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Article

The CRISPR/Cas9 Minipig—A Transgenic Minipig to Produce Specific Mutations in Designated Tissues

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Department of Clinical Medicine, Aarhus University, 8200 Aarhus, Denmark
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Department of Respiratory Diseases and Allergy, Aarhus University Hospital, 8200 Aarhus, Denmark
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Department of Nuclear Medicine & PET, Aarhus University Hospital, 8200 Aarhus, Denmark
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Department of Biomedicine, Aarhus University, 8000 Aarhus, Denmark
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Lars Bolund Institute of Regenerative Medicine, BGI-Qingdao, BGI-Shenzhen, Shenzhen 518083, China
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South Denmark Hospital, 7100 Vejle, Denmark
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Department of Animal Science, Aarhus University, 8830 Tjele, Denmark
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Aarhus Institute of Advanced Studies (AIAS), Aarhus University, 8000 Aarhus, Denmark
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Author to whom correspondence should be addressed.
Academic Editor: Kris L. Vleminckx
Cancers 2021, 13(12), 3024; https://doi.org/10.3390/cancers13123024
Received: 14 May 2021 / Revised: 13 June 2021 / Accepted: 14 June 2021 / Published: 16 June 2021
(This article belongs to the Special Issue CRISPR-Mediated Cancer Modeling)
Research in large animal models has been hampered by the complexity to introduce new gene alterations, but this has been simplified by the discovery of the CRISPR/Cas system. Here, we have cloned a Cas9 minipig to generate a porcine model for pre-clinical research. Six viable piglets were produced and backcrossed to Göttingen minipigs for two generations. Primary cells from different organs were isolated, and multiple gene alterations were performed by CRISPR in vitro. In vivo activation of the Cas9 expression was conducted by viral delivery of the FlpO expression to the skin. Overall, we successfully cloned a Cas9-expressing minipig and confirmed gene alterations introduced by the CRISPR/Cas system to porcine cells.
The generation of large transgenic animals is impeded by complex cloning, long maturation and gastrulation times. An introduction of multiple gene alterations increases the complexity. We have cloned a transgenic Cas9 minipig to introduce multiple mutations by CRISPR in somatic cells. Transgenic Cas9 pigs were generated by somatic cell nuclear transfer and were backcrossed to Göttingen Minipigs for two generations. Cas9 expression was controlled by FlpO-mediated recombination and was visualized by translation from red to yellow fluorescent protein. In vitro analyses in primary fibroblasts, keratinocytes and lung epithelial cells confirmed the genetic alterations executed by the viral delivery of single guide RNAs (sgRNA) to the target cells. Moreover, multiple gene alterations could be introduced simultaneously in a cell by viral delivery of sgRNAs. Cells with loss of TP53, PTEN and gain-of-function mutation in KRASG12D showed increased proliferation, confirming a transformation of the primary cells. An in vivo activation of Cas9 expression could be induced by viral delivery to the skin. Overall, we have generated a minipig with conditional expression of Cas9, where multiple gene alterations can be introduced to somatic cells by viral delivery of sgRNA. The development of a transgenic Cas9 minipig facilitates the creation of complex pre-clinical models for cancer research. View Full-Text
Keywords: CRISPR; cancer; animal models; porcine model; lung cancer; handmade cloning; TP53; STK11; KRAS; Göttingen minipigs CRISPR; cancer; animal models; porcine model; lung cancer; handmade cloning; TP53; STK11; KRAS; Göttingen minipigs
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MDPI and ACS Style

Berthelsen, M.F.; Riedel, M.; Cai, H.; Skaarup, S.H.; Alstrup, A.K.O.; Dagnæs-Hansen, F.; Luo, Y.; Jensen, U.B.; Hager, H.; Liu, Y.; Callesen, H.; Vendelbo, M.H.; Jakobsen, J.E.; Thomsen, M.K. The CRISPR/Cas9 Minipig—A Transgenic Minipig to Produce Specific Mutations in Designated Tissues. Cancers 2021, 13, 3024. https://doi.org/10.3390/cancers13123024

AMA Style

Berthelsen MF, Riedel M, Cai H, Skaarup SH, Alstrup AKO, Dagnæs-Hansen F, Luo Y, Jensen UB, Hager H, Liu Y, Callesen H, Vendelbo MH, Jakobsen JE, Thomsen MK. The CRISPR/Cas9 Minipig—A Transgenic Minipig to Produce Specific Mutations in Designated Tissues. Cancers. 2021; 13(12):3024. https://doi.org/10.3390/cancers13123024

Chicago/Turabian Style

Berthelsen, Martin F., Maria Riedel, Huiqiang Cai, Søren H. Skaarup, Aage K.O. Alstrup, Frederik Dagnæs-Hansen, Yonglun Luo, Uffe B. Jensen, Henrik Hager, Ying Liu, Henrik Callesen, Mikkel H. Vendelbo, Jannik E. Jakobsen, and Martin K. Thomsen. 2021. "The CRISPR/Cas9 Minipig—A Transgenic Minipig to Produce Specific Mutations in Designated Tissues" Cancers 13, no. 12: 3024. https://doi.org/10.3390/cancers13123024

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