Construction and identification of mouse eosinophils CCR3 gene RNA interference lentiviral vector.
- Author:
Xin-hua ZHU
1
;
Bing LIAO
;
Xin-yue WANG
;
Ke LIU
;
Yue-hui LIU
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Base Sequence; DNA; Eosinophils; metabolism; Genetic Vectors; Lentivirus; genetics; Mice; Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Receptors, CCR3; genetics; metabolism; Transfection
- From: Chinese Journal of Otorhinolaryngology Head and Neck Surgery 2013;48(4):316-321
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVEThrough 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.
METHODSFocused 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, SacI, EcoRI, HindIII, BamHI 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.
RESULTSThe 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 4×10(8) TU/ml. CCR3 interference rate was 86.7%.
CONCLUSIONA lentiviral vector of CCR3-gene RNAi was constructed successfully by the genetic engineering technology, and it provides a condition for further research in vitro and vivo.
