Impact Of TCEA3 On Bovine Skeletal Muscle Satellite Cell Differentiation

With the improvement of living standards,people are constantly improving the meat quality of livestock,gradually from the demand quantity into quality requirements,improve animal meat quality becomes the urgent problem of modern animal husbandry breeding.Beef with a bright red color,soft,juicy meat and other characteristics,has become one of the most commonly used meat in daily life.It has deeply satisfaction by Consumer become one of the most frequently purchased meat in daily life.The growth and development of sketetal muscle fiber has definitely pertinence with the meat quality,especially diameter of muscle fiber and density of muscle fiber.The article has been published from the laboratory,we founded that myogenin(MYOG),early growth response-1(EGR-1)expression increases significantly during the differentiation of Bovine muscle-derived satellite cells(MDSCs).It has an influence of the growth of bovine muscle fiber significantly.Research as follows:In our previous high-throughput sequencing analysis,we found that TCEA3 expression increases significantly during the differentiation of bovine MDSCs.In this study,the effect of transcription elongation factor A3(TCEA3)on bovine MDSC differentiation was investigated.(1)This study is in vitro cultured bovine MDSCs experimental material,induce cell differentiation 1 D,3 D,5 D and 7 D with 2% horse serum.Collect cells and culture solution,Western blotting,RT-PCR were used to detect changes in TCEA3 expression in bovine MDSCs at various stages of in vitro differentiation.Differentiated bovine MDSCs at day 0(0 D)were used as the blank control.(2)immunofluorescence assays and cytoplasmic and nuclear protein isolation and purification techniques were used to determine to the expression pattern and protein localization of TCEA3 in bovine MDSCs during in vitro differentiation.(3)TCEA3 expression was upregulated using the CRISPR/Cas9 technique to study its effects on MDSC differentiation in vitro.(4)After transfection with a TCEA3-activating plasmid vector(TCEA3 overexpression),a TCEA3-inhibiting plasmid vector for 24 hours,48 hours respectively.Using immunofluorescence to detect the myotube fusion rate,number of myotubes.Using Western blotting to detect the expression levels of the muscle differentiation-related loci myogenin(MYOG)and myosin heavy chain 3(MYH3).The results show that:(1)TCEA3 expression gradually increased during the in vitro differentiation of bovine MDSCs and peaked on the 5th day of differentiation by western blotting,RT-PCR.(2)TCEA3 was mainly localized in the cytoplasm of bovine MDSCs,and its expression was not detected in the nucleus.The level of TCEA3 was relatively higher in myotubes at a higher degree of differentiation than during early differentiation.(3)After transfection with a TCEA3-activating plasmid vector(TCEA3 overexpression)for 24 hours,the myotube fusion rate,number of myotubes,and expression levels of the MYOG and MYH3 were 1.58-fold?1.76-fold?1.9-fold?1.89-fold greater than those of the control group(p SPg RNA),respectively.After transfection with a TCEA3-activating plasmid vector(TCEA3 overexpression)for 48 hours were 1.8-fold?1.88-fold?1.4-fold?1.8-fold higher than those of the control group(p SPg RNA),respectively.(4)After transfection with a TCEA3-inhibiting plasmid vector for 24 hours,the myotube fusion rate,number of myotubes,and expression levels of MYOG and MYH3 were 0.58-fold?0.7-fold?0.5-fold?0.15-fold those of the control group(p SPg RNA),respectively.After transfection with a TCEA3-inhibiting plasmid vector for 48 hours,the myotube fusion rate,The number of myotubes,MYOG and MYH3 were 0.6-fold?0.5-fold?0.66-fold?0.54-fold those of the control group(p SPgRNA),respectively.Our results indicated,for the first time,that TCEA3 promotes the differentiation of bovine MDSCs and have implications for meat production and animal rearing.At the same time,it provides a strong basis for the research on the treatment of muscle wasting diseases.