Little attention has been paid to find the precise location of transgene integration in the chicken (Gallus gallus domesticus) genome in order to achieve a reliable and persistent expression over time. Identification and characterization of “Genomic Safe Harbor” (GSH) loci could be a major area of interest within the field of transgenic technology. A GSH locus that allows the persistent and reliable expression of a knocked-in gene without epigenetic silencing and disruption of the functions of internal genes would be very important and central to the development of bioreactor transgenic livestock. In this study, using bioinformatics, gene expression atlas, and Hi-C data, GSH regions including cROSA-like and cHIPP-like were predicted in the chicken genome. Then, we used CRISPR/Cas9 technology to integrate the EGFP transgene under the control of CMV or delta CMV promoter in the predicted GSH regions as well as OVA as a non-GSH locus of the chicken somatic cells. In contrast to our expectation, the transgene expression under the control of a CMV promoter in a non-GSH locus outperformed the one integrated into the GSH locus. This result suggested that when the expression of a transgene is controlled by a CMV promoter may act independently from its chromosomal position effects. Replacing the CMV promoter with delta CMV altered the expression level in favor of GSH loci. To decipher the behavior of GSH loci, isogenous clones harboring delta CMV promoter-driven EGFP were isolated. The results revealed that bi-allelic expression levels of EGFP controlled by delta CMV promoter were significantly higher in GSH loci than those of the non-GSH locus. Also, we found that terminally-differentiated somatic cells that have integrated the transgene cassette in the GSH region can express EGFP for more than 6 months. In conclusion, we found that the cROSA-like and cHIPP-like loci as GSH regions could be reliably exploited for consistent production of recombinant proteins in the future.

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