Construct Of RNA Interference Lentiviral Vectors Of Bovine Myostatin Gene And Study On Transfection Of Bovine Fetal Fibroblasts

Myostatin, also known as growth differentiation factor-8, is a member of the TGF-βSuper-family. Myostatin has a negative regulatory role on skeletal muscle growth, development, differentiation and can inhibit cell proliferation and differentiation, regulate the muscle weight, and inhibit intramuscular fat deposition. The loss of MSTN activity will lead to hypertrophic animal muscle tissue, more muscle fibers, larger the diameter and less subcutaneous fat. A large number of studies have shown, the animals with MSTN mutations have widespread skeletal muscle and improved meat quality. Inhibiting the expression of MSTN gene in vivo can improve muscle atrophy and provide new means for the treatments of muscular dystrophy or other muscle wasting diseases.RNA silencing or interference (RNAi) is a remarkable type of gene regulation based on sequence-specific targeting and degradation of RNA. The RNAi encompasses related pathways founding abroad range of eukaryotic organisms, including RNA interference of animal, quelling of fungi, posttranscriptional gene silencing and cosuppression of genes in plants, etc. It has become the expression and regulation of gene function and effective way. RNAi is a technology that can efficiently and specifically reduce the expression of target genes and on study of gene function and gene therapy it has broad application prospects.In this research, two kinds of bovine MSTN gene RNA interference vectors, transient interference vectors and lentiviral vectors were constructed and we detected MSTN mRNA expression levels of bovine fetal fibroblast cells by real-time quantitative PCR. It was the first application of our lab that using the technology of lentiviral vector mediated RNA interference to silence specifically MSTN gene of expression in bovine fetal fibroblast cells. The cell line of MSTN gene knock-out stably was selected then. It was analyzed of MSTN gene expression between transient interference and stability disturbance bovine fetal fibroblast cells. It was laid a good foundation on transgenic cattle production, and also carrying out in cattle in vivo the research of biological function of MSTN gene and the inhibitory effect of MSTN after the lifting of animal muscle tissue growth and differentiation and development. The research covers the following two main components:1. Construction and identification of transient expression vectors4 pairs of siRNA targeting bovine MSTN mRNA sequence were designed and chemically synthesized. Transient expression vectors of MSTN RNAi were obtained after siRNA oligos connecting with the linear vector pSIREN successfully. By restriction enzymes digestion and sequencing, it was confirmed that the fragment has been inserted into the vector correctly. They were named pshRNA-1、pshRNA-2、pshRNA-3 and pshRNA-4. Study the effects of expression vectors on MSTN mRNA expression by transient transfection into bovine fetal fibroblast cells in vitro.The results show that in the transfected bovine fetal fibroblast cells after 48h, the levels of MSTN gene mRNA were significantly decreased, indicating that the designed siRNA sequences having a certain inhibitory effect on MSTN gene. By real-time quantitative PCR experiments detection, the expression levels of MSTN mRNA were 57%、69%、50%�'�25%. The most efficacious vector is pshRNA-2.2. Construction and identification of MSTN RNAi lentiviral vectorsDouble-stranded oligos shRNA targeting MSTN mRNA was inserted into linear pENT/U6 in order to generate the entry vector. After the LR recombination reaction between entry vectors and pLenti6/BLOCK-iT-DEST, the pU6/RNAi expression vectors were constructed. Lentiviral stock delivering MSTN shRNA was generated by co-transfection the optimized Virapower Packaging Mix with pU6/RNAi expression vector into 293FT producer cell line. This MSTN RNAi lentiviral stock was further concentrated by ultra-centrifugation and titration. The bovine fetal fibroblast cells with stable expressing MSTN shRNA were created by Blasticidin resistant selection after lentivirus infection. The effects of gene silencing in MSTN knockdown cells were studied by real time PCR.The results showed that MSTN RNAi lentiviral titer was further rasied by ultra-filtration and ultra-centrifugation and the final titer could achieve to107TU/ml. Using Blasticidin selection, the MSTN RNAi cell line was generated. MSTN mRNA levels were significantly reduced by transient transfection with MSTN RNAi lentiviral in bovine fetal fibroblast cells using real time PCR analysis. the inhibition rates of MSTN mRNA were 60%、77%、55% and 39%. These above results demonstrate MSTN RNAi lentiviral to be a beneficial application for further muscle gene therapy and signal transduction research of TGF-(3 superfamily. A new method is created by generating transgenic bovine by infecting fertilized embryos and zygote with MSTN RNAi lentivirus. The purpose of this research is to establish a lentiviral-mediated system with RNA interference to functionally silence MSTN in bovine fetal fibroblast cells and to use this system to generate a transgenic bovine.