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

Figure

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  Fig. 1 Validation of CreERT2-mediated recombination in pig fibroblasts after 4-hydroxytamoxifen (TM) administration. (A) To test the recombination system, a fibroblast cell line with the CMV-EGFPLoxP construct was transiently transfected with the CreERT2 expression vector by using electroporation and, 24 h after transfection, treated with TM at each indicated concentration for 48 h. EGFP expression of each sample was determined by fluorescence-activated cell sorter analysis. The numbers indicate the percentage of the EGFP-negative population. Puro; puromycin. (B) To examine transient transfection efficiency, a red fluorescent protein (RFP) expression vector was transfected by same method, and its signal was detected by using fluorescence-activated cell sorter (FACS). Approximately 25% of the cells were RFP-positive. (C) When the floxed-population of each sample shown in (A) was normalized to the transfection efficiency shown in (B), approximately 21% of cells were recombined by CreERT2 when activated by TM treatment. Data are presented as mean ± SEM. *p<0.05 between Puro and CreERT2.

• 

  Fig. 2 Establishment of a somatic cell nuclear transfer (SCNT) donor cell line with the CreER-mediated astrocyte-specific recombination system. (A) Strategy for production of transgenic (TG) pig CreERT2-mediated astrocyte-specific recombination system by SCNT and its validation. TM, 4-hydroxytamoxifen. (B) Donor cell lines transfected with LCMV-EGFPLoxP plasmid vector were sorted by using a fluorescence-activated cell sorter (FACS). (C) Morphology of Cloud male No. 5 pGFAP-CreERT2+LCMV-EGFPLoxP donor cells; their EGFP expression was detected by using microscopy (200×). (D) The presence of system vectors in the genome was identified by polymerase chain reaction amplification of two transgenes in genomic DNA samples of constructed donor cell lines. 18s was used as a control.

• 

  Fig. 3 Effect of different donor cell types during preimplantation development (A) on the cleavage pattern and (B) the blastocyst formation pattern of somatic cell nuclear transfer (SCNT) embryos. BL, blastocyst. (C) Summary of embryonic development after SCNT. The cleavage rate was measured on day 2, and blastocyst formation rate was evaluated on day 7 of culture. Values with different superscript letters within a column differ significantly (p < 0.05). Experiment was repeated more than three times. The data presented are means ± SEM. *Percentage of total oocytes. †Percentage of total fused oocytes.

• 

  Fig. 4 Production of transgenic (TG) piglets with pGFAP-CreERT2/EGFPLoxP transgene constructs. (A) Summary of embryo transfer result. J.B.O., just before ovulation. (B) Picture of pGFAP-CreERT2/EGFPLoxP TG piglets. (C) Polymerase chain reaction was performed using genomic DNA isolated from skin tissue of somatic cell nuclear transfer piglets. 18s was amplified as a control. Normal, newborn normal Yucatan piglet tissue.

• 

  Fig. 5 Confirmation of cerebral expression of EGFP and CreERT2 transgenes in the astrocyte-specific CreERT2-LoxP recombination system. (A) Expression of endogenous GFAP and CreERT2 regulated by the exogenous GFAP promoter was confirmed by quantitative polymerase chain reaction analysis. Asterisks indicate significant difference (*p < 0.05) from non-brain tissues. (B) CreERT2 expression in GFAP-positive astrocytes and (C) cerebral expression of the EGFP gene in brain tissues was detected by immunofluorescence staining. Normal, newborn normal Yucatan piglet tissue; GFAP, glial fibrillary acidic protein; EGFP, enhanced green fluorescent protein; DAPI, 4′,6-diamidino-2-phenylindol.

• 

  Fig. 6 Confirmation of pGFAP promoter activity in fibroblasts. (A) Experimental scheme showing expected pre-recombinant and post-recombinant sizes of genomic DNA polymerase chain reaction (PCR) products as amplified using the F and R primer set. (B) Genomic DNA PCR analysis revealed that 4-hydroxytamoxifen (4OH-TM) treatment in fibroblast cells (derived from the No. 2 and No. 3 transgenic pigs) for 48 h did not induce pGFAP-CreERT2-mediated recombination of LoxP sites. Con, control.

• 

  Fig. 7 Induction of CreER-mediated astrocyte-specific recombination system. (A) Scheme of 4-hydroxytamoxifen (4OH-TM) administration. (B) Result of No. 4 pig and control No. 5 pig cerebellum tissue Immunofluorescent staining. No. 4-cerebellum : TM-administrated pig, No. 5-cerebellum : TM nontreatment control. (C) Genomic DNA polymerase chain reaction (PCR) analysis showed that pre-recombinant PCR products were amplified in the cerebral cortex (upper) and cerebellum tissues (bottom) of pGFAP-CreERT2/EGFPLoxP transgenic pigs. Skin-derived genomic DNA was used as a control, representing non-cerebral tissue. (D) Scheme of Semi-nested PCR. After first-round PCR using the F and R primer set, an invisible target PCR product of around 702 bp size was extracted from agarose gel after electrophoresis. Then, a second PCR was performed using F and R′ primers. (E) Detection of recombinant genomic DNA by Semi-nested PCR. ‘C’ means a control and indicates a semi-nested PCR product from the plasmid vector, while ‘V’ represents a single PCR amplification product from thevector.

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