Combinational Treatment of Trichostatin A and Vitamin C Improves the Efficiency of Cloning Mice by Somatic Cell Nuclear Transfer.

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of skillful techniques. Additionally, the efficiencies of cloning have remained low since the successful production of cloned animals, especially mice. There have been many attempts to improve the cloning efficiency, and trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to enhance the efficiency of cloning. Here, we report a dramatically improved cloning method in mice. This somatic cell nuclear transfer method involves usage of Hemagglutinating virus of Japan Envelope (HVJ-E), which enables easy manipulation. Moreover, the treatment using two small molecules, TSA and vitamin C (VC), with deionized bovine serum albumin (dBSA), is highly effective for embryonic development. This approach requires neither additional injection nor genetic manipulation, and thus presents a simple, suitable method for practical use. This method could become a technically feasible approach for researchers to produce genetically modified animals from cultured cells. Furthermore, it might be a useful way for the rescue of endangered animals via cloning

PLAG1 enhances the stemness profiles of acinar cells in normal human salivary glands in a cell type-specific manner

Objectives: Details of the histogenesis of salivary gland tumors are largely unknown. The oncogenic role of PLAG1 in the salivary gland has been demonstrated in vivo. Herein, we demonstrate the roles of PLAG1 in the acinar and ductal cells of normal human salivary glands in an attempt to clarify the early events that occur during the histogenesis of salivary gland tumors. Methods: Normal salivary gland cells with acinar- (NS-SV-AC) and ductal- (NS-SV-DC) phenotypes were transfected with PLAG1 plasmid DNA. Subsequently, the PLAG1 overexpressed and mock cells were examined by cell proliferation, transwell migration, and salisphere formation assays. The expression levels of salivary and pluripotent stem cell markers and differentiation markers were evaluated by quantitative real-time polymerase chain reaction and immunofluorescence. Alterations in transcriptional expressions were investigated via cap analysis of gene expression with gene-enrichment and functional annotation analysis. Results: PLAG1 promoted cell proliferation and transwell migration in the acinar and ductal cells, and markedly enhanced the stemness profiles and luminal cell-like profiles in acinar cells; the stemness profiles were partially increased in the ductal cells. Conclusion: PLAG1 enhanced the stemness profiles in the acinar cells of normal human salivary glands in a cell type-specific manner. Thus, it may be involved in salivary gland tumorigenesis by increasing the stemness character of the normal salivary gland cells

PHB2 Protects Sister-Chromatid Cohesion in Mitosis

SummaryCohesion between sister chromatids is essential for proper chromosome segregation in mitosis. In vertebrate mitotic cells, most cohesin is removed from the chromosome arms [1–4], but centromeric cohesin is protected by shugoshin until the onset of anaphase [5]. However, the mechanism of this protection of centromeric cohesion is not well understood. Here, we demonstrate that prohibitin 2 (PHB2) is involved in the regulation of sister-chromatid cohesion during mitosis in HeLa cells. PHB2 is an evolutionarily conserved protein in eukaryotes and has multiple functions, such as transcriptional regulation and cell viability and development [6–8]. However, its functions in mitosis have not yet been determined. We show that depletion of PHB2 by RNA interference (RNAi) causes premature sister-chromatid separation and defects in chromosome congression accompanied by mitotic arrest by spindle-checkpoint activation. In the absence of PHB2, cohesin is dissociated from centromeres during early mitosis, although the centromeric localization of shugoshin is preserved. Thus, our findings suggest that, in addition to the shugoshin, PHB2 is also required to protect the centromeric cohesion from phosphorylation by Plk1 during early mitosis and that its function is essential for proper mitotic progression

A Single Amino Acid Mutation in SNAP-25 Induces Anxiety-Related Behavior in Mouse

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a presynaptic protein essential for neurotransmitter release. Previously, we demonstrate that protein kinase C (PKC) phosphorylates Ser187 of SNAP-25, and enhances neurotransmitter release by recruiting secretory vesicles near to the plasma membrane. As PKC is abundant in the brain and SNAP-25 is essential for synaptic transmission, SNAP-25 phosphorylation is likely to play a crucial role in the central nervous system. We therefore generated a mutant mouse, substituting Ser187 of SNAP-25 with Ala using “knock-in” technology. The most striking effect of the mutation was observed in their behavior. The homozygous mutant mice froze readily in response to environmental change, and showed strong anxiety-related behavior in general activity and light and dark preference tests. In addition, the mutant mice sometimes exhibited spontaneously occurring convulsive seizures. Microdialysis measurements revealed that serotonin and dopamine release were markedly reduced in amygdala. These results clearly indicate that PKC-dependent SNAP-25 phosphorylation plays a critical role in the regulation of emotional behavior as well as the suppression of epileptic seizures, and the lack of enhancement of monoamine release is one of the possible mechanisms underlying these defects

A genetic resource conservation technology by establishing cultured fibroblast cells derived from two wild mice

[要旨]本実験では、野生種であるアカネズミおよびカヤネズミから少量の組織サンプルを回収し効率よく安定したDNA を回収するために、バロサイクラー（Baro Cycler）を用いてDNA 抽出とその相同性について検索を行なった。さらに、両マウスの尾部から採取した組織より線維芽細胞を樹立し、染色体解析および樹立した細胞の遺伝資源保存を検討した。その結果、尾部組織および樹立線維芽細胞それぞれはシークエンス後のBLAST 検索によってデータベース上にて高い相同性を示した。また、核型解析では、それぞれの核型は高い正常性を示した。さらに、それら体細胞を用いて、電気融合法による異属間体細胞核移植を実施したところ、一部の再構築卵子は2 細胞期胚へと発生することを認めた。以上の結果から、今回樹立した野生マウス由来線維芽細胞に異常は少なく、それら細胞の遺伝子資源の保存が可能であることが示された。 [Abstract] In this study, to improve the DNA extraction efficiencies for a small amount of tissue from two wild mice, we have done a search for DNA homology with Baro Cycler. In addition, fibroblasts were also established from those mice tails. Karyotype analyses were performed on the fibroblasts, and those cells were frozen for the preservation. As a result, the frozen-thawed fibroblast cells have a high number of normal karyotype. Fibroblasts derived from organization and tail confirmed the homology sequence after BLAST search. Furthermore, these somatic cells performed somatic cell nuclear transfer（SCNT）by electrofusion method. These results of reconstructed oocytes several developed to 2-cell stage embryos. In conclusion, abnormalities in this wild mouse derived fibroblast cells were low, and thereforeconservation of genetic resources was possible in these somatic cells.近畿大学先端技術総合研究所紀要編集委員