Nima Dehdilani; Mohsen Fathi Najafi; Hesam Dehghani
Abstract
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 ...
Read More
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 DRG1 and EIF4ENIF1 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.
Mohsen Naeemipour; Mohammadreza Bassami
Abstract
Nowadays, production of recombinant proteins in eukaryotes is gaining good deal of attention. Transgenic chicken as a eukaryotic system has a high potential for producing recombinant proteins. Post-translational changes, especially glycosylation, are characteristic of the eukaryotic proteins. In practice ...
Read More
Nowadays, production of recombinant proteins in eukaryotes is gaining good deal of attention. Transgenic chicken as a eukaryotic system has a high potential for producing recombinant proteins. Post-translational changes, especially glycosylation, are characteristic of the eukaryotic proteins. In practice we need to choose a proper expressing host when considering over-expression of a recombinant protein. Chickens are among the well-considered candidates for such application. Production of transgenic chickens could be achieved in different ways, including application of primordial germ cells. Primordial germ cells are progenitor of sperm and ovum. These cells are round, with a big nucleus and a cytoplasm with lipid and glycogen particles. The first step for having transgenic chickens is isolation and culture of the primordial gem cells. In the present study, these cells were isolated by centrifugation method in presence of ficoll and using magnetic cell sorting, and were cultured in optimal culture medium. These cells were finally characterized with defined methods, like Periodic acid-schiff staining, alkaline phosphates activity assessment, and antibody staining.
