Abstract
Inducible expression systems are indispensable for precise regulation and in-depth analysis of biological process. Binary Tet-On system has been widely employed to regulate transgenic expression by doxycycline. Previous pig models with tetracycline regulatory elements were generated through random integration. This process often resulted in uncertain expression and unpredictable phenotypes, thus hindering their applications. Here, by precise knock-in of binary Tet-On 3G elements into Rosa26 and Hipp11 locus, respectively, a double knock-in reporter pig model was generated. We characterized excellent properties of this system for controllable transgenic expression both in vitro and in vivo. Two attP sites were arranged to flank the tdTomato to switch reporter gene. Single or multiple gene replacement was efficiently and faithfully achieved in fetal fibroblasts and nuclear transfer embryos. To display the flexible application of this system, we generated a pig strain with Dox-inducing hKRASG12D expression through phiC31 integrase-mediated cassette exchange. After eight months of Dox administration, squamous cell carcinoma developed in the nose, mouth, and scrotum, which indicated this pig strain could serve as an ideal large animal model to study tumorigenesis. Overall, the established pig models with controllable and switchable transgene expression system will provide a facilitating platform for transgenic and biomedical research.
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Data availability
The transcriptome sequencing data reported in this study have been submitted to the NCBI Gene Expression Omnibus (GEO) and the accession number is GEO: GSE176519 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE176519). All other related data are represented within supporting data or available from the corresponding author upon reasonable request.
References
Bae, S., Park, J., and Kim, J.S. (2014). Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475.
Beard, C., Hochedlinger, K., Plath, K., Wutz, A., and Jaenisch, R. (2006). Efficient method to generate single-copy transgenic mice by site-specific integration in embryonic stem cells. Genesis 44, 23–28.
Belteki, G., Gertsenstein, M., Ow, D.W., and Nagy, A. (2003). Site-specific cassette exchange and germline transmission with mouse ES cells expressing φC31 integrase. Nat Biotechnol 21, 321–324.
Callesen, M.M., Árnadóttir, S.S., Lyskjaer, I., Ørntoft, M.B.W., Høyer, S., Dagnaes-Hansen, F., Liu, Y., Li, R., Callesen, H., Rasmussen, M.H., et al. (2017). A genetically inducible porcine model of intestinal cancer. Mol Oncol 11, 1616–1629.
Deng, W., Yang, D., Zhao, B., Ouyang, Z., Song, J., Fan, N., Liu, Z., Zhao, Y., Wu, Q., Nashun, B., et al. (2011). Use of the 2A peptide for generation of multi-transgenic pigs through a single round of nuclear transfer. PLoS ONE 6, e19986.
Flisikowska, T., Kind, A., and Schnieke, A. (2016). Pigs as models of human cancers. Theriogenology 86, 433–437.
Floyd, H.S., Farnsworth, C.L., Kock, N.D., Mizesko, M.C., Little, J.L., Dance, S.T., Everitt, J., Tichelaar, J., Whitsett, J.A., and Miller, M.S. (2005). Conditional expression of the mutant Ki-rasG12C allele results in formation of benign lung adenomas: development of a novel mouse lung tumor model. Carcinogenesis 26, 2196–2206.
Friedrich, G., and Soriano, P. (1991). Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev 5, 1513–1523.
Gao, X., Nowak-Imialek, M., Chen, X., Chen, D., Herrmann, D., Ruan, D., Chen, A.C.H., Eckersley-Maslin, M.A., Ahmad, S., Lee, Y.L., et al. (2019). Establishment of porcine and human expanded potential stem cells. Nat Cell Biol 21, 687–699.
Gossen, M., Freundlieb, S., Bender, G., Müller, G., Hillen, W., and Bujard, H. (1995). Transcriptional activation by tetracyclines in mammalian cells. Science 268, 1766–1769.
Groth, A.C., Olivares, E.C., Thyagarajan, B., and Calos, M.P. (2000). A phage integrase directs efficient site-specific integration in human cells. Proc Natl Acad Sci USA 97, 5995–6000.
Hippenmeyer, S., Youn, Y.H., Moon, H.M., Miyamichi, K., Zong, H., Wynshaw-Boris, A., and Luo, L. (2010). Genetic mosaic dissection of Lis1 and Ndel1 in neuronal migration. Neuron 68, 695–709.
Hutchinson, L., and Kirk, R. (2011). High drug attrition rates—where are we going wrong? Nat Rev Clin Oncol 8, 189–190.
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. Nat Biotechnol 25, 1477–1482.
Jackson, E.L., Willis, N., Mercer, K., Bronson, R.T., Crowley, D., Montoya, R., Jacks, T., and Tuveson, D.A. (2001). Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev 15, 3243–3248.
Jin, Y.X., Jeon, Y., Lee, S.H., Kwon, M.S., Kim, T., Cui, X.S., Hyun, S.H., and Kim, N.H. (2014). Production of pigs expressing a transgene under the control of a tetracycline-inducible system. PLoS ONE 9, e86146.
Johnson, L., Mercer, K., Greenbaum, D., Bronson, R.T., Crowley, D., Tuveson, D.A., and Jacks, T. (2001). Somatic activation of the K-ras oncogene causes early onset lung cancer in mice. Nature 410, 1111–1116.
Klymiuk, N., Böcker, W., Schönitzer, V., Bähr, A., Radic, T., Fröhlich, T., Wünsch, A., Keßler, B., Kurome, M., Schilling, E., et al. (2012). First inducible transgene expression in porcine large animal models. FASEB J 26, 1086–1099.
Kurachi, M., Ngiow, S.F., Kurachi, J., Chen, Z., and Wherry, E.J. (2019). Hidden caveat of inducible Cre recombinase. Immunity 51, 591–592.
Lai, L., Kolber-Simonds, D., Park, K.W., Cheong, H.T., Greenstein, J.L., Im, G.S., Samuel, M., Bonk, A., Rieke, A., Day, B.N., et al. (2002). Production of α-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 295, 1089–1092.
Lewandoski, M. (2001). Conditional control of gene expression in the mouse. Nat Rev Genet 2, 743–755.
Li, X., Yang, Y., Bu, L., Guo, X., Tang, C., Song, J., Fan, N., Zhao, B., Ouyang, Z., Liu, Z., et al. (2014). Rosa26-targeted swine models for stable gene over-expression and Cre-mediated lineage tracing. Cell Res 24, 501–504.
Lister, J.A. (2011). Use of phage PhiC31 integrase as a tool for zebrafish genome manipulation. In: Detrich, H.W., Westerfield, M., and Zon, L.I., eds. Methods in Cell Biology. Zebrafish: Genetics, Genomics and Informatics, 3rd Ed. New York: Academic Press. 195–208.
Lunney, J.K., Van Goor, A., Walker, K.E., Hailstock, T., Franklin, J., and Dai, C. (2021). Importance of the pig as a human biomedical model. Sci Transl Med 13, eabd5758.
Meurens, F., Summerfield, A., Nauwynck, H., Saif, L., and Gerdts, V. (2012). The pig: a model for human infectious diseases. Trends Microbiol 20, 50–57.
Plumb, S.J., Argenyi, Z.B., Stone, M.S., and De Young, B.R. (2004). Cytokeratin 5/6 immunostaining in cutaneous adnexal neoplasms and metastatic adenocarcinoma. Am J Dermatopathol 26, 447–451.
Ruan, J., Li, H., Xu, K., Wu, T., Wei, J., Zhou, R., Liu, Z., Mu, Y., Yang, S., Ouyang, H., et al. (2015). Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs. Sci Rep 5, 14253.
Schmidt-Supprian, M., and Rajewsky, K. (2007). Vagaries of conditional gene targeting. Nat Immunol 8, 665–668.
Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872.
Tasic, B., Hippenmeyer, S., Wang, C., Gamboa, M., Zong, H., Chen-Tsai, Y., and Luo, L. (2011). Site-specific integrase-mediated transgenesis in mice via pronuclear injection. Proc Natl Acad Sci USA 108, 7902–7907.
Turan, S., Zehe, C., Kuehle, J., Qiao, J., and Bode, J. (2013). Recombinase-mediated cassette exchange (RMCE)—a rapidly-expanding toolbox for targeted genomic modifications. Gene 515, 1–27.
Vatansever, S., Erman, B., and Gümüş, Z.H. (2019). Oncogenic G12D mutation alters local conformations and dynamics of K-Ras. Sci Rep 9, 11730.
Wang, G., Yang, H., Yan, S., Wang, C.E., Liu, X., Zhao, B., Ouyang, Z., Yin, P., Liu, Z., Zhao, Y., et al. (2015a). Cytoplasmic mislocalization of RNA splicing factors and aberrant neuronal gene splicing in TDP-43 transgenic pig brain. Mol Neurodegener 10, 42.
Wang, K., Jin, Q., Ruan, D., Yang, Y., Liu, Q., Wu, H., Zhou, Z., Ouyang, Z., Liu, Z., Zhao, Y., et al. (2017). Cre-dependent Cas9-expressing pigs enable efficient in vivo genome editing. Genome Res 27, 2061–2071.
Wang, Y., Yang, H.Q., Jiang, W., Fan, N.N., Zhao, B.T., Ou-Yang, Z., Liu, Z.M., Zhao, Y., Yang, D.S., Zhou, X.Y., et al. (2015b). Transgenic expression of human cytoxic T-lymphocyte associated antigen4-Immunoglobulin (hCTLA4Ig) by porcine skin for xenogeneic skin grafting. Transgenic Res 24, 199–211.
Whitworth, K.M., Cecil, R., Benne, J.A., Redel, B.K., Spate, L.D., Samuel, M.S., Prather, R.S., and Wells, K.D. (2018). Zygote injection of RNA encoding Cre recombinase results in efficient removal of LoxP flanked neomycin cassettes in pigs. Transgenic Res 27, 167–178.
Wu, H., Liu, Q., Shi, H., Xie, J., Zhang, Q., Ouyang, Z., Li, N., Yang, Y., Liu, Z., Zhao, Y., et al. (2018). Engineering CRISPR/Cpf1 with tRNA promotes genome editing capability in mammalian systems. Cell Mol Life Sci 75, 3593–3607.
Xie, J., Ge, W., Li, N., Liu, Q., Chen, F., Yang, X., Huang, X., Ouyang, Z., Zhang, Q., Zhao, Y., et al. (2019). Efficient base editing for multiple genes and loci in pigs using base editors. Nat Commun 10, 2852.
Yan, S., Tu, Z., Liu, Z., Fan, N., Yang, H., Yang, S., Yang, W., Zhao, Y., Ouyang, Z., Lai, C., et al. (2018). A Huntingtin knockin pig model recapitulates features of selective neurodegeneration in Huntington’s disease. Cell 173, 989–1002.e13.
Yang, D., Wang, C.E., Zhao, B., Li, W., Ouyang, Z., Liu, Z., Yang, H., Fan, P., O’Neill, A., Gu, W., et al. (2010). Expression of Huntington’s disease protein results in apoptotic neurons in the brains of cloned transgenic pigs. Hum Mol Genet 19, 3983–3994.
Yang, H., Wang, G., Sun, H., Shu, R., Liu, T., Wang, C.E., Liu, Z., Zhao, Y., Zhao, B., Ouyang, Z., et al. (2014). Species-dependent neuropathology in transgenic SOD1 pigs. Cell Res 24, 464–481.
Zhao, J., Lai, L., Ji, W., and Zhou, Q. (2019). Genome editing in large animals: current status and future prospects. Natl Sci Rev 6, 402–420.
Zheng, Q., Lin, J., Huang, J., Zhang, H., Zhang, R., Zhang, X., Cao, C., Hambly, C., Qin, G., Yao, J., et al. (2017). Reconstitution of UCP1 using CRISPR/Cas9 in the white adipose tissue of pigs decreases fat deposition and improves thermogenic capacity. Proc Natl Acad Sci USA 114, E9474–E9482.
Zhou, X., Li, G., Wang, D., Sun, X., and Li, X. (2019). Cytokeratin expression in epidermal stem cells in skin adnexal tumors. Oncol Lett 17, 927–932.
Acknowledgements
This work was supported by the National Key Research and Development Program of China (2017YFA0105103, 2021YFA0805903), the National Natural Science Foundation of China (81941004, 32170542), 2020 Research Program of Sanya Yazhou Bay Science and Technology City (202002011), Major Science and Technology Projects of Hainan Province (ZDKJ2021030), Key Research & Development Program of Hainan Province (ZDYF2021SHFZ052), Youth Innovation Promotion Association of the Chinese Academy of Sciences (2019347), Young Elite Scientist Sponsorship Program by CAST (YESS20200024), Biological Resources Progaramme, Chinese Academy of Sciences (KFJBRP-017-57), Key Research & Development Program of Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory) (2018GZR110104004), China Postdoctoral Science Foundation (2020M682943), Science and Technology Planning Project of Guangdong Province, China (2019A030317010, 2020B1212060052, 2021B1212040016, 2021A1515011110), Science and Technology Program of Guangzhou, China (202007030003) and Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019-I2M-5-025). We thank Du Wu and Yunpeng Ai for assistance with animal feeding and care. We thank Henan Chuangyuan Biotechnology Co. Ltd. for assistance with SCNT, animal feeding, and care.
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Jin, Q., Yang, X., Gou, S. et al. Double knock-in pig models with elements of binary Tet-On and phiC31 integrase systems for controllable and switchable gene expression. Sci. China Life Sci. 65, 2269–2286 (2022). https://doi.org/10.1007/s11427-021-2088-1
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DOI: https://doi.org/10.1007/s11427-021-2088-1