The Generation And Characterization Of Transgenic Mice Carrying Human TGM6

The spinocerebellar ataxias (SCAs) are a heterogeneous group of inherited neurodegenerative disorders. To date, classical genetic studies have revealed33distinct genetic forms of spinocerebellar ataxias, and23causative genes have been cloned. In2010, our group identified two missense mutations in the TGM6gene in two Chinese SCA families, c.1550T>G transition (p.L517W) and c.980A>G transition (p.D327G), suggesting a novel causative gene for SCA, which has been named as SCA35.The molecular pathogenesis among the SCA subtypes may be different. To explore the mechanism of SCA35and the function of TGM6, we generate the transgenic mice carrying the L517W and D327G mutation and wild-type of human TGM6gene. In addition, there has been no report concerning the cellular distribution of TG6in the adult brain, we thus set out to investigate TGM6gene expression in the central nervous system. This data is the basis for further dissecting the physiological function of TGM6.The molecular pathogenesis of the SCA subtypes may be different. There isn’t the report about the physiological function of TGM6gene at home and abroad. To explore the function of TGM6and the mechanism of SCA35, we construct the transgenic mice which carry the L517W and D327G mutation and wild-type of human TGM6gene. In addition, there is few data about TGM6gene expression patterns in the central nervous system. These data is necessary to learn the function of TGM6. Therefore, the thesis investigated the expression patterns in the central nervous system of TGM6and the chemical neuroanatomical characteristics of positive neurons.To generate transgenic mice carrying the mutations of L517W and D327G, and wild-type human TGM6gene, we constructed pCAGGS-TGM6wt-Myc-GFP, pCAGGS-TGM6L517W-Myc-GFP and pCAGGS-TGM6D327G-Myc-GFP transgenic expression vectors, with Myc and GFP tag. These vectors expressed the transgenes effectively in eukaryocytes.The three transgenesic mice expressing human TGM6mutations of L517W and D327G, and the wild-type TGM6were obtained. The three transgenic mice were confirmed by PCR and sequencing, and by detecting expression of GFP and Myc with Western blot, which indicated the expression of transgenes at the protein level. In addition, to show expression of transgenes at the cellular level, we performed double immunological staining of TG6with GFP (or Myc). It showed that TG6immunoreactivity was co-localized with GFP or Myc in the brain. Thus, we have made the transgenic mice carrying human TGM6(wild type and two mutations), which provided a good tool for investigating the function of human TG6in vivo.Immunohistochemistry of TG6in adult brains showed that TG6was abundantly expressed in the diencephalon, brainstem and cerebellum. TG6was located in neurons but not glia cells. Notably, numerous TG6-positive neurons were distributed in the key brain regions involved in regulating locomotion activity including the globus pallidus, subthalamic nucleus, substantia nigra and cerebellum. In addition, we found that the vast majority of TG6-positive cells in the reticular part of substantia nigra were GABAergic inhibitory neurons.