Establishment Of A Transgenic Chimeric Chicken Model And Resistance To Newcastle Disease Virus Research

Transgenic animal models are widely used and often used in animal breeding,gene function identification,and bioreactors.Due to the particular features of poultry embryonic development,the development of poultry transgenes has lagged behind mammals.At present,the main methods for developing transgenic poultry are embryonic stem cell,primordial germ cell,sperm carrier,etc.Their operation is complicated,more technically difficulty and low efficiency rate.Therefore,this study uses a Replication Competent ALV LTR with a Splice Acceptor?RCAS?vector system,which is simple to operate and has a high efficiency rate.RCAS is a retrovirus vector derived from Rous sarcoma virus and has full replication capabilities.Using the RCAS system,the virus with an enhanced green fluorescent protein?EGFP?reporter gene was packaged,and the chimeric transgenic chickens were prepared by optimizing the transduction approaches.Besides,an adeno-associated virus type 2/9?AAV2/9?system was used to prepare an overexpressed EGFP chicken model that was comparable for the transgenic efficiency.Finally,the RCAS system respectively carried two potential genes HN and FAM that may have resistance to the Newcastle disease virus?NDV?.HN is an envelope protein of NDV.FAM is a mouse protein identified by our laboratory that may involve in mitosis.Subsequently,the virus challenge experiments were conducted on the transgenic chimeric chickens.This study provided a method for the functional verification of antiviral genes in animal organisms,and provided a technical platform for the basic research of antiviral transgenic chickens.The specific research content and results are as follows:1.Construction of RCASBP?A?-EGFP expression systemThe EGFP gene fragment was inserted into specific restriction enzyme site Cla I of RCABP?A?vector to construct RCASBP?A?-EGFP.The virus was packaged by transferring vector into DF-1 cell.Fluorescence observation and Western blot were used to detect the efficient expression of EGFP in DF-1 cells.The virus was amplified using DF-1cells and the viral titer was measured as 1.275×10⁸TU/m L?Transducing units/m L?.2.Establishment of an EGFP chimeric chicken model?1?The transduction conditions of chicken embryo age and virus dosage were optimized.While the yolk sac of 8-day-old chicken embryos was injected with RCASBP?A?virus at 10⁶TU,the hatching rate of chicken embryos is highest.?2?The RCASBP?A?-EGFP virus was injected into the yolk sac of 8-day-old chicken embryo at 10⁶TU/egg.The tissue sections were used to detect protein expression by Western blot.The results showed that EGFP was strongly expressed in the heart,liver,kidney,gland stomach,small intestine and trachea,while the spleen,lung,muscular stomach,and bursa weakly expressed EGFP.Shows that the EGFP transgenic chimeric chickens were successfully established.?3?The RCASBP?A?-EGFP virus was injected into the 2-day-old chicks via intravenous injection at2×10⁷TU/bird.The EGFP transgenic chimeric chickens was successfully established.The expression of EGFP at 28dpipi?day post injection?is better than that at 14dpipi.In the chickens at 28dpipi,EGFP was expressed in liver,lung,kidney,muscle,glandular stomach,lymph,the trachea and testis.The liver and glandular stomach more strongly expressed EGFP.?4?AAV2/9-EGFP virus was intravenously injected with 10¹¹vg/bird?Vector genomes/bird?in the 2-day-old chicks.The EGFP overexpressed chicken model was successfully established.The expression of EGFP at 28dpipi is higher than that of the14-day-old chickens.In the chickens at 28dpipi,EGFP was mainly expressed in the heart,liver,lung,kidney,brain,muscle and trachea.3.Study of the transgenic chimeric chickens against NDVThe RCASBP?A?-HN and RCASBP?A?-FAM expression systems were constructed.The stably expressing HN and FAM DF-1 cell lines were respectively constructed by using two systems that showed a certain anti-viral efficency.The viral titers were reduced 10 to100 folds.The yolk sac of 8-day-old chicken embryos was injected with 10⁶TU/egg.The transgenic chimeric chickens were successfully hatched.The HN expressed in muscle and FAM expressed in lung and muscle were detected by Western blot.The genes HN and FAM was detected in the heart,lung,kidney,muscle and other tissues by RT-PCR.Using the virulent strain F48E9 challenging the transgenic chickens,the results showed that compared with the control group,the HN and FAM transgenic chickens had no difference or only a short delay in mortality.The viral titers were slightly decreased in the brain,liver,kidney and glandular stomach of both HN and FAM trsnagenic chickens.In summary,the EGFP transgenic chimeric and overexpressed chicken models were established by using the RCAS system and AAV2/9-EGFP,respectively.By optimizing the transduction conditions,we found that the RCASBP?A?-EGFP virus injection via the yolk sac of 8-day-old chicken embryos was the highest efficiency of transduction and hatch.This roptimal approach was used to prepare HN and FAM transgenic chimeric chickens.In the virus challenge experiments,the HN and FAM transgenic chickens did not showed obvious antiviral effects that might be due to the low expression of HN and FAM in tissues or the themself limited effect of the protein against NDV.This study provided a technical basis for the expression of foreign proteins in vivo and antiviral transgenic chickens.