Comparative Study On The Efficiency Of Adenovirus Vector And Lentivirus Vector In Gene Editing Chichen

Direct injection of chicken embryos using viral vectors is a common method for producing transgenic chickens,which has the advantages of simple operation and high efficiency.However,at present,this method cannot perform targeted modification of genes.This study compared the transfection efficiency of adenovirus vectors and lentiviral vectors delivering enhanced green fluorescent gene（e GFP）in chicken embryos,and combined the viral vectors with the CRISPR/Cas9 gene editing system to knock the e GFP gene into Chicken embryo.This study will lay the foundation for the development of direct injection of viral vectors to prepare gene-edited chickens.Blood vessel microinjection of 5μL adenovirus vector and lentiviral vector into HH14 stage chicken embryos,the survival of the chicken embryos and the viral vector transfection efficiency of chicken embryos were tested.The resμLts significantly reduced the survival rate of chicken embryos,while adenovirus development of chicken embryos;the transfection efficiency of virus vectors on various organs of chicken embryos was different,and it was more preferred to be expressed in the heart,small intestine and muscle of chicken embryos;The transfection efficiency of viral vectors to the heart and gonads of chicken embryos showed a significant titer dependence.The higher the virus titer,the higher the transfection efficiency to chicken embryos.According to the above research resμLts,an adenovirus vector with a titer of 1×10¹⁰TU/m L was selected as the delivery vector for CRISPR/Cas9 knock-in elements,and e GFP gene knock-in was performed on the ATP5E gene locus of chicken embryos.VascμLar microinjection of chicken embryos at the HH14stage to detect the effect of adenovirus injection on the hatchability of chicken embryos,and to detect e GFP in the gonad,heart,liver,spleen,lung,kidney,small intestine,stomach,and muscle of 6-month-old male.The resμLts showed that the microinjection operation significantly reduced the hatching rate of chicken embryos,but there was no significant difference between the injection of PBS m L.MμLtiple tissues of adμLt rooster injected with CRISPR/Cas9 adenovirus vector can express e GFP protein stably for a long time.The direct injection of CRISPR/Cas9 adenovirus vector can achieve e GFP knock-in at the chicken ATP5E site,and the knock-in genes can be continuously and stably expressed.The above experiments laid the foundation for the production of gene-edited chickens by direct injection of CRISPR/Cas9 adenovirus vector.