这项工作部分得到了瓦格宁根大学基金（WUF）、中国研究生奖学金理事会计划以及NWO-VIDI赠款12378的支持。

1. 植物生长和测试条件
<ol><li>在18-22 °C的温度范围内或在受控温室或气候室中种植和维持植物，并在自然光照照下或以16小时/8小时的昼夜为系统。去除辅助分支，使植物更容易管理。</li>
	<li>对于马铃薯，在无菌塑料罐 中保持体外 植物，其中含有 村希格-斯库格 （MS）介质（20克/升蔗糖）， 5 g/L MS 含维生素的盐，8 克/升，pH 5.8），在 18 °C 的气候室受控条件下，使用 16 小时/8 小时的日夜制度，持续两周，然后将其转移到受管制温室的消毒土壤盆中。</li>
</ol>2. 农业渗透
<ol><li>植物材料: 对于 尼科蒂亚娜本塔米亚纳，使用约4-5周大的种子生长植物。对于马铃薯，使用约4-5周大的 体外移植。选择年轻、健康、发育的叶子进行渗透。</li>
	<li>
农用细菌 文化:<ol>
<li>提前准备 YEB 介质（表 1）。用 10 毫升 YEB 介质填充 50 毫升管子，辅以 1μl 乙毒素 （200 mM）、100μl MES 缓冲器 （2-（N-莫索利诺）-硫酸乙烷、1 M） 和适当的抗生素。皮佩特 20μl 甘油库存所需的菌株 （表2） （包含感兴趣的基因） 进入 YEB.同时为此测定中的所有 农用细菌 菌株启动培养。在 28 °C 和 200 rpm 下摇动 1-2 天，孵化细胞，以约1.0 的 OD 600 进行孵化。</li>
		<li>通过离心在3，000 x g下收获细胞10分钟。倒掉超自然物，用新制作的MMA介质（表3）将颗粒重新注入0.3的OD600。 轻轻涡流细胞以恢复它们。对于两种细菌菌株的硬币渗透，将培养物按1:1的比例混合。</li>
		<li>在渗透前，在室温下将培养物放在长凳上1-6小时。同时，给被渗透的植物贴上标签，并标注实验日期。</li>
	</ol>
</li>
	<li>渗透: 使用1毫升无针注射器渗透 到农用细菌 悬架中。小心而缓慢地将叶板与注射器上的悬架一起注射（出于健康和安全原因，在此过程中应佩戴眼睛保护）。每个菌株至少渗透三株植物。每株使用三片叶子作为三叶。 注:通常，1毫升 的农用细菌 悬浮剂可能足够3株 N.本塔米亚纳。对于马铃薯，需要更多的悬浮，因为 索拉努姆 物种的叶子更难渗透。所需的悬浮量取决于 索拉努姆 物种。通过更换手套或在渗透之间用乙醇消毒手套，以及直到接种后的第二天才为植物浇水，避免交叉污染。</li>
	<li>得分: 当细胞死亡表型清晰时，在渗透后3天左右的评分宏观反应（图1）。如果细胞死亡表型是定量的，请使用给定标准（图 2）。简言之，细胞死亡的宏观评分比例主要取决于渗透区域的细胞死亡百分比。细胞死亡的百分比从0%（无症状）到100%（细胞死亡）。中间反应范围从微弱反应，如氯化症到细胞死亡水平的增加。如有需要，还可以使用现代光像设备对宏观评分进行定量评估。</li>
</ol>3. PVX 农业感染
<ol><li>植物材料: 对于 N.本塔米亚纳，使用大约2-3周大的种子生长的植物。对于马铃薯，使用大约2-3周大的 体外移植。对于大规模测试，使用稍老（4-5周）的植物，因为这些植物有越来越多的叶子，以适应更多的 农用细菌 接种点。</li>
	<li>
农用细菌 培养:<ol>
<li>提前准备 YEB 介质。用3毫升YEB填充10毫升管，并辅以适当的抗生素。皮佩特 20μl 甘油库存所需的菌株 （表2） （包含感兴趣的基因） 进入 YEB.同时为此测定中的所有 农用细菌 菌株启动培养。在 28 °C 和 200 rpm 下摇动 1-2 天，孵化细胞，以约1.0 的 OD 600 进行孵化。</li>
		<li>每个 农用细菌 菌株约300μl，并将其传播到LB固体阿加中板，辅以适当的抗生素和孵育细胞在28°C 1-2天。</li>
	</ol>
</li>
	<li>感染: 用铲子收集盘子里的 农用细菌 培养物。在刮刀上浸入 农用细菌 培养物中的木制牙签，刺穿叶子以接种大量细菌。为每株至少接种三株植物。每株使用三片叶子作为三叶。在每片叶子中，为每株进行多个接种位点。</li>
	<li>得分: 接种后两周左右的宏观反应得分（图3）。对于高通量屏幕，记录每个接种点的定性响应（是/否）。然后计算响应站点的百分比，并将它们与控件进行比较。</li>
</ol>

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作者宣称他们没有相互竞争的经济利益。

农业渗透和农用感染等瞬态检测是现代分子植物病理学研究的重要有效方法。尽管存在一些限制，但这些方法满足了工厂对高效、稳健的高通量功能分析的需求。
农用渗透系统是一种在一系列植物物种中广泛使用的功能检测。农业渗透促进将几个转基因基因同时表达相互作用的蛋白质输送到同一细胞中。这有利于通过硬币渗透农用菌株来概括R-AVR关系，用表达匹配R基因的菌株来表达AVR基因。此外，对于已知的R-AVR对，此类硬币胶片可用作正控件。包括这种控制很重要，因为在某些植物基因型中，转化效率可能低于检测响应的阈值。包括负对照，例如含有没有插入基因的载体的农用细菌菌株，对于确定某种植物基因型是否对农用细菌产生非特异性防御反应也至关重要。此功能在马铃薯种质中以一定频率发生，并非所有索拉努姆物种都非常适合这种基于农用细菌的表达系统。一般来说，农用渗透检测在N.本塔米亚纳和大多数马铃薯基因型中效果很好。除了效应学之外，农业渗透技术还有其他各种潜在应用，例如通过共聚焦显微镜从转基因中产生蛋白质和植物细胞中的蛋白质本地化。
PVX 农业感染是一种高度敏感的筛查系统，通常更适合高通量筛查。由于 农用细菌 现在只存在于本地，因此，由于PVX病毒接管了转基因的进一步传播，对这种细菌的非特异性反应现在不是很令人不安。然而，植物可能对PVX具有抗药性，或具有极端抗药性（ER）反应，在这种情况下，农业感染方法不合适。PVX农用感染方法的另一个局限性是兴趣基因的插入大小。观察到的反应表型可能因伤口周围强烈的黑色坏死而不同，到接种点附近的微弱坏死。在 N.本塔米亚纳 和 索拉努姆 物种中，PVX农业感染被公认为比农业渗透更敏感。
考虑到不同测试植物基因型的遗传背景可能有一些限制（见上文），我们通常通过PVX农业感染和农业渗透得出类似的结论。这些结果与其他检测结果（如蛋白质渗透29 和ELISA3）也相当。考虑到两个系统的优点和局限性，我们建议使用这两种方法来补充或确认独立的结果。

效应学，一种高通量功能基因组学方法最近已成为识别作物中抗药性（R）基因和匹配病原体1-4的avirence（Avr）基因的有力工具。与R基因更耗时的稳定转化相比，效应定向策略基于病原体基因序列的瞬时检测。
自基因组学时代以来，植物病原体的基因组得到了广泛的探索。例如，对于包括最具破坏性的植物病原体在内的卵母细胞，已经生成并分析了大量序列，以寻找在与植物5-10相互作用期间发挥作用的基因。一类病原蛋白代表效应器，它操纵宿主细胞结构和功能，或促进感染（毒性因素）或触发防御反应（病毒因子）11-13。含有R基因的植物细胞中AVR基因的表达通常会导致超敏感细胞死亡反应（HR）14，15。在植物表达的R和Avr基因可以完成使用瞬态表达系统，如农用细菌的图姆法西恩为基础的瞬态转化（农业渗透）16。这种瞬态转化也可以与病毒表达系统（农业感染）17，18相结合应用。
对于农业过滤，最常用的剂是 A.tmefaciens， 一种广泛的多能植物的宿主范围病原体。 A. 肿瘤 含有肿瘤诱导（Ti）质粒。在细菌的毒力机制被激活后，从Ti质粒转移DNA（T-DNA）将转移到植物细胞中。这可以在受伤的植物细胞中触发，由释放的低分子量酚类化合物和单糖在微酸环境中19。在 农用细菌 悬浮物渗透到主要静脉定义的叶板中后，毒性基因被激活。然后，叶板中的植物细胞将被转化，并表达T-DNA区域所含的转基因。
农业感染是基于伤口接种的农用细菌，它调解病毒转移到植物细胞。然后，在没有农用细菌的情况下，病毒进一步扩散到邻近的植物组织。对于农业感染，可以使用多种植物病毒。RNA病毒是基因表达的理想载体，因为它们可以在受感染的植物中繁殖到非常高的水平。在植物RNA病毒中，马铃薯病毒X（PVX）被广泛用于效应学屏幕。为了便于插入基因的功能测试，含有PVX基因组的二元载体被克隆成A.tumefaciens20的T-DNA。。T-DNA被转移到植物细胞后，T-DNA中所含的PVX基因组从35S促进者转录。然后病毒颗粒在受感染的植物中系统地传播，导致插入基因的表达。这种基于农用细菌和PVX的方法称为PVX农业感染。
在这里，我们展示了农业渗透和PVX农业感染检测的例子。作为宿主植物，我们使用马铃薯种质（索拉努姆部分佩托塔），为此，效应学方法已被开创和证明成功3，4。我们还使用尼科蒂亚娜本塔米亚纳，这是著名的模型工厂在索拉纳塞斯植物14，21，22。

<table border="0" cellpadding="0" cellspacing="0" width="955">
	<colgroup>
		<col />
		<col />
		<col span="2" />
	</colgroup>
	<tbody>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Beef extract</td>
			<td style="width:267px;">Sigma-Aldrich</td>
			<td style="width:219px;">B4888</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Bacteriological peptone</td>
			<td style="width:267px;">Oxoid</td>
			<td style="width:219px;">LP0037</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Yeast extract</td>
			<td style="width:267px;">Oxoid</td>
			<td style="width:219px;">LP0021</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">MgSO4</td>
			<td style="width:267px;">Sigma-Aldrich</td>
			<td style="width:219px;">208094</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">MS salts (without vitamins)</td>
			<td style="width:267px;">Duchefa Biochemie</td>
			<td style="width:219px;">M0221</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">MES</td>
			<td style="width:267px;">Duchefa Biochemie</td>
			<td style="width:219px;">M1503</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">LB broth powder</td>
			<td style="width:267px;">Sigma-Aldrich</td>
			<td style="width:219px;">L3022</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Acetosyringone</td>
			<td style="width:267px;">Sigma-Aldrich</td>
			<td style="width:219px;">D134406</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Syringe (1 ml)</td>
			<td style="width:267px;">BD Plastipak</td>
			<td style="width:219px;">300013</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Incubator</td>
			<td style="width:267px;">Infors HT</td>
			<td style="width:219px;">Multitron II</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Centrifuge</td>
			<td style="width:267px;">Heraeus</td>
			<td style="width:219px;">Multifuge 3S-R</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Spectrophotometer</td>
			<td style="width:267px;">Eppendorf</td>
			<td style="width:219px;">Biophotometer 6131</td>
			<td style="width:219px;"></td>
		</tr>
	</tbody>
</table>

agroinfiltration and pvx agroinfection in potato and nicotiana benthamiana

农业渗透和PVX农业感染是植物候选基因功能分析的两种有效的瞬态表达检测。农用渗透最常用的剂是 农用细菌， 这是许多迪科特植物物种的病原体。这意味着农业渗透可以应用于许多植物物种。在这里，我们介绍我们的协议和预期的结果时，应用这些方法的马铃薯（索拉努姆管），其相关的野生块茎轴承 索拉努姆 物种（索拉努姆 节 佩托塔）和模型植物 尼科蒂亚娜本塔米亚纳。除了对单个基因（如抗药性（R）或抗病毒（Avr）基因）进行功能分析外，农用渗透检测非常适合通过简单地将 R 和 Avr 转基因引入同一细胞来回顾与特定宿主病原体相互作用相关的 R-AVR 相互作用。然而，一些植物基因型可以提高非特异性防御反应 的农用细菌，正如我们观察到的，例如几个马铃薯基因型。与农业渗透相比，PVX农用感染的AVR活性检测更敏感，功能屏幕的吞吐量更高，对 农用细菌的非特异性防御反应的敏感度较低。但是，PVX 可能发生非特定防御，并且由于病毒引起的极端耐药性而有可能错过响应。尽管有这些限制，但根据我们的经验，农业渗透和PVX农业感染都是适当和互补的检测，可以同时用于确认彼此的结果。

图1显示了马铃薯和N.本塔米亚纳的7种不同效应剂（E1-E7）的农业渗透有代表性的实验。细胞死亡出现在渗透后约3天的渗透叶板。细胞死亡表型的程度需要与控制器进行比较。农用细菌株AGL1（pVirG）23的混合物含有双增生-R3a和pK7WG2-AVR3a用作正对照24，25，而 AGL1（pVirG） 用作负控制。图1A和1B分别显示了马铃薯基因型MaR8（马斯滕布罗克R8）26和N.本塔米亚纳的农业过滤的良好例子，它们非常适合农业过滤。叶板中含有与阳性控制相同的细胞死亡，而负控制或 pBINplus-R3a 则不会发生细胞死亡反应。在MaR8中，两个效应器AVR3a（E2）和AVR4（E3）诱发细胞死亡，而其他效应器（E1和E4-E7）则不会。 在N.本塔米亚纳，没有一个测试的效应器能诱导细胞死亡反应。图1C和1D显示了野生马铃薯索拉努姆泊位483-1和索拉努姆雷切210-5的农业渗透的例子，这些例子对农用渗透技术不太适应。在S.伯斯托蒂483-1中，叶组织对负控件以及所有测试的效应器表现出非特异性坏死。在S.rechei 210-5中，渗透的叶板显示细胞死亡对阳性控制的反应非常微弱。
图2 显示了一系列的评分尺度，可用于量化对农业渗透 性农用细菌的反应。细胞死亡的百分比以0-100%的比例描述。观察到的表型范围从宏观上不可见的症状（0%），到显示氯化和细胞死亡水平增加的一系列中间反应，最高可达汇合细胞死亡（100%）。
图3显示了马铃薯PVX农用感染后的一个具有代表性的实验。通常，在拔牙接种后两周左右，这些部位可以发现细胞死亡扩大。如两个图面板所示，扩大细胞死亡存在于用pGR106-CRN2（使用效应器皱褶器Crn2进行正控制）的拔牙接种部位，这是来自P.侵扰者27的一般细胞死亡诱导基因。除了对伤害的轻微反应外，在用pGR106空（负控制）接种牙齿的部位没有注意到细胞死亡扩大。在图3A中，代表马铃薯基因型MaR3（马斯滕布鲁克R3），两个pGR106效应器（E1和E2）没有诱发细胞死亡。在图3B中，介绍了在野生索拉努姆万坎本斯354-1的效果筛选的阳性结果:细胞死亡反应观察到两个pGR106效应器（E1-E3，代表引出）28。
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/50971/50971fig1highres.jpg" src="/files/ftp_upload/50971/50971fig1.jpg"> 图1。马铃薯和本塔米亚纳的农业渗透的例子。植物包括 （A） 马铃薯基因型马 R8 （马斯滕布罗克 R8）， （B） N. 本塔米亚纳， （C）索拉努姆伯托尔蒂483-1 和 （D）索拉努姆雷切210-5 被渗透与混合物农用菌株AGL1（pVirG）23含有双增-R3a和pK7WG2-AVR3a（正控制 ）、AGL1（pvirG）（ 负控制）、双增-R3a、 和七个效应器（E1-E7）。<a href="https://www.jove.com/files/ftp_upload/50971/50971fig1highres.jpg" target="_blank">单击此处查看更大的图像</a>。
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/50971/50971fig2highres.jpg" src="/files/ftp_upload/50971/50971fig2.jpg"> 图2。农业渗透反应量化。照片显示细胞死亡的代表性评分表，从0%（无症状）到100%（共生细胞死亡）不等。中间反应范围从微弱反应，如氯化症到细胞死亡水平的增加。 <a href="https://www.jove.com/files/ftp_upload/50971/50971fig2highres.jpg" target="_blank">单击此处查看更大的图像</a>。
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/50971/50971fig3highres.jpg" src="/files/ftp_upload/50971/50971fig3.jpg"> 图3。马铃薯中PVX农业感染的例子。马铃薯基因型 MaR3 （马斯滕布罗克 R3）（A）和 索拉努姆万卡班本斯 354-1（B） 拔牙接种 pGR106-CRN2 （正控制）， pGR106 空 （负控制） 和 pGR106-E1-2 （效果）， 或 pGR106-E1-E3， <a href="https://www.jove.com/files/ftp_upload/50971/50971fig3highres.jpg" target="_blank">单击此处查看更大的图像</a>。
表1。叶布介质
<table border="1"><tbody>
<tr>
<td>1 L</td>
			<td>蒸馏H2O</td>
		</tr>
<tr>
<td>5 克</td>
			<td>蔗糖</td>
		</tr>
<tr>
<td>5 克</td>
			<td>牛肉提取物</td>
		</tr>
<tr>
<td>5 克</td>
			<td>细菌肽</td>
		</tr>
<tr>
<td>1 克</td>
			<td>酵母提取物</td>
		</tr>
<tr>
<td>2 毫升</td>
			<td>MgSO4 (1 M)</td>
		</tr>
</tbody></table>表2。用于农业渗透和PVX农业感染的载体和菌株。可以使用多个二进制向量。下面列表中的载体允许高表达候选基因，并且在我们手中运作良好。我们更喜欢使用农用菌株GV3101（pMP90）29在N.本塔米亚纳，并发现AGL130含有帮助质粒pVirg（pBBR1MCS-5.virGN54D）23更适合土豆31。其他组已经分析了各种模型植物的其他菌株，包括N.本塔米亚纳，但不是在马铃薯32。
<img alt="Table 2" fo:content-width="5in" fo:src="/files/ftp_upload/50971/50971table2highres.jpg" src="/files/ftp_upload/50971/50971table2.jpg">
GW: 网关版本36
表3。妈妈介质。 <img alt="Table 3" fo:content-width="5in" fo:src="/files/ftp_upload/50971/50971table3highres.jpg" src="/files/ftp_upload/50971/50971table3.jpg">
将pH口调整为5.6。

Watch this Scientific Journal Video about Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana at JoVE.com

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

农业渗透和PVX农业感染是植物中基因瞬时异位表达的常规功能检测。这些方法是有效电理学策略（快速抗药性和抗病毒基因发现）的有效分析，对分子植物病理学的现代研究至关重要。它们满足了工厂对强健的高通量功能分析的需求。

马铃薯和尼科蒂亚娜本塔米亚纳的农业渗透和PVX农业感染

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

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Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

20.2K 次观看.  Wageningen University. 农业渗透和PVX农业感染是植物中基因瞬时异位表达的常规功能检测。这些方法是有效电理学策略（快速抗药性和抗病毒基因发现）的有效分析，对分子植物病理学的现代研究至关重要。它们满足了工厂对强健的高通量功能分析的需求。

视频: Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

病毒诱导的基因沉默（VIGS）烟草benthamiana和番茄

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

科研

生物学

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

高级别瞬态表达重组蛋白的植物高效的农杆菌

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

优化和利用<em&gt;农杆菌</em&gt;介导的瞬态蛋白生产中<em&gt;烟草</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

大会和"剪切环"中的应用:一种新型内皮模型轨道，单向和定期流体流动和剪应力

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

荧光互补成像检测在烟草烟叶中瞬时测定蛋白质-蛋白质相互作用动力学

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

Beneficial plant-associated bacteria play an important role in promoting growth and preventing disease in plants. The application of plant growth-promoting rhizobacteria (PGPR) as biofertilizers or biocontrol agents has become an effective alternative to the use of conventional fertilizers and can increase crop productivity at low cost. Plant-microbe interactions depend upon host plant-secreted signals and a reaction hereon by their associated bacteria. However, the molecular mechanisms of how beneficial bacteria respond to their associated plant-derived signals are not fully understood. Assessing the transcriptomic response of bacteria to root exudates is a powerful approach to determine the bacterial gene expression and regulation under rhizospheric conditions. Such knowledge is necessary to understand the underlying mechanisms involved in plant-microbe interactions. This paper describes a detailed protocol to study the transcriptomic response of B. mycoides EC18, a strain isolated from the potato endosphere, to potato root exudates. With the help of recent high-throughput sequencing technology, this protocol can be performed in several weeks and produce massive datasets. First, we collect the root exudates under sterile conditions, after which they are added to B. mycoides cultures. The RNA from these cultures is isolated using a phenol/chloroform method combined with a commercial kit and subjected to quality control by an automated electrophoresis instrument. After sequencing, data analysis is performed with the web-based T-REx pipeline and a group of differentially expressed genes is identified. This method is a useful tool to facilitate new discoveries on the bacterial genes involved in plant-microbe interactions.

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

The goal of the protocol presented here is to study the transcriptomic response of endosphere-isolated Bacillus mycoides to potato root exudates. This method facilitates the identification of important bacterial genes involved in plant-microbe interactions and is in principle applicable to other endophytes and plants, with minor adjustments.

植物-微生物相互作用:丝状芽孢杆菌对马铃薯根系分泌物的转录反应

Beneficial plant-associated bacteria play an important role in promoting growth and preventing disease in plants. The application of plant ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

使用两步PCR和下一代16S rRNA基因测序进行微生物群分析

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing need in developing efficient methods for recombinant antibody production. As of 2019, there were more than 70 FDA-approved monoclonal antibodies, and there is exponential growth potential. Despite their promise, limiting factors for widespread use are manufacturing costs and complexity. Potentially, plants offer low-cost, safe, and easily scalable protein manufacturing&#160;strategies. Plants like Nicotiana benthamiana not only can correctly fold and assemble complex mammalian proteins but also can add critical post-translational modifications similar to those offered by mammalian cell cultures. In this work, by using native GFP and an acid-stable variant of green fluorescent protein (GFP) fused to human monoclonal antibodies, we were able to visualize the entire transient antibody expression and purification process from N. benthamiana plants. Depending on the experiment&#39;s purpose, native GFP fusion can ensure easier visualization during the expression phase in the plants, while acid-stable GFP fusion allows for visualization during downstream processing. This scalable and straightforward procedure can be performed by a single researcher to produce milligram quantities of highly pure antibody or antibody fusion proteins in a matter of days using only a few small plants. Such a technique can be extended to the visualization of any type of antibody purification process and potentially many other proteins, both in plant and other expression systems. Moreover, these techniques can benefit virtual instructions and be executed in a teaching laboratory by undergraduate students possessing minimal prior experience with molecular biology techniques, providing a foundation for project-based exploration with real-world applications.

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

We describe here a simple method for expression, extraction, and purification of recombinant human IgG fused to GFP in Nicotiana benthamiana. This protocol can be extended to purification and visualization of numerous proteins that utilize column chromatography. Moreover, the protocol is adaptable to the in-person and virtual college teaching laboratory, providing project-based exploration.

在尼科蒂亚娜本 thamiana中瞬态表达IgG融合蛋白的生产

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing ...