Preliminary Study On The Preparation Of Myostatin(MSTN) Knockout Chicken By Direct Injection Of CRISPR/Cas9 Adenovirus

Myostatin(MSTN),also known as growth differentiation factor 8(GDF-8),is a member of the TGF-? superfamily.MSTN make a down regulation to muscle cells growth and MSTN gene mutation will result in animal double muscle effect which improve the domestic animal meat production.Since CRISPR/Cas9 is simple,low cost and high effectively,it has'been developed as a genetic modification technique in recent years.Direct in vivo injection of chicken embryos,which injects gene editing tools and delivery vectors(such as liposomes,viruses,etc.)into chicken embryos,directly transfects PGC in vivo to produce chimeric chicken embryos,and realizes the preparation of genetically edited chickens.In this study,the effects of different opening sites,sealing conditions and injection sites on the survival and hatchability of chicken embryos were studied.Adenovirus direct injection method and CRISPR/Cas9 technology were combined to select chicken MSTN gene as target site,and gRNA was designed to knock out the MSTN gene in chicken embryos,which laid the foundation for preparing chicken myostatin gene knockout by adenovirus direct injection.The effects of different opening methods,sealing conditions and injection location on the exchange rate and hatchability of chicken embryo village were studied.The transfection efficiency of GFP adenovirus in different locations of chicken embryos was investigated by equatorial opening,LSD-high elastic sealant sealing and peripheral intravenous injection.The results showed that eggs hatched with equatorial opening,sealant sealing and peripheral intravenous injection had a low impact on hatching rate,and the transfection efficiency of GFP virus in eggs met the requirements of subsequent experiments.The designed gRNA adenovirus and CRSIPR/Cas9 adenovirus were co-transfected into chicken embryonic fibroblasts,and the cleavage efficiency was detected by T7EI digestion.Then CRSIPR/Cas9 adenovirus and gRNA adenovirus were injected into chicken embryos using the optimized equatorial opening,LSD-high elastic sealant sealing and peripheral intravenous injection.After 12 days of incubation,gonadal DNA was extracted and the knockout efficiency of MSTN gene was detected by PCR.The results showed that adenovirus could be used as delivery vector to obtain efficient editing effect of CRISPR gene,and the chimeric chicken embryos with large fragment of MSTN gene could be obtained by direct injection of CRISPR adenovirus.