Highly Efficient Transgenesis in Ferrets Using CRISPR/Cas9-Mediated Homology-Independent Insertion at the ROSA26 Locus

Miao Yu; Xingshen Sun; Scott R Tyler; Bo Liang; Anthony M Swatek; Thomas J Lynch; Nan He; Feng Yuan; Zehua Feng; Pavana G Rotti; Soon H Choi; Weam Shahin; Xiaoming Liu; Ziying Yan; John F Engelhardt

doi:10.1038/s41598-018-37192-4

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Highly Efficient Transgenesis in Ferrets Using CRISPR/Cas9-Mediated Homology-Independent Insertion at the ROSA26 Locus

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Highly Efficient Transgenesis in Ferrets Using CRISPR/Cas9-Mediated Homology-Independent Insertion at the ROSA26 Locus

Miao Yu, Xingshen Sun, Scott R Tyler, Bo Liang, Anthony M Swatek, Thomas J Lynch, Nan He, Feng Yuan, Zehua Feng, Pavana G Rotti, …

Scientific reports, Vol.9(1), pp.1971-1971

02/13/2019

DOI: 10.1038/s41598-018-37192-4

PMCID: PMC6374392

PMID: 30760763

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Abstract

The domestic ferret (Mustela putorius furo) has proven to be a useful species for modeling human genetic and infectious diseases of the lung and brain. However, biomedical research in ferrets has been hindered by the lack of rapid and cost-effective methods for genome engineering. Here, we utilized CRISPR/Cas9-mediated, homology-independent insertion at the ROSA26 "safe harbor" locus in ferret zygotes and created transgenic animals expressing a dual-fluorescent Cre-reporter system flanked by PhiC31 and Bxb1 integrase attP sites. Out of 151 zygotes injected with circular transgene-containing plasmid and Cas9 protein loaded with the ROSA26 intron-1 sgRNA, there were 23 births of which 5 had targeted integration events (22% efficiency). The encoded tdTomato transgene was highly expressed in all tissues evaluated. Targeted integration was verified by PCR analyses, Southern blot, and germ-line transmission. Function of the ROSA26-CAG- tdTomato EGFP (ROSA-TG) Cre-reporter was confirmed in primary cells following Cre expression. The Phi31 and Bxb1 integrase attP sites flanking the transgene will also enable rapid directional insertion of any transgene without a size limitation at the ROSA26 locus. These methods and the model generated will greatly enhance biomedical research involving lineage tracing, the evaluation of stem cell therapy, and transgenesis in ferret models of human disease.

Details

Title: Subtitle: Highly Efficient Transgenesis in Ferrets Using CRISPR/Cas9-Mediated Homology-Independent Insertion at the ROSA26 Locus
Creators: Miao Yu - College of Life Science, Ningxia University, Yinchuan, Ningxia, 750021, China
Xingshen Sun - Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, 52242, USA
Scott R Tyler - Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, 52242, USA
Bo Liang - Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, 52242, USA
Anthony M Swatek - Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, 52242, USA
Thomas J Lynch - Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, 52242, USA
Nan He - Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, 52242, USA
Feng Yuan - Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, 52242, USA
Zehua Feng - Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, 52242, USA
Pavana G Rotti - Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, 52242, USA
Soon H Choi - Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, 52242, USA
Weam Shahin - Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, 52242, USA
Xiaoming Liu - College of Life Science, Ningxia University, Yinchuan, Ningxia, 750021, China. xiaoming-liu@uiowa.edu
Ziying Yan - Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, 52242, USA. Ziying-yan@uiowa.edu
John F Engelhardt - Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, 52242, USA. john-engelhardt@uiowa.edu
Resource Type: Journal article
Publication Details: Scientific reports, Vol.9(1), pp.1971-1971
DOI: 10.1038/s41598-018-37192-4
PMID: 30760763
PMCID: PMC6374392
NLM abbreviation: Sci Rep
ISSN: 2045-2322
eISSN: 2045-2322
Publisher: England
Grant note: P30 ES005605 / NIEHS NIH HHS R24 HL123482 / NHLBI NIH HHS P01 HL051670 / NHLBI NIH HHS R24 DK096518 / NIDDK NIH HHS R01 DK047967 / NIDDK NIH HHS P30 DK054759 / NIDDK NIH HHS
Language: English
Date published: 02/13/2019
Academic Unit: Roy J. Carver Department of Biomedical Engineering; Anatomy and Cell Biology; Surgery; Radiation Oncology; Cardiothoracic Surgery; Internal Medicine
Record Identifier: 9984025420002771

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