Construction And Identification Of Lentivial RNA Interference Vector Of Sheep Myostatin Receptor Gene

Objective: To construct the lentiviral RNA interference (RNAi) vector of sheep myostatin gene and evaluate the effects of silencing myostatin gene expression by shRNA. Methods: Firstly retrieve purpose gene sequences in the Genebank to get myostatin sequence of sheep. Design and synthesize four pairs of oligonucleotide sequence target myostatin, and synthesize high-expression vector according to the ambion shRNA design system. Synthetic whole gene sequences, which is put into pMD-18T carrier. Find mutation points by sequencing, repair the mutating gene sequence through the overlapping PCR, then use XhoI and EcoRI enzyme cut the sequent, and connect to pCDNA3.1(+).high-expression vector is constructed by sequencing. Transfect shRNA and high-expression vector into HEK293cells, the gene silencing efficacy of the four targets was compared by real-time PCR, ShRNA - 86C's interference efficiency is best, gene silencing efficiency is 60.13%, ShRNA - 86C is screened and vector pDONR221 execute BP recombination reaction, so to obtain an entry vector containing interference sequence. Then the entry vector containing interference sequence and the pLenti6/BLOCK-iT-DEST vector execute LR recombination reaction to get the lentiviral expression vector with interference sequence. qPCR detection of the physical titer of the virus. Results: Successfully constructed Lentivector-mediated-shRNA, and the titer is 8.8×10~9TU/ml. Provide a stable infected cells vector to study the impact of myostatin gene on muscle growth.