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Int. J. Mol. Sci. 2015, 16(8), 17838-17856; doi:10.3390/ijms160817838

Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations

1
Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima 890-8544, Japan
2
Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan
3
Animal Genome Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, Ibaraki 305-8602, Japan
4
Division of Basic Molecular Science and Molecular Medicine, School of Medicine, Tokai University, Kanagawa 259-1193, Japan
5
Department of Cardiovascular Research, Graduate school of Medicine, Shinshu University, Nagano 390-8621, Japan
6
Department of Pediatric Dentistry, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan
7
Division of Pediatric Dentistry, Department of Oral Health Sciences, Course for Oral Life Science, Graduate School of Medical and Dental Sciences, Niigata University, Niigata 951-8514, Japan
8
Division of Biomedical Engineering, National Defense Medical College Research Institute, Saitama 359-8513, Japan
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed.
Academic Editor: Izuho Hatada
Received: 9 June 2015 / Revised: 9 June 2015 / Accepted: 24 July 2015 / Published: 3 August 2015
(This article belongs to the Special Issue Genome Editing)
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Abstract

Some reports demonstrated successful genome editing in pigs by one-step zygote microinjection of mRNA of CRISPR/Cas9-related components. Given the relatively long gestation periods and the high cost of housing, the establishment of a single blastocyst-based assay for rapid optimization of the above system is required. As a proof-of-concept, we attempted to disrupt a gene (GGTA1) encoding the α-1,3-galactosyltransferase that synthesizes the α-Gal epitope using parthenogenetically activated porcine oocytes. The lack of α-Gal epitope expression can be monitored by staining with fluorescently labeled isolectin BS-I-B4 (IB4), which binds specifically to the α-Gal epitope. When oocytes were injected with guide RNA specific to GGTA1 together with enhanced green fluorescent protein (EGFP) and human Cas9 mRNAs, 65% (24/37) of the developing blastocysts exhibited green fluorescence, although almost all (96%, 23/24) showed a mosaic fluorescent pattern. Staining with IB4 revealed that the green fluorescent area often had a reduced binding activity to IB4. Of the 16 samples tested, six (five fluorescent and one non-fluorescent blastocysts) had indel mutations, suggesting a correlation between EGFP expression and mutation induction. Furthermore, it is suggested that zygote microinjection of mRNAs might lead to the production of piglets with cells harboring various mutation types. View Full-Text
Keywords: α-Gal epitope; α-1,3-galactosyltransferase; CRISPR/Cas9; microinjection; mRNA; mosaicism; biallelic KO; indel mutations; isolectin BS-I-B4 α-Gal epitope; α-1,3-galactosyltransferase; CRISPR/Cas9; microinjection; mRNA; mosaicism; biallelic KO; indel mutations; isolectin BS-I-B4
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Sato, M.; Koriyama, M.; Watanabe, S.; Ohtsuka, M.; Sakurai, T.; Inada, E.; Saitoh, I.; Nakamura, S.; Miyoshi, K. Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations. Int. J. Mol. Sci. 2015, 16, 17838-17856.

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