925-928
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish the mouse embryonic fibroblast (MEF) cells lines with stable expression of MiR-100-3p inhibitor,which lays a foundation for its function research of the role of miR-100 in the interaction of environmental pollutants and Src homology 2 domain tyrosine phosphatases (SHP-2) gene mutation.Methods Designed and synthesized a particular DNA fragment as hsa-MiR-100-3p inhibitor,which was then cloned into the hU6-MCS-Ubiquitin-ECFP -IRES-puromycin vector.This vector was then co-transfected with lentiviral packaging vectors pHelper1.0 and pHelper2.0 into 293T to produce lentiviral particles.Finally,MEF cells were infected with the above-mentioned lentiviral particles and the stable cell lines expressing hsa-miR-100-3p inhibitor were selected by puromycin.The expression of green fluorescent protein (GFP) and the expression of miR-100 were detected which indicated its inhibition effect of lentiviral vector in MEF cells.Results The construction of the recombinant plasmid was confirmed by sequencing.The subsequent infection confirmed that the packaging of lentiviruses was successfully.MEF cells successfully infected with hsa-miR-100-3p inhibitor.Flow cytometry indicates the expression of green fluorescent protein (significantly inhibition the expression of miR-100) has reached 97.4％ and 95.1％,as well as proved by RT-PCR.Conclusion We successfully constructed the has-miR-100-3p lentiviral silencing vector,and established the stable inhibition cell lines which lay a foundation for its function research of miR-100.
