我们要感谢安德烈Kremsreiter优秀的技术援助。麦迪逊 - 我们实验室的工作是由美国国立卫生研究院GM096088和威斯康星大学的拉斯信托基金的支持。

1。酵母发光法方法使用酿酒酵母的菌株BYYT。这是BY4741（ 马塔，ura3D0，his3D1，leu2D0，lys2D0，yvc1 :: HIS3，tok1 :: kanMX4）的yvc1-tok1衍生 。转化细胞与列伊可选水母发光蛋白表达质粒pEVP1/Aeq及市建局可选鼠TRPV4表达质粒p416GPDV4，如Batiza 等 26 Loukin 等 24所描述的质粒建设和改造27使用标准方法。酵母菌株和质粒可根据要求提供。 <ol><li>文化2毫升酵母细胞中过夜，以30℃摇床中LEU-URA-"DCD"辍学介质28（在CMD-LEU-URA培养基29的硫酸盐耗尽变种），辅以1M山梨醇后对数生长期。接种200微升这些文化进1.8毫升新鲜培养基中添加2微米luciferi的Ñ​​腔肠素。在室温下另外24小时生长在黑暗中无晃动。 </li><li>测量培养物的摩尔渗透压浓度（通常在1400毫渗透摩尔浓度）和其他解决方案（见下文）用蒸汽压渗透压力计。 </li><li>分装20μL新鲜培养成12毫米光度计管单管光度计。 </li><li>为低渗休克，添加200微升的溶液中，含有25mM NaEGTA（pH值7.2）或名称（pH 7.2）中和500-100 mM氯化钠，（1,200-400的毫渗透摩尔浓度总渗透压），这取决于所需的冲击的程度。连续监测荧光前后渗透压downshock后，至少120秒，中断只有短暂的稀释操作。注册光度计输出一台台式电脑作为相对发光单位（RUL）上。 </li></ol>虽然不是转基因TRPV4的研究中，我们使用上述协议的一个变体的自动化系统中，研究了大量的酵母菌株的反应。 Ø研究F中的反应低30或酵母deletome（共4906酵母单基因deletants集合）的使用微板光度计高渗31的刺激，并与相应的软件分析已经成功。 2。和3。卵母细胞电生理学方法电采用的基本方法32。 PCR扩增的野生型或突变型TRPV4采用高保真PfuUtra聚合酶和集成到pGH19 33的开放式阅读框架。这引起该ORF的爪蟾 β-珠蛋白基因和T7 RNA聚合酶启动子的下游的5&#39;和3&#39;非翻译区之间具有精确的插入。线性化的3&#39;端非编码区的下游，并用来作为在体外 T7 RNA聚合酶反应的模板构建物。使用标准的分子生物学技术34。 使用阶段的第五和第六的卵母细胞来自十蟾 。畜牧业，部分ovariecto我，defolliculation和卵黄膜去除遵循标准程序32,33,35。使用德拉蒙德Nanoinject II自动微量注射器注入2-40纳克每卵母细胞的cRNA的解决方案。注入〜30卵母细胞中的每个组实验。 TRPV4-的cRNA注射后，孵育在ND96介质中的卵母细胞用庆大霉素（100微克/毫升）和1μM的钌红14。 2。两个电极电压钳<ol><li>从精密的一次性100微升分别用吸管拉马拉微量硼硅玻璃录音移液器。 </li><li>将两个电压测量和电流注入吸管电极拉到有〜1微米直径的尖端光圈。 </li><li>用2M的KCl回填电极产生0.1-0.2MΩ的电阻。它们安装在HS2A的探头，连同一个VG-2Ax100虚拟接地浴钳，通过一个Digidata 1440数字化仪连接到GeneClamp 500放大器接口。获得<html>使用pClamp10软件的数据。 </li><li>将卵母细胞在1ml浴中制造的塑料腔室安装在解剖显微镜以20X放大率的阶段进行测试。槽液是虚拟接地由一个3米氯化钾琼脂桥和氯化银线放在靠近卵母细胞，并连接到一个VG-2A的虚拟地面的阶段。 </li><li>建构室连续灌注塑料管材作为解决方案的入口和出口。连接入口管连接在一个装配灌注系统，它是由六个50毫升注射器壳，每个填充有不同的解决方案的歧管。任何特定的溶液比重的进给率控制在〜2-3使用"凯克"斜夹在油管毫升/分钟。 </li><li>安装两个电极与他们的显微操作的探头。通过显微操作进行卵母细胞的电极穿透。 </li></ol> 3。直接Mechanosensitivity的膜片钳考试"&gt;膜片钳的基本方法，包括吸移管的制备，GΩ的密封形成，并且形成于细胞或切除的模式是那些在Hamill 等人，36和康恩32。 <ol><li>补的硼硅酸盐玻璃移液管具有约1微米直径的开口的末端（气泡数5-6）与98毫米氯化钾，1毫摩尔MgCl 2和10mM K +-HEPES，pH值7.2。 </li><li>通过塑料管连接到一个5毫升注射器，并通过一个T型接头连接的膜片钳吸移管，也压力计。使用计算机来处理这两个电极和压力计输出。在10千赫获取数据，然后在1千赫进行分析回放通过一个8极Bessel滤波器。 </li><li>不同的补丁可以从相同的卵母细胞反复被切除。这种做法使得TRPV4的分子活动的检查更加有效和可靠。 </li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+patch-clamp+techniques+for+high-resolution+current+recording+from+cells+and+cell-free+membrane+patches.">Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.</a> Pflugers Arch. 391, 85-100 (1981).</li><li>Maroto, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPC1+forms+the+stretch-activated+cation+channel+in+vertebrate+cells.">TRPC1 forms the stretch-activated cation channel in vertebrate cells.</a> Nat. Cell Biol. 7, 179-185 (2005).</li></ol>

作者宣称，他们有没有竞争的财务权益。

正如引言所述，通常用于研究TRPV4功能的方法有时会导致不一致和争议。两套此处描述的方法提供了一些优点，它可以补充现有的方法。虽然我们只描述上TRPV4的研究中，该方法可以被扩展以研究其他离子通道为好。 1。酵母发光法方法出芽酵母具有天然的Ca 2 +内流响应是受影响的脂质组合物30的突变敏低渗休克。这个信号可以被擦除与外部EGTA。这样的螯合剂，但是，不从异源表达的TRPV4 24擦除信号，表明该信道被表示在一个内部膜，最有可能是内质网中，从其中的Ca 2 +时的渗透释放到细胞质中震撼。的异源表达的交通渠道尚未研究在酵母。应这种方法扩展到其它TRP通道或其它公认的机械敏感性离子通道的研究中，螯合测试需要进行与本机系统的存在下被记住。 2A。双电极电压钳不同的膜片钳实验中，将两电极电压钳实验上进行完整的卵母细胞，在除去卵黄膜之前。既镜检和容量值表示质膜不是均匀的球形，但高内陷卵黄信封的相对非弹性球体的下方。因此，造成hyponicity的机械应力不扩大球形质膜的只是一个各向同性的通胀紧张。从压抵约束卵黄膜和/或远离既定胞质附着在日膜的应力有可能的结果Ë面对渗透压增高。 TRPV4的表达，并可能某些其它信道，是有毒的卵母细胞，大概是由于Ca 2 +的渗漏。因此重要的是连续培养TRPV4-的cRNA注射中的通道阻断剂，钌红的存在后卵母细胞。 TRPV4表达的程度表明变异性，这是不完全根据实验对照。 "功能获得的"突变TRPV4通道，那些表现显著自发开放，有系统地早期注射后比其野生型对应23表示。 2B。膜片钳 非洲爪蟾卵母细胞表达原生机械敏感性通道，其整流，在向内的方向（〜50 PS向内〜10 PS向外）。一项研究表明，它是TRPC1 37的表达。这个不断表示本地通道提供一个校准TRPV4，杂其中logous表达可能并不总是成功的补丁观察。 遇到TRPV4的可能性也愈高卵母细胞中培养一段较长时间后，通常3-4天的cRNA注射后。进行一个有效的办法是先进行双电极电压钳实验（上图），并确保该卵母细胞表达的自发TRPV4宏观电流，然后进行取样品补丁从相同的前去除卵黄膜卵母细胞。捕获TRPV4活动的概率，也可以使用"macropatches"，安装在移液管具有较大的孔（气泡数6-7）增强。火抛光这些移液器32似乎是在实现GΩ封很有帮助。密封形成后，切除修补程序可能表现出非常高的性能，噪音非常小，通常与生物膜有关，并没有原生通道的活性。这最有可能在dicates双膜形成。一个简短的暴露在空气中，通过瞬时解除半月板显微以上的吸管通常可以打破不必要的附加层。备选地，外层可以通过轻轻触摸针对的硬化硅酮密封悬浮在浴中一个线程的吸管孔中被除去。

跨国激进党（瞬时受体电位）包括服务于感觉功能1,2阳离子通道七个亚科。在哺乳动物中，TRPV，跨国激进党的香草亚科，有六个品种。 TRPV4（类型4）3,4响应于热，某些化学品，渗透膨胀，或剪切应力。该TRPV4基因反复分离候选基因和/或表达克隆5-8。后一种方法遵循了基因产物的反应，低渗透压。 TRPV4表达于几乎所有的器官和功能的开发，生理学，或许多不同的细胞类型3,4的病理。 引人注目的是&gt; 50人常染色体显性TRPV4基因突变，引起周围神经病变和/或骨骼发育不良（在骨骼发育异常）9-11。骨骼发育不良的范围从轻微brachyomia 3型（3型侏儒症），spondylometaphyseal发育不良科兹洛夫斯基类型，以塞弗重新发育不良，有的引起新生儿或胚胎死亡。虽然机制的所有举止似乎不可能，没有解释的多样性，复杂性，多变性，而这些疾病4偶尔重叠。 像其他TRP通道1，TRPV4是一个四聚体。在大鼠或人TRPV4，每个子单元包括871个残基。其核心内容是六跨膜螺旋一（S1〜S6），这是有可能安排在类似电压门控K +通道的方式。还有，S1至S4形成外围域和S5和S6形成渗透毛孔领域。之间S5和TRPV4的S6是一个短孔螺旋后跟序列LDLFKLTIGMGDL，其中四个想必会聚以形成阳离子的过滤器。 470个残基的N-末端胞质段包含6个锚蛋白重复序列​​，已知结合的蛋白质或小分子配体。的C-末端152个残基的胞质片段包括其中结合OT其它可能位点的钙调蛋白结合序列她元素3。 TRPV4是，基本上不包括阴离子1阳离子通道。而其生理功能是转导的刺激与Ca 2 +内流，这也是可渗透的其它阳离子与埃森曼IV渗透性序列，在p利于钙的二价:P 娜 〜7 12。单通道电导在整流〜90 PS向外〜40 PS向内6,13,14。异源表达的电流（下图）可以通过低渗膨胀，剪切应力，或温暖15被激活。它也是由多不饱和脂肪酸16,17和合成phorbal酯4αPDD18激活。目前，最有效的激动剂是GSK1016790A 19和拮抗剂是GSK2193874，显效10 -9〜10 -8 M 20，这两个发现通过高通量的，小分子画面。 TRPV4研究的两个关键领域仍扑朔迷离:（1）即使TRPV4主要是研究其mechanosensitivity，其分子基础是有争议的。一个模型描述了低渗透压不知何故激活磷脂酶A2（PLA2），以产生不饱和脂肪酸（PUFA）花生四烯酸（AA），这是转换由一个环氧化物酶，以环氧二十碳三烯酸（EET）和EET的结合激活TRPV4 16 17。然而，TRPV4本身已被证明能直接响应膜拉伸14（下图），提供了一个简单的解释。 （2）TRPV4突变病理学是扑朔迷离。在该基础上，人们需要知道的疾病是否是由于损失，减少，或信道活动的增加。即使在这里，文学是很不明朗。虽然多个骨骼发育异常等位基因据报道，有较高的活动，（功能获得的，GOF）4,21，数据报道，有活动减少（功能丧失的，LOF）10,22。 14个等位基因的系统研究发现它们全部GOF突变（下）23。有些是外场所的说法似乎矛盾的TRPV4-/表型-基因敲除小鼠，这是可行的或肥沃，只有轻微的缺陷，尽管TRPV4功能完全丧失。 这些争议的部分有方法的起源。实验室使用不同的方法，或一种方法的变体，并使用不同的判断标准。最常见的是，TRPV4短暂表达在培养的哺乳动物细胞（HEK，CHO，HeLa细胞）和内部的[Ca 2 +]的上升时的激动剂或低渗性刺激被注册到的Ca 2 +敏感的荧光染料Fura-2的。在此荧光测定法的过度依赖已被诟病1。在不同人群中的表达水平，分布在其中，以及在有效的染料浓度，需要加以控制和记录。更可靠的是直接的电生理检查。即使是这样，由于commonlŸ练，也并非没有问题。因为在单个培养的细胞中的表达水平是难以控制的，全细胞电流有大的变化。此外，由于电流很小，可靠的统计数据将不得不依赖于大样本的大小，往往不实用。膜片钳考试却很少被执行。一些这样的唱片展团活动爆发，使统计评价具有挑战性16,17。 为了更好地理解分子mechanosensitivity，我们已经开发了两个额外的系统来检查TRPV4。 （1）要隔离TRPV4远离其一贯的哺乳动物的合作伙伴，我们在芽殖酵母24表示大鼠TRPV4。 TRPV4在这遥远的进化方面的功能表现表明，它仍然可以应对渗透力不其一贯的合作伙伴。因为酵母是没有多不饱和脂肪酸，如AA或EET，其基因组有没有磷脂酶A2或表氧化酶基因，这种EXPRE裂变也表明，对于TRPV4感知力它们不是必需的。有TRPV4在分子生物学上最适合真核生物还允许高效的正向或反向遗传操作25。 （2）为深入生物物理TRPV4的分析，我们在非洲爪蟾卵母细胞表达的TRPV4。不同的培养细胞，其产生子nA到NA（10 -10〜10 -9 A）的电流，卵母细胞表达在μA的（10 -6〜10 -4 A）的电流。在噪音大得多信号可以更好地量化和更自信的比较。 TRPV4，所以表达，也可以检测作为使用膜片钳单个分子。单卵母细胞可重复修补采样，再次使定量更可靠。这些研究表明，TRPV4通道本身可直接通过膜拉伸力14被激活。分析还表明，14代表骨骼发育不良等位基因是功能获得的所有突变。另外，DEGR此构的Ca 2 +渗漏的ee值平行的骨骼疾病23的严重性。 因为他们的新颖性和实用性，这两种方法的详细程序在这里集会，允许重复在未来的TRPV4或类似途径的研究。

<table border="0" cellpadding="0" cellspacing="0" width="595">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vapor Pressure Osmometer</td>
			<td style="width:93px;">Wescor</td>
			<td style="width:119px;">Wescor 5500</td>
			<td style="width:167px;">Logan, UT, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sirius Single Tube Luminometer </td>
			<td style="width:93px;">Titertek-Berhold</td>
			<td style="width:119px;">Sirius </td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microplate luminometer</td>
			<td style="width:93px;">Titertek-Berthold </td>
			<td style="width:119px;">Mithras LB940</td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Luminometry software</td>
			<td style="width:93px;">Titertek-Berthold</td>
			<td style="width:119px;">MikroWin2000</td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:216px;">Patch clamp and software</td>
			<td style="width:93px;">Axon Instrument</td>
			<td style="width:119px;">HS2A headstage, VG-2Ax100 virtual grounded bath clamp, GeneClamp 500 amplifier, digidata 1440 digitizer, pClamp10 software </td>
			<td>Union City, CA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">manometer</td>
			<td style="width:93px;">Bio-Tec</td>
			<td style="width:119px;"> </td>
			<td>Winooski, VT, USA</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pipet puller</td>
			<td style="width:93px;">Sutter Instrument Co</td>
			<td style="width:119px;">Model P-97</td>
			<td>Novata, CA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microinjector</td>
			<td style="width:93px;">Drummond Scientific</td>
			<td style="width:119px;">Nanoinject II</td>
			<td>Broomall, PA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Material:</td>
			<td style="width:93px;"> </td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">luciferin coelenterazine</td>
			<td style="width:93px;">Invitrogen</td>
			<td style="width:119px;"> </td>
			<td>Carlsbad, CA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">T7 RNA polymerase reaction</td>
			<td style="width:93px;">Ambion</td>
			<td style="width:119px;">mMessage mMachine</td>
			<td>Austin, TX, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gentamicin</td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ruthenium red </td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">micropipets </td>
			<td style="width:93px;">Drummond</td>
			<td style="width:119px;">100 microliter micropipets</td>
			<td>Broomall, PA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">high-fidelity PfuUtra polymerase </td>
			<td style="width:93px;">Stratagene</td>
			<td style="width:119px;"> </td>
			<td>La Jolla, CA, USA</td>
		</tr>
	</tbody>
</table>

yeast luminometric and xenopus oocyte electrophysiological examinations of the molecular mechanosensitivity of trpv4

TRPV4（瞬时受体电位，香草素家族，类型4）是广泛表达于脊椎动物的组织和由几个刺激，包括通过机械力活化。某些TRPV4基因突变导致遗传性复杂骨或神经元病症的人。野生型或突变型TRPV4转基因通常表达在培养的哺乳动物细胞并通过Fura-2的荧光和由电极检测。在mechanosensitivity与疾病的分子基础的机制方面，目前的文献是混乱和争议。为了补充现有的方法中，我们描述了另外两种方法来检查TRPV4的分子特性。 （1）大鼠TRPV4和水母发光蛋白基因转化为芽殖酵母。转化人口的低osmtic冲击产生一个发光计量信号，由于水母发光蛋白与钙离子结合，通过TRPV4通道释放。在这里，TRPV4从其一贯哺乳动物伴侣蛋白分离并揭示了其自身的mechanosensitivity。 （2）TRPV4的的cRNA注入爪蟾卵母细胞。后培养一段合适的宏观TRPV4电流检测用双电极电压钳。在去除惰性渗透剂从卵母细胞浴目前的上涨是指示mechanosensitivity的。从卵母细胞中的微安（10 -6〜10 -4 A）的电流是比从培养的细胞中的subnano到毫微安（10 -10〜10 -9 A）的电流大得多，产生更清晰的量化和更自信评估。微观电流反映个别通道蛋白质的活性也膜片钳下直接注册，在对小区或切模式。同卵母细胞提供了多个补丁的样品，允许更好的数据复制。适用于补丁吸力可以激活TRPV4直接评估mechanosensitivity。这些方法也应该是在其他类型的TRP通道的研究是有用的。

在一个典型的发光法的实验中，一系列400-1,200毫渗量低渗震荡回落是通过添加200微升不同的低渗透压冲击的解决方案，以20微升的文化在1400毫渗量来实现。转化了空p416GPDV4质粒或1，其中包含TRPV4基因有突变的离子过滤器（ 图1）24的酵母细胞:在实验的RLU进行比较，该两个阴性对照的TRPV4活性是显而易见的。 在一个典型的双电极电压钳实验中，卵母细胞的初步沐浴在含有流动等渗浴溶液（以mM计）66 KMeSO 4，1.8巴（内消旋4）2，5 K +-HEPES（pH 7.2）中和100山梨糖醇。峰值电流是在100毫秒的测试脉冲结束时从-20毫伏，每10秒的保持电位评估为20毫伏。为了激发低渗响应流程变更为山梨糖醇缺乏一个类似的解决方案。 Ŧ<html>他的低渗的响应是返回后山梨醇可逆以及可阻挡由TRP通道阻断剂钌红（ 图2A）。 在一个典型的膜片钳实验中，对称的溶液被使用， 即该溶液洗澡的补丁以及移液管填充溶液是相同的（98毫米氯化钾，1毫摩尔MgCl 2和10mM K +-HEPES，pH 7.2）中。在这里，由内向外补丁从鼠TRPV4表达的卵母细胞切除保持在+50 mV时。从注射器中产生吸力的脉冲。简要吸管，数百毫秒的持续时间，施加在几十毫米汞柱，引出〜40 PS酉电导原产于卵母细胞的膜37和异源表达的TRPV4（ 图2B）14〜90 PS酉电导。 <html> les/ftp_upload/50816/50816fig2.jpg"宽度="600px的"/&gt; 图1。大鼠TRPV4在酵母中表达，以响应低渗休克。 （A）示出的实验方法。（B）750毫渗量低渗休克（箭头）将触发大量发光的增加（相对发光单位，RLU）在TRPV4转化，而 ​​不是在一个空质粒或质粒转化轴承TRPV4在其离子滤网（M680K）（C）低渗休克和峰值响应之间的剂量-反应关系的突变（平均值±标准差，n = 3）。从2.4×10 6细胞每24测量。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50816/50816fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 0PX"/&gt; 图2。 TRPV4活动的电生理检查异源表达的爪蟾卵母细胞（A）全卵母细胞宏观电流响应审查，双电极电压钳低渗刺激。从卵母细胞表达非常高水平的野生型TRPV4峰值电流时响应于所述切除100毫秒电压的步骤（从-20至+20 mV的每10秒）（插图）（注40毫微克的cRNA后5天）从250毫渗量沐浴液（开条），并加入3微米钌红（RUR;填吧）100毫米山梨醇。明显是在回归等渗溶液或追加通道阻滞剂（RUR）的重复峰值电流增大时低渗性刺激，其跌幅（B）直接活化的野生型TRPV4通过拉伸膜膜片钳下可见。的平均质量的一个补丁从TRPV4-expreesssing卵母细胞切除，显示激活原​​始痕迹的样本VATION 60毫米汞柱抽吸（底部的迹线）应用于切除内到外的补丁在+50 mV时举行。最上面的轨迹显示在一个较快的时基显示关闭（C）之间的单一电流转换和两个开放水平（O 1和O 2）14。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50816/50816fig2highres.jpg" target="_blank">点击这里查看大图</a> 。

Watch this Scientific Journal Video about Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4 at JoVE.com

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

以补充常用的方法来研究TRPV4的两个方法被描述:其mechanosensitivity可以通过在低渗挑战的转基因酵母的Ca 2 +-水母发光蛋白发光法进行研究。它也可以用在TRPV4-RNA检测注入非洲爪蟾卵母细胞的全细胞双电极电压钳或膜片钳在对小区或切模式。

酵母发光计量和<em&gt;爪蟾</emTRPV4的分子Mechanosensitivity的&gt;卵母细胞电生理检查

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including ...

yeast-luminometric-xenopus-oocyte-electrophysiological-examinations

Nanyang Technological University

Research

JoVE Journal

Biology

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

10.0K 次观看.  University of Wisconsin – Madison. 以补充常用的方法来研究TRPV4的两个方法被描述:其mechanosensitivity可以通过在低渗挑战的转基因酵母的Ca 2 +-水母发光蛋白发光法进行研究。它也可以用在TRPV4-RNA检测注入非洲爪蟾卵母细胞的全细胞双电极电压钳或膜片钳在对小区或切模式。 

视频: Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

一个现场的固定化酵母细胞可视化的快速技术

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

科研

生物学

Molecular Evolution of the Tre Recombinase

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. 
We started with Cre, a 38-kDa recombinase, that recognizes a 34-bp double-stranded DNA sequence known as loxP. Because Cre can effectively eliminate genomic sequences, we set out to tailor a recombinase that could remove the sequence between the 5'-LTR and 3'-LTR of an integrated HIV-1 provirus. As a first step we identified sequences within the LTR sites that were similar to loxP and tested for recombination activity. Initially Cre and mutagenized Cre libraries failed to recombine the chosen loxLTR sites of the HIV-1 provirus. As the start of any directed molecular evolution process requires at least residual activity, the original asymmetric loxLTR sequences were split into subsets and tested again for recombination activity. Acting as intermediates, recombination activity was shown with the subsets. Next, recombinase libraries were enriched through reiterative evolution cycles. Subsequently, enriched libraries were shuffled and recombined. The combination of different mutations proved synergistic and recombinases were created that were able to recombine loxLTR1 and loxLTR2. This was evidence that an evolutionary strategy through intermediates can be successful. After a total of 126 evolution cycles individual recombinases were functionally and structurally analyzed. The most active recombinase -- Tre -- had 19 amino acid changes as compared to Cre. Tre recombinase was able to excise the HIV-1 provirus from the genome HIV-1 infected HeLa cells (see "HIV-1 Proviral DNA Excision Using an Evolved Recombinase", Hauber J., Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany). While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of "molecular surgery" and molecular medicine.

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of molecular surgery and molecular medicine.

TRE重组酶的分子进化

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within ...

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

补丁钳爪蟾卵母细胞中表达的离子通道

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

可视化的RNA中的本地化爪蟾卵母细胞

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

生产转基因非洲爪蟾限制性内切酶介导的整合和核移植

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

条形码酵母图书馆竞争力的基因屏幕

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

高活性酵母核糖体的色谱分离纯化

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

绿色怪物承载多基因缺失的酵母菌株的产生过程

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

该<em&gt;爪蟾</em&gt;卵母细胞切割开凡士林隙电压钳技术与荧光

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

酵母发光计量和

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