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

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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.

OriginalsprogEngelsk
Artikelnummer3024
TidsskriftCancers
Vol/bind13
Nummer12
ISSN2072-6694
DOI
StatusUdgivet - jun. 2021

Bibliografisk note

Funding Information:
Funding: This research was funded by AUFF NOVA (AUFF-E-2015-FLS-9-8), the Danish Cancer Society (R146-A9394), Ellegaard Göttingen Minipigs Research Foundation grants, Aage og Johanne Louis-Hansen’s fund, Fabricant Einar Willumsens memorial fund, Director Emil C. Hertz and wife Inger Hertz’s fund, Helge Peetz and Verner and wife Vilma Peetz fund, Karen Elise Jensen’s fund and from The Aarhus University Research Foundation, Denmark. MFB was co-funded by The Graduate School of Health, Aarhus University.

Publisher Copyright:
© 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Copyright:
Copyright 2021 Elsevier B.V., All rights reserved.

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