Construction And Efficiency Study On RNAi Binary Expression Vector Of MSTN

Myostatin, also called growth and diferentiation factor-8 (GDF-8), belongs to the transforming growth factor-β(TGF-β)super-family, was firstly cloned from skeletal muscle cDNA library in mice. Myostatin null mice and naturally myostatin mutant double-muscling cattles show that myostatin was an inhibitor of skeletal muscle growth.RNA interference(RNAi), a phenomenon of post-transcriptional gene silending (PTGS) triggered by dsRNA that can block the expression of corresponding gene. Now RNAi has been used as a new method to investigate gene function.Here the author constructed the chicken's myostatin gene RNAi binary vector pEGFP-N1-MSTN₁-MSTN₂, transfected it into myoblast that cultured in vitro then exerted the function that to block expression of MSTN gene in myoblast.In the chapter one the author compared the myostatin cDNA of Big-Bone chicken that had been cloned in our laboratory with the cDNA of myostatin in GenBank to find the homologous region of tnem through BLAST 2 SEQUENCE system firstly, then designed one pair of primers with the primer primer 5.0 system, synthesized two pairs of primers by TaKaRa Biotechnology (Dalian) Co., Ltd, which added different restrictive enzymes at 5'port. Two same 393bp cDNA fragment of myostatin gene was obtained through PCR, then carried on two fragments into TA clone vector and named two positive clones as pMD-MSTN-1 and pEGM-MSTN-2. Through sequence analysis with BLAST 2 SEQUENCE system separately, the obtained nucleotide sequence of myostatin cDNA was proved to be 100% homology with the big bone chicken equence of myostatin cDNA in GenBank. Digested the pMD-MSTN-1 and the middle carrier vector of pHANNIBAL with two corresponding restrictive enzymes and connected the objective nucleotide in pMD-MSTN-1 and pHANNIBAL, named the positive clone as pHAN-MSTN₁. Digested the pEGM-MSTN-2 and pHAN- MSTN₁ with the double restrictive enzymes and connected the objective nucleotide in pEGM-MSTN-2 with pHAN-MSTN₁, named positive clone as pHAN-MSTN₁-MSTN₂. Digested the expression vector of pEGFP-N1 and pHAN-MSTN₁-MSTN₂ with two corresponding restrictive enzyme, carried on the connection. After the list, named the recombinant plasmid that Digested by two corresponding restrictive enzymes and proved correct as pEGFP-N1-MSTN1-MSTN2. Finally construct myostatin gene RNAi binary vector successfully.In the chapter two, culture myoblast in leg muscles of 14 days Big-bone Chicken embryo in vitro. When the density of myoblast up to 10⁵ utilized lipidosome transfect the pEGFP-N1-MSTN1-MSTN2 to myoblast that cultured in vitro. Observed the hyperplasia, differentiation, fuse rate of myoblast and detected the quantity of myostatin protein expression that were transfected, we found that the number, fuse rate, hyperplasia, differentiation and protein quantity of myoblast were all increased.The result indicated that pEGFP-N1-MSTN1-MSTN2 can block expression of myostatin in myoblast and then promoted myoblast hyperplasia and differentiation.