Construction Of ShRNA Expression Vector Targeting MSTN Gene And Its Interference In Vivo

Myostatin(MSTN),also known as myostatin and growth differentiation factor 8(GDF-8),belongs to the TGF-superfamily.Its function is mainly as a negative regulator of skeletal muscle growth and limiting muscle hyperdevelopment.The deletion or mutation of this gene causes the proliferation and hypertrophy of muscle cells and lead to double muscled trait in cattle and sheep.In recent years,studies have found that MSTN not only regulates muscle growth and development,but also affects fat formation and bone development.MSTN also plays a key role in muscle homeostasis and is related to insulin sensitivity.The in-depth study of the MSTN gene is of great significance in the field of animal husbandry and medicine.Therefore,the relationship between the MSTN gene and various diseases and its role in body metabolism have received widespread attention.RNA interference(RNAi)refers to a phenomenon that is highly conserved during the evolution process,and is caused by double-stranded RNA(ds RNA),and the homogeneous m RNA is efficiently and specifically degraded.This technology can easily,quickly,efficiently,and specifically suppress gene expression,so it has attracted the attention of researchers in various fields.In this article,p SIREN-MSTN-sh RNA expression vector targeting MSTN gene will be constructed and transfected into mice by using different in-vivo transfection methods.The interference effect will be evaluated.All the results will lay a foundation for the subsequent research on the function of MSTN gene.Firstly,we designed si RNA based on the m RNA sequence of the MSTN gene.According to selected three efficient si RNAs,DNA sequence coding corresponding sh RNAs,were systhesized and inserted into the plasmid p SIREN.Then the p SIREN-MSTN-sh RNA expression vector was delivered to mice by intramuscular injection.Real-time PCR and Western blot were used to detect the m RNA and protein expression levels of MSTN in mousemuscle tissue after transient interference.The sequencing results showed that the p SIREN-MSTN-sh RNA expression vector was successfully constructed.All three recombinant plasmids can significantly reduce the m RNA and protein levels of MSTN in muscle tissue.Among the three MSTN RNAi plasmids,p SIREN-M664 and p SIREN-M819 expression vectors have better interference effect than the other one.Compared with the control group,MSTN m RNA expression in experimental groups was reduced by 65% and71% respectively,and the protein expression levels were also significantly reduced.Therefore,in this article,we will use the intramuscular injection method,PEI/sh RNA complex and liposome-mediated transfection method to introduce the interference plasmid(p SIREN-M664 and p SIREN-M819)into mice to study its interference effect.The results show that the intramuscular injection of mice in vivo interference experiment has the best inhibition effect.When the recombinant plasmid was introduced into mice by intramuscular injection,each mouse was injected with 100 ?g of plasmid each time,once a week for a total of 4 injections.Three days after the last injection,the mice were killed by neck dissection,weighed and dissected.The results showed that compared with the p SIREN-NC control group,the body weight of mice treated with p SIREN-M664 and p SIREN-M819 interference plasmid increased by 8.75% and 4.38%,respectively.Western blot analysis of MSTN protein expression in mouse muscle tissue after interference showed that the interference plasmids p SIREN-M664 and p SIREN-M819 can significantly inhibit the expression of MSTN protein in mice.HE staining results of muscle tissue sections showed thickening of muscle fibers.Compared with the control group,the muscle fiber area of p SIREN-M664 treated group increased by 25%.In summary,this study successfully constructed the p SIREN-MSTN-sh RNA expression vector targeting the MSTN gene,and obtained the interference plasmid p SIREN-M664,which has a good inhibitory effect in mice.It lays a foundation for the subsequent regulation ofMSTN gene expression by RNAi technology to improve and treat MSTN-related diseases.