Ferdowsi University of Mashhad

Document Type : Research Articles


1 Division of Biotechnology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran

2 Stem Cell Biology and Regenerative Medicine Research Group, Research Institute of Biotechnology, Ferdowsi University of Mashhad, Iran

3 Education and Extension Organization (AREEO), Agricultural Research, Razi Vaccine and Serum Research Institute, Mashhad Branch, Mashhad, Iran

4 Department of Basic Sciences, 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 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. 


Fishman, V., Battulin, N., Nuriddinov, M., Maslova, A., Zlotina, A., Strunov, A., et al. (2019) 3D organization of chicken genome demonstrates evolutionary conservation of topologically associated domains and highlights unique architecture of erythrocytes’ chromatin. Nucleic Acids Research 47:648–665.
Hilliard, W. and Lee, K.H. (2021) Systematic identification of safe harbor regions in the CHO genome through a comprehensive epigenome analysis. Biotechnology & Bioengineering 118:659–675.
Hippenmeyer, S., Youn, Y.H. Moon, H.M., Miyamichi, K. Zong, H., Wynshaw-Boris, A., et al. (2010). Genetic mosaic dissection of Lis1 and Ndel1 in neuronal migration. Neuron 68: 695–709.
Irion, S., Luche, H., Gadue, P., Fehling, H.J., Kennedy, M. and Keller, G. (2007) Identification and targeting of the ROSA26 locus in human embryonic stem cells. Nature Biotechnology 25: 1477–1482.
Kimura, Y., Hisano, Y., Kawahara, A. and Higashijima, S. (2014) Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering. Scientific Report 4:6545.
Kobayashi, T., Kato-Itoh, M., Yamaguchi, T., Tamura, C., Sanbo, M., Hirabayashi, M. et al. (2012). Identification of rat Rosa26 locus enables generation of knock-in rat lines ubiquitously expressing tdTomato. Stem Cells Development 21:2981–2986.
Lee, E.-S., Moon, S., Abu-Bonsrah, K.D., Kim, Y.K., Hwang, M.Y., Kim, Y.J. et al. (2019) Programmable nuclease-based integration into novel extragenic genomic safe harbor identified from Korean population-based CNV analysis. Molecular Therapy Oncolytics 14:253–265.
Li, X., Yang, Y., Bu, L., Guo, X., Tang, C., Song, J. et al. (2014) Rosa26-targeted swine models for stable gene over-expression and Cre-mediated lineage tracing. Cell Research 24:501–504.
Li, Y.-S., Meng, R.-R., Chen, X., Shang, C.-L., Li, H.-B., Zhang, T.-J., et al. (2019) Generation of H11-albumin-rtTA Transgenic Mice: A tool for inducible gene expression in the liver. G3. 9:591–599.
Liu, T., Hu, Y., Guo, S., Tan, L., Zhan, Y., Yang, L., et al. (2018) Identification and characterization of MYH9 locus for high efficient gene knock-in and stable expression in mouse embryonic stem cells. PLoS One 13 (2):e0192641.
Ma, L., Wang, Y., Wang, H., Hu, Y., Chen, J., Tan, T., et al. (2018) Screen and verification for transgene integration sites in pigs. Scientific Report 8:1–11.
Oleg E. Tolmachov, Subkhankulova, T. and Tolmachov, T. (2013) Silencing of transgene expression: a gene therapy perspective. Gene therapy tools and potential applications, francisco martin molina, intechopen, doi: 10.5772/53379. available from: https://www.intechopen.com/chapters/43164
Papapetrou, E.P., Lee, G., Malani, N., Setty, M., Riviere, I., Tirunagari, L.M.S., et al. (2011) Genomic safe harbors permit high β -globin transgene expression in thalassemia induced pluripotent stem cells. Nature Biotechnology 29: 73–78.
Pryzhkova, M. V., Xu, M.J. and Jordan, P.W. (2020) Adaptation of the AID system for stem cell and transgenic mouse research. Stem Cell Research. 49.
Rizzi, N., Rebecchi, M., Levandis, G., Ciana, P. and Maggi, A. (2017) Identification of novel loci for the generation of reporter mice. Nucleic Acids Research 45(6): 37.
Ruan, J., Li, H., Xu, K., Wu, T., Wei, J. and Zhou, R. (2015) Highly efficient CRISPR / Cas9- mediated transgene knockin at the H11 locus in pigs. Scientific Reports 5:1–10.
Sadelain, M., Papapetrou, E.P. and Bushman, F.D. (2011) Safe harbours for the integration of new DNA in the human genome. Nature Reviews Cancer 12:51–58.
Shin, S., Kim, S.H., Shin, S.W., Grav, L.M., Pedersen, L.E., Lee, J.S., et al. (2020) Comprehensive analysis of genomic safe harbors as target sites for stable expression of the heterologous gene in HEK293 cells. ACS Synthetic Biology 9:1263–1269.
Stanford, W.L., Cohn, J.B., Cordes, S.P. and Lunenfeld, S. (2001) Gene-trap mutagenesis: past, present and beyond. Nature reviews genetics 2:756–768.
Tasic, B., Hippenmeyer, S., Wang, C., Gamboa, M., Zong, H., Chen-Tsai, Y., et al. (2011) Site-specific integrase-mediated transgenesis in mice via pronuclear injection. Proceedings of the National Academy of Sciences 108: 7902–7907.
Voong, L.N., Xi, L., Sebeson, A.C., Xiong, B., Wang, J.-P. and Wang, X. (2016) Insights into nucleosome organization in mouse embryonic stem cells through chemical mapping. Cell 167:1555-1570.
Wu, M., Wei, C., Lian, Z., Liu, R., Zhu, C., Wang, H., et al. (2016) Rosa26 -targeted sheep gene knock-in via CRISPR-Cas9 system. Scientific Reports. 6:1–7.
Yang, D., Song, J., Zhang, J., Xu, J., Zhu, T. and Wang, Z. (2016) Identification and characterization of rabbit ROSA26 for gene knock-in and stable reporter gene expression. Scientific Reports 6:1–8.
Yang, D., Song, J., Zhang, J., Xu, J., Zhu, T., Wang, Z., et al. (2016) Identification and characterization of rabbit ROSA26 for gene knock-in and stable reporter gene expression. Scientific Reports 6: 251-61.
Zambrowicz, B.P., Imamoto, A., Fiering, S., Herzenberg, L.A., Kerr, W.G. and Soriano, P. (1997) Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells. Proceedings of the National Academy of Sciences 94: 3789–3794.
Zhao, Y., Wang, J., Liang, F., Liu, Y., Wang, Q., Zhang, H., et al. (2019) NucMap: a database of genome-wide nucleosome positioning map across species. Nucleic Acids Research. 47: 163–169.
Zhu, F., Gamboa, M., Farruggio, A.P., Hippenmeyer, S., Tasic, B., Schüle, B., et al. (2014) DICE, an efficient system for iterative genomic editing in human pluripotent stem cells. Nucleic Acids Research. 42(5):e34.