Construction and identification of mouse eosinophils CCR3 gene RNA interference lentiviral vector
10.3760/cma.j.issn.1673-0860.2013.04.012
- VernacularTitle:趋化因子受体3 RNA干扰慢病毒载体的构建及对嗜酸粒细胞作用的初步研究
- Author:
Xin-Hua ZHU
1
;
Bing LIAO
;
Xin-Yue WANG
;
Ke LIU
;
Yue-Hui LIU
Author Information
1. 330006,南昌大学第二附属医院耳鼻咽喉头颈外科
- Keywords:
RNA interference;
Receptors,CCR3;
Lentivirus;
Eosinophils
- From:
Chinese Journal of Otorhinolaryngology Head and Neck Surgery
2013;48(4):316-321
- CountryChina
- Language:Chinese
-
Abstract:
Objective Through construction of a lentiviral expression vector of chemokine receptor 3 (CCR3)RNA interference (RNAi) of mouse,to further study the function of CCR3 gene on eosinophils.Methods Focused on the CCR3 gene sequences,RNAi target sequences were designed,then the target sequences of Oligo DNA were synthesized and annealed to double stranded DNA,which was subsequently connected to pLVX-shRNA2-m vector digested by MluⅠ,Sac Ⅰ,EcoR Ⅰ,Hind m,BamH Ⅰ and Xho Ⅰ,short hairpin RNA lentiviral vectors were constructed.Short hairpin RNA lentiviral vectors were constructed.293T cells and eosinophils were transfected by shRNA lentiviral vector,and virus titer was determined.The expression of the CCR3 gene in eosinophils was identified by quantitative-PCR.Results The lentiviral vector of shRNA-mCCR3-oligonucleotide chain was inserted correctly.Infection efficiency of 293T cells observed under fluorescence microscope was more than 90％,the virus titer was 4 × 108TU/ml.CCR3 interference rate was 86.7％.Conclusion A lentiviral vector of CCR3-gene RNM was constructed successfully by the genetic engineering technology,and it provides a condition for further research in vitro and vivo.
