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

Martin Fogtmann Berthelsen; 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; Martin Kristian Thomsen

doi:10.3390/cancers13123024

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

Martin Fogtmann Berthelsen, 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, Martin Kristian Thomsen^*

^*Corresponding author af dette arbejde

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avis › Tidsskriftartikel › Forskning › peer review

12 Citationer (Scopus)

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 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 KRAS^G12D 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.

Originalsprog	Engelsk
Artikelnummer	3024
Tidsskrift	Cancers
Vol/bind	13
Nummer	12
ISSN	2072-6694
DOI	https://doi.org/10.3390/cancers13123024
Status	Udgivet - jun. 2021

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10.3390/cancers13123024Licens: CC BY

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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. (2021). The CRISPR/Cas9 Minipig—A Transgenic Minipig to Produce Specific Mutations in Designated Tissues. Cancers, 13(12), Artikel 3024. https://doi.org/10.3390/cancers13123024

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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}",

year = "2021",

month = jun,

doi = "10.3390/cancers13123024",

language = "English",

volume = "13",

journal = "Cancers",

issn = "2072-6694",

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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

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

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DO - 10.3390/cancers13123024

M3 - Journal article

C2 - 34208747

AN - SCOPUS:85107897352

SN - 2072-6694

VL - 13

JO - Cancers

JF - Cancers

IS - 12

M1 - 3024

ER -

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

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