Cloning And Expression Of FoxD3 CDNA From Xuefeng Black-bone Chicken And Construction Of FoxD3 Chimeric Chicken With Gene Editing

As the popular gene editing method,CRISPR/Cas9 system consisted of Cas9 protein and guide RNA(g RNA).At present,gene editing mediated by CRISPR/Cas9 has become one of the research hotspots in biological research and genetic manipulation,which owed to its advantages of simple designing,low price,high targeting to genes and high efficiency.Forkhead frame-transcription factor 3(FOXD3),a member of the forkhead frame-transcription factor family,occupies a central role in melanocyte fate determination.FoxD3 has been reported to regulate the expression of Mitf,Pax3 and other genes involving in melanin synthesis and migration.In this study,FoxD3 chimera chicken was constructed through gene editing,and the function of related genes was further studied.The main results are as follows:1.FoxD3 cDNA cloning and comparative expression array of skin and muscle in Xuefeng black-boned chickens.The cloned FoxD3 gene cDNA of Xuefeng black-bone chicken has a total length of 1197 bp.compared with the original FoxD3 sequence(NC＿006095.5)published by NCBI,the results show that there is no variation in FoxD3 gene cDNA of Xuefeng black-bone chicken.At m RNA level,FoxD3 expression in muscle was significantly higher than that in skin(P<0.05).2.Optimizing the way of windowing and sealing the fertilized eggs.The hatchability of the four kinds of window sealing methods,i.e.air chamber window medical glue sealing window,air chamber window thin tube sealing powder sealing window,equatorial window medical glue sealing window and equatorial window thin tube sealing powder sealing window,are 0%,2.5%,9% and 24% respectively.the equatorial window thin tube sealing powder sealing window is significantly better than the other three methods(P<0.01).3.Constructing Foxd3 chimeras via CRISPR/Cas9 technology.The knockout group produced 3 chicks,and two of them showed gene knockout phenotype during the later growth and development.After sequencing,it was confirmed that Fxo D3 was knocked out and Foxd3 chimera was successfully constructed.In total,FoxD3 of Xue-Feng black bone chicken was cloned and expressed in tissues in our study,furthermore,FoxD3 knockout was performed on fertilized eggs with equatorial fenestration tube and powder fenestration window,and chimera of gene knockout was obtained,which provided technical support for targeted gene knockout of CRISPR/Cas9.It provides technical support and data support for the follow-up study of Xue-Feng black bone chicken melanin-related genes and the cultivation of new strains.