Preliminary Study On Transgenic Chickens With Oviduct-specific Expression Of ?-IFN

Objective:With the continuous progress of transgenic technology,the research on transgenic animals has also made new progress.At present,the production capacity of using conventional genetically modified organisms(such as bacteria,insects,etc.)to produce pharmaceutical proteins in Glycosylation is greatly limited.The research on the production of pharmaceutical proteins in Glycosylation by using transgenic animal bioreactors has gradually become a research hotspot.Oviduct bioreactor in transgenic chicken have some advantages,such as shorter generation Period,lower feeding cost,faster reproduction and higher yield,making it a very ideal tool for producing pharmaceutical protein.The oviduct-specific expression of interest protein is finally located in egg white,with higher ratio of ovalbumin,whose components are simpler,being beneficial for the separation and purification of interest protein.Therefore,chicken oviduct bioreactor is promising for application in the field of biopharmaceuticals.The purpose of this study was to integrate the human interferon Gamma into the chicken genome by the means of the PiggyBac transposon system to generate transgenic chicken with oviduct-specific expression.Methods:(1)According to the previous research experience and the laboratory conditions,this study was firstly to establish a stable surrogate eggshell culture for chicken embryos,and optimize the amount ratio of transfection regent and plasmid DNA for subcavity injection of chicken embryonic disk;(2)transfecting HEK 293T cells and primary chicken oviduct epithelial cells respectively with pLenti6.4-cERE/cOVP-EGFP and pLenti6.4-CMV-EGFP vectors,which have been previously constructed in our laboratory,to ve:rify the activity of cERE/cOVP promoter;(3)Using pLenti6.4-CMV-y-hIFN,pLenti6.4-cERE/cOVP-EG FP and PUC57-P2A-RFP vectors(as templates,cERE/cOVP,y-hIFN and P2A-RFP fragments were respectively amplified by PCR,and y-hIFN-P2A-RFP fragments was formed by overlapping PCR,and then cERE/cOVP and y-hIFN-P2A-RPP fragments were respectively inserted between NheI/MfeI and AscI sites of the promoter-less PB002G transposon vector to construct recombinant plasmid PB002G-cERE/cOVP-y-hIFN-P2A-RFP for oviduct-specific expression.The y-hIFN-P2A-RFP expression cassette was subcloned between MfeI and AscI sites of the PB-dual-promotor transposon vector to construct the PB-Dual-CMV-y-hIFN-P2A-RFP vector.PB002G-cERE/cOVP-?-hIFN-P2A-RFP and PB-Dual-CMV-y-hIFN-P2A-RFP were respectively transfected HEK 293T cells and primary chicken oviduct epithelial cells to detect the expression of y-hIFN and RFP.(4)Using in vivo jetPEI transfection reagent,PB002G-cERE/cOVP-y-hIFN-P2A-RFP and PBase transposase plasmids were combined for injection to subgerminal cavity of chicken blastoderm.After 14 days of injection,chicken embryonic tissues and organs are taken to extract genomic DNA,and PCR was carried out to detect integration of?-hIFN and EGFP in the genome of the chicken embryos.Results:(1)The suitable amount of transfect regeant and plasmids for microinjection was 2.5ul.(2)After transfection of pLenti6.4-cERE/c OVP-EGFP,the cERE/cOVP promoter can successfully initiate EGFP expression in HEK 293T cells and chicken primary oviduct epithelial cells.(3)PB002G-cERE/cOVP-y-hIFN-P2A-RFP and PB-Dual-CMV-y-hIFN-P2A-RFP transposon vectors were successfully constructed and transfected into HEK 293T cells and chicken primary oviduct epithelial cells respectively.The expression of y-hIFN and RFP genes was detected by observing fluorescence expression with microscope,RT-PCR,immunofluorescence staining and Western Blot.(4)Amoung 45 injected embryos,17 embryos survived for 14 days,and the survival rate was 37.78%.Genomic DNA was extracted from the heart and gonad tissues of chicken embryos,and PCR detection indicated that the integration of y-hIFN gene was detected in embryonic heart tissues of 4 embryos and in gonad tissues of 2 embryos,the same result was found both in heart and gonad tissues of 1 embryo.This study preliminarily proved that transposon vector can mediate the integration of y-hIFN gene in chicken embryo,which laid a foundation for the study of transgenic chicken with oviduct-specific expression of y-hIFN mediated by PiggyBac transposon.