25 PRODUCTION OF TRANSGENIC PIGS WITH CreER-MEDIATED ASTROCYTIC-SPECIFIC RECOMBINATION SYSTEM FOR NEUROLOGICAL DISEASE MODELS

Pigs are one of the most suitable alternative laboratory models than other animals, because they have similar cardiovascular, renal and gastrointestinal organs with those of human. However, in the case of genetically engineered animals, early development of embryos is inhibited by expression of foreign genes, there are many cases of miscarriage or birth early mortality. To overcome these problems, we constructed pig glial fibrillary acidic protein (GFAP) promoter-Cre recombinase fused to a mutated ligand-binding domain of the human oestrogen receptor (CreERT2) and enhanced green fluorescent protein (EGFP)-LoxP transgenes for tamoxifen(TM)-inducible CreERT2-mediated recombination. We then established donor transgenic pig fibroblasts with pGFAP-CreERT2; LCMV-EGFPLoxP transgenes for somatic cell nuclear transfer (SCNT). We produced the SCNT embryos using a Cloud male #5 pGFAP-CreERT2+LCMV-EGFPLoxP donor cell line that was verified in vitro. It was transferred into a surrogate mother and then 5 pGFAP-CreERT2; EGFPLoxP TG piglets were born. By immunofluorescence staining and semi-nested PCR analysis, it was proved that CreER-mediated astrocytic-specific recombination system was operated in some cerebral astrocytic cells after TM-administration to TG pig #4. Additionally, we obtained brain magnetic resonance imaging (MRI) images using 3T-tesla MRI. Brain compartment volume (total brain, grey matter, white matter, cerebellum, brainstem, lateral ventricle, thalamus, midbrain, pons, medulla oblongata, hypophysis) was no significant differences between normal pig and pGFAP-CreERT2; EGFPLoxP transgenic (TG) pig. In summary, we verified the pGFAP promoter-driven CreERT2-LoxP recombination system in TG pig generated by SCNT depending on the TM administration. We suggest that this technology will be a useful tool for studying physiology of astrocytes and generating TG pig model of neurological disease such as Huntington’s disease, Alzheimer’s disease and brain tumour.