91 Expression Pattern of Neuron-Specific Red Fluorescence Protein in Nervous System of Transgenic Dog with Human Synapsin I Promoter

Abstract

Neurodegenerative disorders, such as Alzheimer’s disease, affect neurons in large areas within the central nervous system. The selective expression of disease-causing and therapeutic genes in susceptible regions and cell types is critical to the generation of animal models. In our previous studies, we reported production of a transgenic dog by nuclear transfer using donor cells that had been stably transfected by vector containing the red fluorescence protein (RFP) and human synapsin I (SynI) promoter, a neuron-specific promoter. Here, we evaluated whether RFP expression in transgenic dog with SynI (SynI dog) had high neuronal specificity and strong transcriptional activity. For confirmation of neuron-specific RFP expression, tissue samples were obtained from a killed 4-year-old SynI dog. Quantification of RFP in heart, kidney, liver, lung, spleen, cerebrum, cerebrum, cerebellum, midbrain, hippocampus, peripheral nerves, skin, and spinal cord of SynI dog was analysed using ELISA (Cell Biolabs, San Diego, CA, USA). Moreover, the distribution of RFP activity in neural system of the SynI dog was determined by immunohistochemistry. The first antibody, rabbit polyclonal RFP antibody (1:200, ab62341, Abcam, Cambridge, MA, USA) was applied, and antibody labelling was visualised by incubation with avidin-biotinylated horseradish peroxidase complex (1:1,000; ABC Elite, Vector Laboratories, Burlingame, CA, USA). In the 4-year-old SynI dog, RFP was observed only in neuronal tissues including brain, spinal cord, and peripheral nerves, but was not detected in non-neuronal tissue such as heart, kidney, and skin. In addition, robust RFP expression was observed in the neurons of the peripheral nerve, spinal cord, and prefrontal cortex. In the hippocampus and cerebellum, the RFP-expressing cells appeared heterogeneous in hippocampus neurons and purkinje cells. In conclusion, we report that human SynI promoter is functional in neural cells of dogs. A neural specific-transgene expressed dog could be applied as a research tool in the study of neurodegenerative disorders.

This research was supported by RDA (#PJ010928032017), Korea IPET (#316002-05-2-SB010), NRF (#2016R1D1A1B03932198), and Research Institute for Veterinary Science, the BK21 plus program.