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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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          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 nonmeiotic allele introgression in livestock using custom endonucleases.

              Wenfang Tan, Daniel Carlson, Cheryl Lancto … (2013)
              We have expanded the livestock gene editing toolbox to include transcription activator-like (TAL) effector nuclease (TALEN)- and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-stimulated homology-directed repair (HDR) using plasmid, rAAV, and oligonucleotide templates. Toward the genetic dehorning of dairy cattle, we introgressed a bovine POLLED allele into horned bull fibroblasts. Single nucleotide alterations or small indels were introduced into 14 additional genes in pig, goat, and cattle fibroblasts using TALEN mRNA and oligonucleotide transfection with efficiencies of 10-50% in populations. Several of the chosen edits mimic naturally occurring performance-enhancing or disease- resistance alleles, including alteration of single base pairs. Up to 70% of the fibroblast colonies propagated without selection harbored the intended edits, of which more than one-half were homozygous. Edited fibroblasts were used to generate pigs with knockout alleles in the DAZL and APC genes to model infertility and colon cancer. Our methods enable unprecedented meiosis-free intraspecific and interspecific introgression of select alleles in livestock for agricultural and biomedical applications.
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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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