32 NEURON-SPECIFIC EXPRESSION OF THE RED FLUORESCENCE PROTEIN IN CLONED DOGS

Abstract

The pathogenesis of neuronal degenerative disease as Alzheimer's disease (AD) has been a subject of intensive research for the last few decades worldwide. But despite such effort, treatment or preventive measures for AD have so far made no breakthrough. One of the contributing factors that hindered the progress of research is the lack of appropriate AD models. Mouse models have limitations for AD research because the irreconcilable species gap between the rodent and human has impeded the research itself as well as the application of the findings from the rodent studies to human cases. As an alternative, here we performed a preliminary study to develop novel neuronal degenerative disease models using a canine transgenic somatic cell nuclear transfer (SCNT) technology. The aim of this study is to produce a transgenic dog that expresses neuron-specific transgene in the brain by SCNT. In this study, we chose human synapsin 1 promoter as primarily neuron selective, driving the red fluorescent protein transgene. For SCNT, synapsin 1-red fluorescence protein (SYN1-RFP) was introduced into female beagle adipose-derived stem cell via lentiviral vector infection. The SYN1-RFP cells were injected into enucleated in vivo-matured dog oocytes and fused by electric stimulation. The fused couplets (80/94, 85.1%) were chemically activated and transferred into the uterine tube of 5 naturally oestrus-synchronized surrogates. Three of them (60%) maintained pregnancy and subsequently gave birth to 3 cloned pups (SYN1-RFP A, SYN1-RFP B, SYN1-RFP C) by natural delivery or cesarean section. Birth weights of the offspring ranged from 120 to 280 g and SYN1-RFP C is still alive, healthy and does not show any abnormalities. The microsatellite analysis shows that all SYN1-RFP puppies originated from the SYN1-RFP cells used in SCNT and mitochondrial DNA analysis shows that the puppies had been derived from the oocyte donors. In order to investigate the result in multiple transgene insertions, SYN-RFP puppies were screened by Southern blot analysis using DNA extracted from skin biopsies. Transgene copy number was estimated by Southern blot analysis. The SYN-RFP A and B that died at 3 days after birth had approximately 5 and 2 copies of the transgene integrated, respectively, whereas the alive SYN-RFP C has 1 copy. SYN-RFP B was particular in that it did not express RFP in the entire body, but samples collected postmortem showed expression of the RFP transgene under the human synapsin 1 promoter in neural cells in the brain of SYN-RFP B. In conclusion, we report here that (1) the human synapsin promoter is functional in neural cells of dog brain and (2) a neural-specific-transgene-expressed dog was generated for the first time by transgenic SCNT technique. Furthermore, the SYN-RFP dog has great potential to understand the function of a neuronal degenerative disease model dog. This study was supported by MKE (Grant # 10033839-2011-13), RNL Bio, IPET and TS Corporation.