Nima Dehdilani - Division of Biotechnology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran
Mohsen Fathi Najafi - Education and Extension Organization (AREEO), Agricultural Research, Razi Vaccine and Serum Research Institute, Mashhad Branch, Mashhad, Iran
Hesam Dehghani - Division of Biotechnology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran

To achieve a reliable and persistent expression, the transgene should be precisely integrated into the genome safe harbor (GSH) loci. Little attention has been paid to find the safe harbor loci of the chicken (Gallus gallus domesticus) genome. Identification and characterization of GSH loci that allow the persistent and reliable expression of knock-in genes could be a major area of interest within the field of transgenic technology and is central to the development of transgenic livestock. Randomly integrated transgenes might encounter position effects and epigenetic silenc­ing, so unstable phenotypes, as well as unreliable and unpredictable expression of the knock-in transgene could occur. In contrast to random gene insertion, site-specific gene targeting provides a superior strategy that exploits homologous recombination to insert a transgene of interest into a pre-determined locus. In this study, based on bioinformatics, gene expression atlas, and Hi-C analyses, the GSH region was predicted in the chicken genome between DRG۱ and EIF۴ENIF۱  genes. To do so, we introduce a fast and easy-to-use pipeline that allows the prediction of orthologue GSH loci in all organisms, especially chickens. In addition, the procedure to design targeting vectors for targeting these predicted GSH regions is described in detail. 

Genome safe harbor loci, Genetically engineered birds, transgenic chicken, CRISPR/Cas۹, Gene expression atlas, Hi-C map