Production of transgenic pigs using a pGFAP-CreER(T2)/EGFP(LoxP) inducible system for central nervous system disease models
10.4142/jvs.2018.19.3.434
- Author:
Seon Ung HWANG
1
;
Kiyoung EUN
;
Junchul David YOON
;
Hyunggee KIM
;
Sang Hwan HYUN
Author Information
1. Laboratory of Veterinary Embryology and Biotechnology, Veterinary Medical Center and College of Veterinary Medicine, Chungbuk National University, Cheongju 28644, Korea. shhyun@cbu.ac.kr
- Publication Type:Original Article
- Keywords:
genetically modified animals;
glial fibrillary acidic protein;
nuclear transfer techniques;
swine
- MeSH:
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
- From:Journal of Veterinary Science
2018;19(3):434-445
- CountryRepublic of Korea
- Language:English
-
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.
