Reserch And Optimization Of Transgenic Chicken Prepared By Direct Virus Injection

Since the emergence of transgenic animals,it has aroused widespread concern in the scientific community.With the development of science and medicine,transgenic animal bioreactors such as bovine mammary gland bioreactors have been further studied.Because of their unique reproduction and the stability of the egg environment,chickens This makes the transgenic chicken oviduct bioreactor an ideal bioreactor for drug protein production.In order to explore the method of transgenic chicken preparation,this study conducted a systematic study.The lentiviral vector microinjection method is the main method for preparing transgenic chickens.In order to explore the factors that affect the hatching of microinjected transgenic chickens and the effect of different opening positions on hatchability,blunt end openings and equatorial openings are adopted.The results showed that the hatching rate of the control group was 90.00%;the hatching rate of the blunt-end open group was 53.33%,and the hatching rate of the equatorial plane group was 80.00%;it can be seen that the hatching rate of the equatorial plane group was significantly higher than that of the blunt-end open group.In order to explore the influence of injection methods on the survival rate of chicken embryos and hatching rate of eggs,this experiment carried out a comparative experiment of subembryonic cavity and vascular injection.The results are as follows: the hatching rate of the control group was 93.33%;the incubation rate of the subembryonic injection group was 33.33%,blood vessels The incubation rate of the injection group was 53.33%.It can be seen that the incubation rate of the vascular injection group is significantly higher than that of the subembryonic cavity group.In this study,a lentiviral vector was designed,and transgenic chickens were prepared by microinjection.After blood collection,there were 10 positive chickens identified by Elisa,but the highest protein content was no more than 100ng/m L.In order to enable chickens to produce more proteins of interest and to achieve precise gene editing of chickens using viral vector methods,this experiment used adenovirus as a CRISPR/Cas9 delivery system to perform knock-in detection tests on chicken embryo oviduct cells and chicken embryo fibroblasts.Attempted to inject recombinant adenovirus directly into chicken embryos to produce target gene knockout chickens,establishing a chicken CRISPR/Cas9 recombinant adenovirus system.After extracting the genome of the cells transfected with adenovirus CRISPR/Cas9 for PCR amplification and digestion,the results showed that the targeting efficiency of adenovirus delivery CRISPR/Cas9 in CEF was 40.7%.Three donor adenoviruses(HDR,HMEJ and MMEJ)were designed to co-infect CEF with CRISPR/Cas9-tale adenovirus,and the knock-in efficiency in CEF was determined.The experiment found that HMEJ had the highest knock-in efficiency.The method of delivering the body to the insertion site to further improve the efficiency of KI,constructed a fusion protein of Cas9 and transcription activator-like effector(TALE),and amplified three kinds of HMEJ donors with TALE targeting sequences by PCR Different-HMEJ donors,namely 5’-BaitHMEJ,Inside-Bait-HMEJ,5’-Bait-HMEJ,found that Inside-Bait-HMEJ had the highest knock-in efficiency in chicken embryo fibroblasts by single cell PCR.Explored the knock-in efficiency of Cas-TALE+HMEJ,CasTALE+Inside-Bait-HMEJ adenovirus in fallopian tube cells,Western blot detection,the experiment showed that Cas-TALE+HMEJ,CasTALE+Inside-Bait-HMEJ can all Knock into the fallopian tube cells to produce the protein of interest.Elisa experiment showed that CasTALE+Inside-Bait-HMEJ has higher knock-in efficiency.In this experiment,transgenic chickens were successfully prepared by microinjection of blood vessels into the equatorial plane of the lentiviral vector.It was verified that adenovirus as a CRISPR/Cas9 delivery system can knock in chicken embryo fibroblasts and chicken oviduct cells,which provides future production of transgenic chickens.reference.