| Two-electrode Voltage-clamp Recordings in Xenopus laevis Oocytes：Reconstitution of Abscisic Acid Activation of SLAC1 Anion Channel via PYL9 ABA Receptor
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Author:
Date:
2017-01-20
[Abstract] Two-Electrode Voltage-Clamp (TEVC) recording in Xenopus laevis oocytes provides a powerful method to investigate the functions and regulation of ion channel proteins. This approach provides a well-known tool to characterize ion channels or transporters expressed in Xenopus laevis oocytes. The plasma membrane of the oocyte is impaled by two microelectrodes, one for voltage sensing and the other one for current injection. Here we list a protocol that allows robust reconstitution of multi-component signaling pathways. This protocol has been used to study plant ion channels, including the SLAC1 channel (SLOW ANION CHANNEL-ASSOCIATED 1), in particular SLAC1 activation by either the protein kinase OST1 (OPEN STOMATA 1), Ca2+-dependent protein kinases (CPKs) or the ...
[摘要] 在非洲爪蟾卵母细胞中记录的双电极电压钳（TEVC）提供了一种强大的方法来研究离子通道蛋白的功能和调节。这种方法提供了一种众所周知的用于表征在非洲爪蟾卵母细胞中表达的离子通道或转运蛋白的工具。卵母细胞的质膜由两个微电极引起，一个用于电压感测，另一个用于电流注入。在这里，我们列出了一个允许多组分信号通路强制重组的协议。该方案已经用于研究植物离子通道，包括SLAC1通道（SLOW ANION CHANNEL-ASSOCIATED 1），特别是SLAC1通过蛋白激酶OST1（OPEN STOMATA 1），Ca 2 +依赖性蛋白激酶（CPK）或GHR1（GUARD细胞过氧化氢抗性1）跨膜受体样蛋白。显示了通过“单体”ABA（脱落酸）受体RCAR1 / PYL9（PYRABACT INRESISTANCE1 [PYR1] / PYR1-样[PYL] / ABA受体[RCAR]的调节因子]重建SLAC1阴离子通道的脱落酸活化的数据。通过共表达脱落酸信号核心的四个组分。该方案也适用于研究其他离子通道功能和调节机制，以及转运蛋白。背景 ...
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| Generation of Mitochondrial-nuclear eXchange Mice via Pronuclear Transfer
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Author:
Date:
2016-10-20
[Abstract] The mitochondrial paradigm for common disease proposes that mitochondrial DNA (mtDNA) sequence variation can contribute to disease susceptibility and progression. To test this concept, we developed the Mitochondrial-nuclear eXchange (MNX) model, in which isolated embryonic pronuclei from one strain of species are implanted into an enucleated embryo of a different strain of the same species (e.g., C57BL/6 and C3H/HeN, Mus musculus), generating a re-constructed zygote harboring nuclear and mitochondrial genomes from different strains. Two-cell embryos are transferred to the ostia of oviducts in CD-1 pseudopregnant mice and developed to term. Nuclear genotype and mtDNA haplotype are verified in offspring, and females selected as founders for desired MNX colonies. By ...
[摘要] 从植物组织提取的缩合单宁是酚醛树脂的合适替代物。它们的可能影响它们的化学反应性的分子结构可以通过在酸硫解和MALDI-TOF质谱之后使用HPLC-UV来评估。用半胱胺盐酸盐在酸性甲醇中溶解植物提取物导致缩合的单宁寡聚体的单体单元的释放，其可以通过与分析标准比较通过反相HPLC-UV进一步定量。使用2,5-二羟基苯甲酸作为基质和K sup +作为阳离子化试剂的MALDI-TOF质谱分析突出了单宁的分子结构特征（例如单体单元序列）低聚物。该方法允许估计平均和最大（可观察）聚合度，单体单元的类型和单宁单体的糖基化和/或酯化的存在。
[背景] 缩合单宁是由可从几种植物组织（例如软木树皮）中提取的黄烷-3-醇单体单元组成的多酚低聚物。它们已被认为是树脂配方（例如木材粘合剂和泡沫材料）中合成酚醛树脂的合适替代品。在缩合鞣酸中检测到的最常见的黄烷-3-醇单体，其羟基化模式和立体化学不同，如图1所示。
![]() "src ="/attached/image/20161012/20161012024340_4416.jpg"/>
图1.在缩合单宁结构中确定的最常见的单体
低聚物中单体单元的具体结构和聚合度强烈影响单宁的化学反应性和物理性质，例如与醛的缩合反应速率，重金属螯合能力和水溶液的粘度溶液（Pizzi和Stephanou，1994; ...
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| In utero Electroporation of Mouse Cerebellar Purkinje Cells
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Author:
Date:
2016-06-05
[Abstract] In utero electroporation (IUE) of mouse cerebellar Purkinje cells allows high expression levels of transgenes without toxicity (Nishiyama et al., 2012). This technique is suitable for co-transfection of multiple plasmid genes. Therefore, it is useful to express various sets of genes such as drug-inducible Cre/loxP constructs and CRISPR/Cas9 genome editing constructs (Takeo et al., 2015). Murine Purkinje cells arise from subventricular zone of fourth ventricle at embryonic day (E) 10-12. IUE at E11.5 into fourth ventricle results the most efficient transfection into Purkinje cells.
[摘要] 在小鼠小脑浦肯野细胞的子宫电穿孔（IUE）中，允许没有毒性的转基因的高表达水平（Nishiyama等，2012）。 该技术适用于多重质粒基因的共转染。 因此，表达各种基因如药物诱导型Cre / loxP构建体和CRISPR / Cas9基因组编辑构建体是有用的（Takeo等，2015）。 小鼠浦肯野细胞起因于胚胎期（E）10-12的第四脑室下室。 IUE在E11.5进入第四脑室导致最有效的转染到浦肯野细胞。
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