Construction and identification of mouse eosinophils CCR3 gene RNA interference lentiviral vector / 中华耳鼻咽喉头颈外科杂志

ABSTRACT

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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, 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.</p><p><b>RESULTS</b>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 4×10(8) TU/ml. CCR3 interference rate was 86.7%.</p><p><b>CONCLUSION</b>A 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.</p>