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

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The CRISPR/Cas9 Minipig—A Transgenic Minipig to Produce Specific Mutations in Designated Tissues. / Berthelsen, Martin Fogtmann; Riedel, Maria; Cai, Huiqiang et al.

I: Cancers, Bind 13, Nr. 12, 3024, 06.2021.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

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@article{f1f00d4102094a5692cea2bd8dd947ac,
title = "The CRISPR/Cas9 Minipig—A Transgenic Minipig to Produce Specific Mutations in Designated Tissues",
abstract = "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 G{\"o}ttingen 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.",
keywords = "Animal models, Cancer, CRISPR, G{\"o}ttingen minipigs, Handmade cloning, KRAS, Lung cancer, Porcine model, STK11, TP53",
author = "Berthelsen, {Martin Fogtmann} and Maria Riedel and Huiqiang Cai and Skaarup, {S{\o}ren H.} and Alstrup, {Aage K.O.} and Frederik Dagn{\ae}s-Hansen and Yonglun Luo and Jensen, {Uffe B.} and Henrik Hager and Ying Liu and Henrik Callesen and Vendelbo, {Mikkel H.} and Jakobsen, {Jannik E.} and Thomsen, {Martin Kristian}",
note = "Publisher Copyright: {\textcopyright} 2021 by the authors. Licensee MDPI, Basel, Switzerland. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.",
year = "2021",
month = jun,
doi = "10.3390/cancers13123024",
language = "English",
volume = "13",
journal = "Cancers",
issn = "2072-6694",
publisher = "M D P I AG",
number = "12",

}

RIS

TY - JOUR

T1 - The CRISPR/Cas9 Minipig—A Transgenic Minipig to Produce Specific Mutations in Designated Tissues

AU - Berthelsen, Martin Fogtmann

AU - Riedel, Maria

AU - Cai, Huiqiang

AU - Skaarup, Søren H.

AU - Alstrup, Aage K.O.

AU - Dagnæs-Hansen, Frederik

AU - Luo, Yonglun

AU - Jensen, Uffe B.

AU - Hager, Henrik

AU - Liu, Ying

AU - Callesen, Henrik

AU - Vendelbo, Mikkel H.

AU - Jakobsen, Jannik E.

AU - Thomsen, Martin Kristian

N1 - Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.

PY - 2021/6

Y1 - 2021/6

N2 - 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.

AB - 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.

KW - Animal models

KW - Cancer

KW - CRISPR

KW - Göttingen minipigs

KW - Handmade cloning

KW - KRAS

KW - Lung cancer

KW - Porcine model

KW - STK11

KW - TP53

UR - http://www.scopus.com/inward/record.url?scp=85107897352&partnerID=8YFLogxK

U2 - 10.3390/cancers13123024

DO - 10.3390/cancers13123024

M3 - Journal article

C2 - 34208747

AN - SCOPUS:85107897352

VL - 13

JO - Cancers

JF - Cancers

SN - 2072-6694

IS - 12

M1 - 3024

ER -