J Vet Sci.  2018 May;19(3):434-445. 10.4142/jvs.2018.19.3.434.

Production of transgenic pigs using a pGFAP-CreER(T2)/EGFP(LoxP) inducible system for central nervous system disease models

• Hwang SU
• Eun K
• Yoon JD
• Kim H
• Hyun SH

• KMID: 2412134
• DOI: http://doi.org/10.4142/jvs.2018.19.3.434

Abstract

Transgenic (TG) pigs are important in biomedical research and are used in disease modeling, pharmaceutical toxicity testing, and regenerative medicine. In this study, we constructed two vector systems by using the promoter of the pig glial fibrillary acidic protein (pGFAP) gene, which is an astrocyte cell marker. We established donor TG fibroblasts with pGFAP-CreER(T2)/LCMV-EGFP(LoxP) and evaluated the effect of the transgenes on TG-somatic cell nuclear transfer (SCNT) embryo development. Cleavage rates were not significantly different between control and transgene-donor groups. Embryo transfer was performed thrice just before ovulation of the surrogate sows. One sow delivered 5 TG piglets at 115 days after pregnancy. Polymerase chain reaction (PCR) analysis with genomic DNA isolated from skin tissues of TG pigs revealed that all 5 TG pigs had the transgenes. EGFP expression in all organs tested was confirmed by immunofluorescence staining and PCR. Real-time PCR analysis showed that pGFAP promoter-driven Cre fused to the mutated human ligand-binding domain of the estrogen receptor (CreER(T2)) mRNA was highly expressed in the cerebrum. Semi-nested PCR analysis revealed that CreER(T2)-mediated recombination was induced in cerebrum and cerebellum but not in skin. Thus, we successfully generated a TG pig with a 4-hydroxytamoxifen (TM)-inducible pGFAP-CreER(T2)/EGFP(LoxP) recombination system via SCNT.

Keyword

genetically modified animals; glial fibrillary acidic protein; nuclear transfer techniques; swine

MeSH Terms

Animals, Genetically Modified
Astrocytes
Central Nervous System*
Cerebellum
Cerebrum
DNA
Embryo Transfer
Embryonic Development
Estrogens
Female
Fibroblasts
Fluorescent Antibody Technique
Glial Fibrillary Acidic Protein
Humans
Nuclear Transfer Techniques
Ovulation
Polymerase Chain Reaction
Pregnancy
Real-Time Polymerase Chain Reaction
Recombination, Genetic
Regenerative Medicine
RNA, Messenger
Skin
Swine*
Tissue Donors
Toxicity Tests
Transgenes
DNA
Estrogens
Glial Fibrillary Acidic Protein
RNA, Messenger