J Vet Sci.  2016 Sep;17(3):261-268. 10.4142/jvs.2016.17.3.261.

Transgenesis for pig models

• Yum SY
• Yoon KY
• Lee CI
• Lee BC
• Jang G

• KMID: 2413124
• DOI: http://doi.org/10.4142/jvs.2016.17.3.261

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.

Keyword

conditional expression; knockout; genome editing; pig; transgenesis

MeSH Terms

Animals
Animals, Genetically Modified/*genetics
Gene Transfer Techniques/*veterinary
*Models, Animal
Sus scrofa/*genetics

Figure

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  Fig. 1 Gene expression by cassette exchange via cyclic recombinase (Cre). (A) Floxed blasticidin-resistant gene by loxP and lox2272 were integrated into porcine cells. (B) Donor DNA (puromycin-linked RFP gene) and Cre recombinase were co-transfected and blasticidin gene was then exchanged. (C) Genomic polymerase chain reaction (PCR) on recombinant target genes confirmed cassette exchange by Cre recombinase. 1, DNA ladder; 2, wild type cells; 3, blasticidin integrated cells; 4, cassette exchanged cells; (−), negative control.

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  Fig. 2 Dre-rox recombination in porcine cells and embryos. (A) DNA construction and PCR-detection regions. (B) With or without Dre recombinase transfection in porcine skin fibroblasts — upper without Dre, lower with Dre. (C) Validation of DNA excision by PCR. (D) Target gene expression by Dre recombinase injection into the cloned embryos from donor cells with transfection.

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  Fig. 3 Gene integration and expression by PhiC31 recombinase. (A) Porcine fibroblasts with the attP-blasticidin gene were generated. AttB-DNA and PhiC31 recombinase were co-transfected into the fibroblasts and recombination occurred. (B) After recombination, the fibroblast expressed eGFP. (C) Recombination was confirmed by genomic PCR. 1, control fibroblasts; 2, attP-transfected fibroblasts; 3, recombinated fibroblasts by PhiC31.

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  Fig. 4 Conditional gene expression with or without doxycycline. (A) Illustration of Tet-on gene expression by doxycycline. (B) RFP expression (left; with doxycycline) and non-expression (right; without doxycyline) in porcine fibroblasts after transfection of tet-on RFP vector.

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  Fig. 5 Illustration of the deletion of a specific gene in the endogenous gene (CMAH) in the porcine cell line. Cas9 and sgRNA were transfected into porcine fibroblasts and mutations were analyzed by T7E1 assay and sequencing.

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