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      Transgenesis for pig models

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          Abstract

          Animal models, particularly pigs, have come to play an important role in translational biomedical research. There have been many pig models with genetically modifications via somatic cell nuclear transfer (SCNT). However, because most transgenic pigs have been produced by random integration to date, the necessity for more exact gene-mutated models using recombinase based conditional gene expression like mice has been raised. Currently, advanced genome-editing technologies enable us to generate specific gene-deleted and -inserted pig models. In the future, the development of pig models with gene editing technologies could be a valuable resource for biomedical research.

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          Most cited references37

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          Establishment in culture of pluripotential cells from mouse embryos.

          Sydney M. Evans, H Kaufman (1981)
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            Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos.

            John Driver, Benjamin P Beaton, Kristin M. Whitworth … (2014)
            Targeted modification of the pig genome can be challenging. Recent applications of the CRISPR/Cas9 system hold promise for improving the efficacy of genome editing. When a designed CRISPR/Cas9 system targeting CD163 or CD1D was introduced into somatic cells, it was highly efficient in inducing mutations. When these mutated cells were used with somatic cell nuclear transfer, offspring with these modifications were created. When the CRISPR/Cas9 system was delivered into in vitro produced presumptive porcine zygotes, the system was effective in creating mutations in eGFP, CD163, and CD1D (100% targeting efficiency in blastocyst stage embryos); however, it also presented some embryo toxicity. We could also induce deletions in CD163 or CD1D by introducing two types of CRISPRs with Cas9. The system could also disrupt two genes, CD163 and eGFP, simultaneously when two CRISPRs targeting two genes with Cas9 were delivered into zygotes. Direct injection of CRISPR/Cas9 targeting CD163 or CD1D into zygotes resulted in piglets that have mutations on both alleles with only one CD1D pig having a mosaic genotype. We show here that the CRISPR/Cas9 system can be used by two methods. The system can be used to modify somatic cells followed by somatic cell nuclear transfer. System components can also be used in in vitro produced zygotes to generate pigs with specific genetic modifications. © 2014 by the Society for the Study of Reproduction, Inc.
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              Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases.

              J. Hauschild, B. Petersen, Y. Santiago … (2011)
              Zinc-finger nucleases (ZFNs) are powerful tools for producing gene knockouts (KOs) with high efficiency. Whereas ZFN-mediated gene disruption has been demonstrated in laboratory animals such as mice, rats, and fruit flies, ZFNs have not been used to disrupt an endogenous gene in any large domestic species. Here we used ZFNs to induce a biallelic knockout of the porcine α1,3-galactosyltransferase (GGTA1) gene. Primary porcine fibroblasts were treated with ZFNs designed against the region coding for the catalytic core of GGTA1, resulting in biallelic knockout of ∼1% of ZFN-treated cells. A galactose (Gal) epitope counter-selected population of these cells was used in somatic cell nuclear transfer (SCNT). Of the resulting six fetuses, all completely lacked Gal epitopes and were phenotypically indistinguishable from the starting donor cell population, illustrating that ZFN-mediated genetic modification did not interfere with the cloning process. Neither off-target cleavage events nor integration of the ZFN-coding plasmid was detected. The GGTA1-KO phenotype was confirmed by a complement lysis assay that demonstrated protection of GGTA1-KO fibroblasts relative to wild-type cells. Cells from GGTA1-KO fetuses and pooled, transfected cells were used to produce live offspring via SCNT. This study reports the production of cloned pigs carrying a biallelic ZFN-induced knockout of an endogenous gene. These findings open a unique avenue toward the creation of gene KO pigs, which could benefit both agriculture and biomedicine.
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                Author and article information

                Journal
                Journal ID (nlm-ta): J Vet Sci
                Journal ID (iso-abbrev): J. Vet. Sci
                Journal ID (publisher-id): JVS
                Title: Journal of Veterinary Science
                Publisher: The Korean Society of Veterinary Science
                ISSN (Print): 1229-845X
                ISSN (Electronic): 1976-555X
                Publication date (Print): September 2016
                Publication date (Electronic): 20 September 2016
                Volume: 17
                Issue: 3
                Pages: 261-268
                Affiliations
                [1 ]Laboratory of Theriogenology and Biotechnology, Department of Veterinary Clinical Science, College of Veterinary Medicine and the Research Institute of Veterinary Science, Seoul National University, Seoul 08826, Korea.
                [2 ]Department of Biotechnology & Laboratory Animals, Shingu College, Seongnam 13174, Korea.
                [3 ]Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 08826, Korea.
                [4 ]Emergence Center for Food-Medicine Personalized Therapy System, Advanced Institutes of Convergence Technology, Seoul National University, Suwon 16229, Korea.
                [5 ]Farm Animal Clinical Training and Research Center, Institutes of GreenBio Science Technology, Seoul National University, Pyeongchang 25354, Korea.
                Author notes
                Corresponding author: Tel: +82-2-880-1280; Fax: +82-2-873-1269; snujang@ 123456snu.ac.kr

                †The first two authors equally contributed to this work.

                Article
                DOI: 10.4142/jvs.2016.17.3.261
                PMC ID: 5037292
                PubMed ID: 27030199
                SO-VID: b81d4898-0f6e-4d0a-a174-245728a57f2e
                Copyright © © 2016 The Korean Society of Veterinary Science.
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date received : 29 December 2015
                Date accepted : 12 February 2016
                Funding
                Funded by: Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries, CrossRef http://dx.doi.org/10.13039/501100003668;
                Award ID: 109023-05-5-CG000
                Funded by: National Research Foundation of Korea, CrossRef http://dx.doi.org/10.13039/501100003725;
                Award ID: 2011-0014941
                Categories
                Subject: Review

                ScienceOpen disciplines: Veterinary medicine
                Keywords: conditional expression,knockout,genome editing,pig,transgenesis
                Data availability:
                ScienceOpen disciplines: Veterinary medicine
                Keywords: conditional expression, knockout, genome editing, pig, transgenesis

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