这项研究得到了台湾的赠款 (科学技术部, 多数 100-2923-002-002-MY3 和 106-2313-b-002-011-MY3 H.J.L.)、捷克共和国 (南波希米亚大学赠款机构、GAJU 赠款 065/2017/P y.h. L) 和西班牙 (西班牙经济和竞争力部, 赠款 CGL2012-36251 和 CGL2015-64727-P 王啸波和加泰罗尼亚政府, 赠款 2014 SGR 619 王啸波);它还得到欧洲经济和区域发展基金 (菲德基金王啸波) 的财政支助。

1. dsRNA 的合成和制备
<ol><li>确定目标基因 3 ' 未翻译区域中的 dsRNA 目标点。dsTub 用于靶向α蛋白 (盆) 基因 (基因库加入号: KX228233), dsEGFP 作为负 dsRNA 控制是设计从序列的增强绿色荧光蛋白 (EGFP;基因库加入号码: LC311024)。</li>
	<li>进行标准 PCR 扩增, 合成 dsRNA 模板与基因特异引物包含 T7 启动程序序列 (5 '-TAATACGACTCACTATAGGG-3 ')。PCR 扩增条件和引物信息是由林et报告的。(2017)12 (请参见材料表)。</li>
	<li>按照制造商的说明, 使用 T7 转录套件 (参见材料表) 进行 dsRNA 准备。 注意:要产生高数量的 dsRNA, 混合两个反应在一个管 (总共40µL), 并孵化在37°c 过夜。</li>
	<li>将2µL 的 DNase (参见材料表) 添加到 dsRNA 样本中, 15 分钟37摄氏度, 以消化 DNA 模板。</li>
	<li>添加200µL 萃取试剂 (参见材料表) 和40µL 氯仿, 然后是涡流。</li>
	<li>离心机为10分钟在 1.3万 x g 在4°c, 然后收集上清 (150 µL) 在一个新的离心管。</li>
	<li>在冰上加入150µL 异丙醇并孵化15分钟, 沉淀 dsRNA。</li>
	<li>离心机15分钟在 1.3万 x g 在4°c, 然后去除上清。</li>
	<li>添加200µL 70% 乙醇, 以洗涤 dsRNA 丸。离心机为10分钟在 1.3万 x g 在4°c。</li>
	<li>除去乙醇, 重复乙醇洗涤步骤。</li>
	<li>用离心真空浓缩器去除乙醇和干 dsRNA 颗粒3分钟, 然后并用重悬 dsRNA 无 RNase 水。</li>
	<li>用微体积紫外-可见光分光光度计 (参见材料表) 确定纯化 dsRNA 的数量, 并根据制造商的说明调整到所需浓度 (例如, 0.25 µg/µL, 以准备dsRNA lipoplexes)。</li>
	<li>将 dsRNA 样品存放在摄氏-20 摄氏度, 长达3月。</li>
</ol>2. dsRNA Lipoplexes 的制备
<ol><li>通过添加4µL 5% 葡萄糖溶液稀释4µL 的转染试剂 (参见材料表)。通过添加4µL 5% 葡萄糖溶液稀释4µL 的 dsRNA (1 µg)。</li>
	<li>立即将稀释脂质体试剂溶液加入稀释后的 dsRNA 溶液中。漩涡简单地混合, 然后孵育15分钟在25摄氏度。 注意:不要以相反的顺序混合解决方案。</li>
	<li>dsRNA lipoplexes 已经准备好使用了。使用1小时内准备, 并在1小时后丢弃。 注意:根据制造商的指示, 应尽快使用 lipoplexes。</li>
</ol>3. 从血淋巴和中肠果汁中收集胞外酶
<ol><li>
昆虫盐水缓冲液 (10x 库存)
	<ol>
<li>混合900毫升双蒸馏水, 109.3 克氯化钠, 15.7 克氯化钾, 6.3 克 CaCl2, 8.3 g 氯化镁2· 6H2O。</li>
		<li>将 pH 值调整为 6.8, 并填充至1000毫升。</li>
	</ol>
</li>
	<li>
血淋巴收集
	<ol>
<li>麻醉在冰上的蟑螂, 直到他们不移动3分钟。</li>
		<li>用两个手指 (拇指和食指) 抓住蟑螂, 把蟑螂的腹侧打开。</li>
		<li>使用解剖剪刀切断后腿髋部的尖端。 注意:切断髋和转子之间的连接交叉口。另一部分的髋部切除术对收集血淋巴的效率较低。</li>
		<li>用中指轻轻挤压腹部, 用10µL 微从切口收集血淋巴出血。</li>
		<li>从几个蟑螂 (5 个人) 的血淋巴池中离心管。 注意:把离心管放在冰上以防黑化。从一只雄性蟑螂身上获得的血淋巴平均容积为 2-3 µL。</li>
		<li>十年代用台式离心机简单地旋转血淋巴。</li>
		<li>转移所需血淋巴量 (即, 10 µL), 并将其与50µL 1x 昆虫盐水缓冲液混合。</li>
		<li>离心机为10分钟在 1000 x g 在4°c 下包囊。将上清液转移到干净的离心管上。</li>
		<li>通过测量 A28015, 用微体积紫外-可见光分光光度计定量测定总蛋白浓度。</li>
		<li>调整每个样品具有相同的蛋白质浓度 (6 毫克/µL 的总蛋白)。</li>
	</ol>
</li>
	<li>
中肠果汁系列
	<ol>
<li>麻醉把蟑螂放在冰上。用昆虫引脚将蟑螂腹侧用冷1x 昆虫盐缓冲器固定在解剖板上。</li>
		<li>用细镊子解剖腹部。将整个肠道 (包括前, 中, 后肠道) 移到新鲜的盘子里, 1x 昆虫盐缓冲。</li>
		<li>取出前肠和后肠道, 迅速将中肠转移至含有100µL 昆虫盐水缓冲液的离心管。</li>
		<li>midguts 从六只蟑螂在同一管, 和漩涡十年代。</li>
		<li>离心机为10分钟, 在 1000 x g 在4°c, 包囊和肠道组织的旋转。将上清液转移到干净的离心管上。</li>
		<li>通过测量 A28015, 用微体积紫外-可见光分光光度计定量测定总蛋白浓度。</li>
		<li>调整每个样品具有相同的蛋白质浓度 (6 毫克/µL 的总蛋白)。</li>
	</ol>
</li>
</ol>4. dsRNA 降解试验
<ol><li>新鲜地准备4µL 赤裸 dsRNA 或 dsRNA lipoplexes (1 µg)。</li>
	<li>混合 dsRNA 溶液与10µL 的昆虫盐水缓冲 (控制), 提取酶的血淋巴, 或中肠果汁。</li>
	<li>添加2µL EGTA (20 毫米) 或无 RNase 水, 以分离样品作为控制酶活性的抑制。</li>
	<li>孵育1小时或更长 (6, 12, 或24小时) 在25°c。</li>
	<li>加入200µL 萃取试剂和40µL 氯仿, 然后涡流。</li>
	<li>离心机为10分钟在 1.3万 x g 在4°c, 然后收集上清 (150 µL) 在一个新的离心管。</li>
	<li>在冰上加入150µL 异丙醇并孵化15分钟, 沉淀 dsRNA。</li>
	<li>离心机15分钟在 1.3万 x g 在4°c, 然后去除上清。</li>
	<li>添加200µL 70% 乙醇, 以洗涤 dsRNA 丸。离心机为10分钟在 1.3万 x g 在4°c。</li>
	<li>除去乙醇, 重复乙醇洗涤步骤。</li>
	<li>用离心真空浓缩器将乙醇和干 dsRNA 颗粒去除3分钟并用重悬 dsRNA, 10 µL 无 RNase 水。</li>
	<li>在1.5% 琼脂糖凝胶上通过凝胶电泳检查 dsRNA 的完整性。</li>
</ol>5. dsRNA 的口服管理
<ol><li>在广告随意饮食中保持蟑螂 (狗周)。在 dsRNA 摄入实验前一天, 从蟑螂那里夺走了水的供应。</li>
	<li>用挠性钳抓住它的翅膀 (图 1,步骤 5) 来抓蟑螂。 注意:不要抓蟑螂的身体部位。</li>
	<li>准备 dsRNA lipoplexes, 以及裸 dsRNA, 以供喂养, 如第2节所述。 注意:dsRNA lipoplexes 应在进食前准备新鲜。</li>
	<li>饲料4µL dsRNA lipoplexes 或裸 dsRNA (250 吴) 与微。</li>
	<li>慢慢地吸管 dsRNA 溶液的水滴靠近蟑螂的口器, 让蟑螂完全摄取水滴。</li>
	<li>每天进行两次摄取实验 (1 小时后灯亮起, 1 小时熄灯前) 持续8天或16天。 注意:所有蟑螂获得水只有通过手动喂养 dsRNA 溶液或控制试剂 (例如, 核酸酶免费水, 葡萄糖溶液, 脂质体试剂) 在摄取实验。</li>
	<li>在16天的 dsRNA 实验后, 给蟑螂提供水瓶。</li>
</ol>6. 评估击倒
<ol><li>在选择的时间点 (2, 9, 17 天后 dsRNA 摄取化验), 收集 dsEGFP 和 dsTub 处理的昆虫和解剖所选择的组织 (例如, 中肠)。</li>
	<li>进行定量实时 pcr (qRT pcr)。qRT-PCR 扩增条件和引物信息的报告, 林et al。(2017)12。</li>
	<li>每天评估控制和 dsRNA 治疗的昆虫, 以检查死亡率和清除不再移动的蟑螂。</li>
</ol>

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作者没有什么可透露的。

本协议提出了一种有效的 rna 干扰方法, 通过口服 dsRNA lipoplexes, 包括防止核糖核酸消化在中肠果汁的德国蟑螂。正如在其他昆虫种类的研究中所显示的, 通过口服 dsRNA 的干扰效应很差, 主要是由于 dsRNA8,9,10的退化。该协议产生脂质体, 作为保护性的工具, 在口头交付 dsRNA 抗降解的肠道。此外, dsRNA lipoplexes 的制备方法简单, 易于应用于口服 rna 干扰, 特别是对肠道内具有较强核糖核酸活性的昆虫。
与其他的研究相比, 喂养相对大量的裸 dsRNA 在不同的昆虫10,18,19, 这个协议使用相对较低数量的 dsRNA (每天0.5 µg) 口服管理, 这导致了B.) 中肠靶基因表达的显著耗竭该议定书的关键方面包括精确的喂养方法和少量 dsRNA 的累计摄取量。首先, 该饲养技术适用于连续摄取试验, 并最大限度地减少每种昆虫摄取 dsRNA 量的变化。第二, 在中肠中,浴盆表达式的损耗效应从9天的40% 日增加到了17天的 60%, dsTub lipoplexes 的连续口服管理 (图 3A)。尽管裸 dsRNA 在 1 h (图 2A) 内的中肠果汁的前体孵化中迅速退化, 但裸 dsRNA 连续喂养16天也导致了浴盆表达式的大量耗尽。在中肠 (图 3A) 中。此外, dsRNA lipoplexes 持续喂养时间延长16天, 是造成明显致死效应的要求。
尽管脂质体载体 (参见材料表) 最初是用于体外将 DNA 转染成昆虫细胞, 但在我们的昆虫模型中, 应用这种脂质体试剂作为口服 dsRNA 的载体是没有问题的。许多研究也成功地证明了利用不同的脂质体试剂来改善其他昆虫种类的干扰性沉默, 这是为 DNA 或 RNA 分子设计的17,20,21。然而, 由于其与磷脂结构的生物相容性, dsRNA 在昆虫肠道中的吸收机制还没有得到完全的研究, 脂质体的输送系统可以提高 dsRNA 对细胞的吸收, 这是因为它具有22。因此, 在脂质体中封装的 dsRNA 的口服交付可能是一种适当的策略, 以实现有效的 rna 干扰, 由于 RNases 退化的延迟 (图 2)。
该协议的一个缺点可能是脂质体载体中的少量 dsRNA, 因此可能需要更长的连续喂养时间。dsRNA 与脂质体 (0.25 µg: 1 µL) 之间的这种特殊比例的限制是由于商业脂质体试剂的设计, 根据制造商的建议。报告了在制备大直径尺寸的脂质体纳米粒子和 lipoplexes 表面不同电荷的配方, 以增加 siRNA 和 rna 干扰沉默效率的封装,23,24。因此, 不同脂质体纳米粒子也可用于改善喂养程序。

rna 干扰已被证明是一种有效的方法来击倒基因表达通过一个机制的转录后沉默途径触发的 dsRNA 分子在许多真核生物1。在过去的十年中, rna 干扰已经成为一个有用的工具来研究基因从发展到行为的功能, 通过消耗特定基因的表达, 通过注射和/或喂养 dsRNA 在昆虫的各种分类器2,3。由于损耗效应的特殊性和鲁棒性, 目前正在将 rna 干扰的应用视为害虫控制管理的潜在策略4,5。然而, 在昆虫种类之间, rna 干扰的效率差别很大, 这取决于不同基因的靶向和传递方法。越来越多的证据表明, 由 ribonucleases 降解的 dsRNA 的不稳定性是 rna 干扰5、6的有限功效的一个关键因素。例如, Manduca 天蛾中的低干扰敏感度被解释为: 与血淋巴混合的 dsRNA 在1小时的7中迅速退化。同样, 中肠碱性核酸的存在, 有效地降低了摄取 dsRNA, 在不同的昆虫顺序中与低 rna 干扰效率有很强的相关性8,9,10。
dsRNA 的口头交付对于在害虫防治战略中应用 rna 干扰特别有趣, 但尚未开发出一种延缓中肠核酸降解 dsRNA 的方法, 这将有可能确保有效通过喂食进行 rna 干扰。然而, dsRNA 的口服的对停止响应是通过喂养大量的 dsRNA ,例如50µg在家蚕, 或连续喂养8天 (总共8µg dsRNA) 的蝗虫物种。德国蟑螂,德国 germinica, 对通过 dsRNA11,12,13,14的注入而对 rna 的干扰非常敏感, 但对 dsRNA 通过喂食没有响应。最近, 林等。(2017) 证明了脂质体载体封装的 dsRNA 成功地进行了 rna 干扰, 以击倒中肠中的α蛋白基因表达, 并触发德国蟑螂15的显著死亡率。由于中肠 dsRNA 的降解是口服 rna 干扰的限制因素, 脂质体载体作为一种保护 dsRNA 免受降解的载体, 在肠道内具有较强核酸酶活性的其他昆虫中很容易被应用。值得注意的是, 选择特定转染试剂的原因 (参见材料表) 我们作为脂质体载体在目前的协议是, 它已被测试的昆虫细胞线转染较少的毒性, 根据制造商的说明。通过对不同脂质体转染系统的比较 Gharavi et 。(2013)16中, 转染小干扰 RNA (siRNA) 的效率大约与其他昆虫17、18中用于 dsRNA 传送系统的其他商用系统之间的效果大致相同..此外, 我们的喂养方法是足够小心, 以确保每只蟑螂摄取适量的 dsRNA, 结果是稳健的和确认的。总之, 本协议和结果表明, 使用 dsRNA lipoplexes 提高了 dsRNA 的稳定性, 为 rna 干扰的策略设计开辟了大门, 这是今后害虫防治的一个有前途的途径。

<table border="0" cellpadding="0" cellspacing="0" style="width:1124px;" width="1123">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">GenJe Plus DNA in vitro Transfection reagent</td>
			<td style="width:133px;">SignaGen</td>
			<td style="width:157px;">SL100499</td>
			<td style="width:481px;">for lipoplexes preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blend Taq plus</td>
			<td style="width:133px;">TOYOBO&#160;</td>
			<td style="width:157px;">BTQ-201</td>
			<td>for PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast SYBR Green Master Mix</td>
			<td style="width:133px;">ABI&#160;</td>
			<td style="width:157px;">4385612</td>
			<td>for qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">FirstChoice RLM-RACE Kit</td>
			<td style="width:133px;">Invitrogen</td>
			<td style="width:157px;">AM1700</td>
			<td>for 3&#39; UTR identification&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">MEGAscript T7 Transcription Kit</td>
			<td style="width:133px;">Invitrogen</td>
			<td style="width:157px;">AMB13345</td>
			<td>for dsRNA synthesis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TURBO DNase</td>
			<td style="width:133px;">Invitrogen</td>
			<td style="width:157px;">AM2239</td>
			<td>remove DNA template from dsRNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRIzol</td>
			<td style="width:133px;">Invitrogen</td>
			<td style="width:157px;">15596018</td>
			<td>for dsRNA or total RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RQ1 RNase-Free Dnase</td>
			<td style="width:133px;">Promega</td>
			<td style="width:157px;">M6101</td>
			<td>remove DNA template from total RNA&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">chloroform&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">C2432</td>
			<td>for dsRNA or total RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">2-Propanol</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">I9516</td>
			<td>for dsRNA or total RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>24102</td>
			<td>for dsRNA or total RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Diethyl pyrocarbonate, DEPC</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">D5758</td>
			<td>for RNase free water preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">glucose solution</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">G3285</td>
			<td>for lipoplexes preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride, NaCl</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">S7653</td>
			<td>insect saline buffer formula</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Potassium chloride, KCl</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">P9333</td>
			<td>insect saline buffer formula</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:352px;">Calcium chloride, CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C1016</td>
			<td>insect saline buffer formula</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Magnesium chloride hexahydrate, MgCl2.6H2O</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">M2670</td>
			<td>insect saline buffer formula</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">EGTA&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">E3889</td>
			<td>enzyme inhibitor&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dissecting scissor</td>
			<td style="width:133px;">F.S.T.</td>
			<td style="width:157px;"></td>
			<td>cockroach dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fine tweezers</td>
			<td style="width:133px;">F.S.T.</td>
			<td style="width:157px;"></td>
			<td>cockroach dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">flexible tweezer</td>
			<td style="width:133px;">F.S.T.</td>
			<td style="width:157px;"></td>
			<td>cockroach holding&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pipetman</td>
			<td style="width:133px;">RAININ</td>
			<td style="width:157px;">P10</td>
			<td>sample preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microcentrifuge tube</td>
			<td style="width:133px;">Axygen</td>
			<td>MCT175C, PCR02C</td>
			<td>sample preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">pipette tip&#160;</td>
			<td style="width:133px;">Axygen</td>
			<td></td>
			<td>sample preparation</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">vortexter</td>
			<td style="width:133px;">Digisystem</td>
			<td style="width:157px;">vm1000</td>
			<td>sample preparation</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Minispin centrifuge</td>
			<td style="width:133px;">The Gruffin Group</td>
			<td style="width:157px;">GMC 206</td>
			<td>for liquid spin down&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td style="width:133px;">ALC</td>
			<td style="width:157px;">PK121R</td>
			<td>sample preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pH meter&#160;</td>
			<td style="width:133px;">JENCO</td>
			<td style="width:157px;">6071</td>
			<td>for pH adjust</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">micro-volume spectrophotometer</td>
			<td style="width:133px;">Quawell</td>
			<td style="width:157px;">Q3000</td>
			<td>nucleic acid quantitative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">PCR Thermal cycler</td>
			<td style="width:133px;">ABI&#160;</td>
			<td style="width:157px;">2720</td>
			<td>for template PCR or&#160; dsRNA synthesis incubation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">quantitative real-time PCR</td>
			<td style="width:133px;">ABI&#160;</td>
			<td style="width:157px;">StepOne plus</td>
			<td>gene expression quantitative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifugal Vacuum Concentrators</td>
			<td style="width:133px;">eppendorf</td>
			<td style="width:157px;">5301</td>
			<td>for dsRNA or total RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multipette&#160;</td>
			<td style="width:133px;">eppendorf</td>
			<td style="width:157px;">xstream</td>
			<td>for real-time PCR sample loading&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose I</td>
			<td style="width:133px;">amresco</td>
			<td style="width:157px;">0710</td>
			<td>for nucleic acid electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">tub gene specfifc forward preimer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>GGG ACA AGC CGG AGT GCA GA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">tub gene specfifc reverse preimer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>TCC TGC TCC TGT CTC GCT GA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">dsTub template forward primer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>TAA TAC GAC TCA CTA TAG GGA CAA GCC GGA GTG CAG&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">dsTub template reverse primer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>TAA TAC GAC TCA CTA TAG GGT CCT GCT CCT GTC TCG CTG&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">dsEGFP template forward preimer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>TAA TAC GAC TCA CTA TAG GGT ATG GTG AGC AAG GGC GAG GAG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">dsEGFP template reverse preimer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>TAA TAC GAC TCA CTA TAG GGT GGC GGA TCT TGA AGT TCA CC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">tub qPCR forward primer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>GGA CCG CAT CAG GAA ACT GGC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">tub qPCR reverse preimer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>CCA CAG ACA GCC TCT CCA TGA GC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">ef1 qPCR forward primer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>CGC TTG AGG AAA TCA AGA AGG A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">ef1 qPCRreverse preimer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>CCT GCA GAG GAA GAC GAA G</td>
		</tr>
	</tbody>
</table>

practical use of rna interference: oral delivery of double-stranded rna in liposome carriers for cockroaches

rna 干扰在揭示众多基因的生物学功能方面得到了广泛的应用, 并被设想为一种害虫控制工具, 其主要基因表达被打乱。虽然报告了不同的方法, 如注射, 喂养和浸泡, 成功地交付双链 RNA (dsRNA), 通过口服 dsRNA 的 rna 干扰的效率在不同的昆虫群中是高度可变的。德国蟑螂, 德国的大蠊, 对 dsRNA 的注射高度敏感, 正如此前发表的许多研究显示的那样。本研究描述了用脂质体载体包裹 dsRNA 的方法, 足以延缓中肠果汁对 dsRNA 的降解。值得注意的是, 脂质体包覆的 dsRNA 的连续喂养大大降低了中肠蛋白的表达, 导致了蟑螂的死亡。总之, dsRNA lipoplexes 的配制和利用, 保护 dsRNA 免受核酸的危害, 是今后害虫防治的一种实用方法。

在图 1中介绍了 dsRNA 口头交付协议的简化方案, 其中显示了准备 dsRNA lipoplexes 的关键步骤。
为了研究脂质体载体对 dsRNA 降解的保护作用, 对B...................dsTub lipoplexes 在中肠果汁中进行了一种前体检测, dsRNA随后对1.5% 琼脂糖凝胶进行了分析。图 2显示了在B. 小蠊(图 2A) 中的中肠果汁中存在强 RNA 核酸酶活动, 而脂质体载体能够保护 dsRNA 抗降解至少在前体内1小时内孵化 (图 2A, B)。因此, 口服 dsRNA lipoplexes 每天两次, 以增加中肠组织对 rna 干扰的敏感性在以下实验。
通过测量在连续摄取 dsRNA (图 3A) 后不同时间点的中肠内的浴盆mRNA 表达式的损耗效应, 评估了 dsRNA 口服后的 rna 干扰反应的有效性。在连续摄取 dsTub lipoplexes 8 天后, 中肠中的浴盆表达明显耗尽。虽然连续喂养的 dsRNA 持续了16天, 但中肠的浴盆表达在裸 dsTub 和 dsTub lipoplexes 组中明显耗尽 (分别约为24和 60%), 相比之下对照。图 3B显示在口服 dsTub lipoplexes 16 天后死亡率的持续、显著增加。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57385/57385fig1.jpg"> 图 1: 为蟑螂 dsRNA lipoplexes 的口服分娩简化方案.准备 dsRNA lipoplexes 的关键步骤为步骤1至4。送料技术 (步骤 5) 显示在照片中。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57385/57385fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57385/57385fig2.jpg"> 图 2:前体dsRNA 由 B... 淋巴 (血液) 或中肠果汁.(A) 1 µg 裸或脂质体-cojugated dsTub, 分别以盐水缓冲剂和从血淋巴 (血液) 或中肠果汁中提取酶的方法孵化1小时。裸 dsTub, 但不是脂质体共轭 dsTub, 完全降解时, 1x 中肠果汁孵化。指出了原中肠果汁 (1x) 的不同稀释因子。由于 EGTA 处理的控制, 离子螯合抑制了降解。(B) 对脂质体封装的 dsTub (1 µg) 的时间依赖性保护, 以防止血淋巴或中肠果汁中的体内降解。注意, 脂质体共轭 dsTub 在24小时后与中肠果汁一起孵化后完全降解。血淋巴和中肠果汁用于培养不同时间段的原始浓度。结果由林et调整。(2017)12.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57385/57385fig2large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57385/57385fig3.jpg"> 图 3: 通过 dsRNA 的摄取对B... ..。(A) 定量实时 pcr (qRT pcr) 用于确定德国蟑螂在不同喂养处理后中肠内的相对浴盆表达式。在2天, 不同治疗方法的表达水平规范化为葡萄糖组。价值是从三独立实验 (n = 3) 中的平均值, 每个都有 3-5 生物复制。在不同治疗组中, 条形图上的不同字母表示在p < 0.05 (方差分析后 Tukey HSD 后) 之间存在显著差异。(B) 吞食裸 dsTub 或 dsTub lipoplexes 8 天 (8 d) 或16天 (16 d) 的蟑螂存活率 (每组12至15人; n = 3 个独立实验)。价值观是卑鄙的. 治疗组的不同字母表明, 在p < 0.05 (克鲁斯卡尔-沃利斯测试) 中, 存活率存在显著差异。结果由林et调整。(2017)12.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57385/57385fig3large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>

Watch this Scientific Journal Video about Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches at JoVE.com

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

rna 干扰的实际应用: 口服双链 rna 在脂质体载体为蟑螂

本手稿通过口服双链 RNA 包裹在脂质体中, 证明了德国蟑螂中肠内基因表达的耗竭。

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool ...

practical-use-rna-interference-oral-delivery-double-stranded-rna

Nanyang Technological University

Research

JoVE Journal

Biology

9.3K 次观看.  National Taiwan University. 本手稿通过口服双链 RNA 包裹在脂质体中, 证明了德国蟑螂中肠内基因表达的耗竭。

视频: Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

在体外转录和Gaussia荧光素酶基因的上限由HeLa细胞转染

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

科研

生物学

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA纯化的染色质隔离（CHIRP）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

CRISPR / CAS系统内切酶的底物生成

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

使用屏幕在车身尺寸规例中的线虫基因介导的RNA干扰取食策略<em&gt; C.线虫</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

核糖体RNA的蚊子肠道宏基因组RNA-seq的枯竭

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been made in identifying the components of the editosome complex that catalyze RNA editing. However, it is still not clear how those proteins work together. Chemical compounds obtained from a high-throughput screen against the editosome may block or affect one or more steps in the editing cycle. Therefore, the identification of new chemical compounds will generate valuable molecular probes for dissecting the editosome function and assembly. In previous studies, in vitro editing assays were carried out using radio-labeled RNA. These assays are time consuming, inefficient and unsuitable for high-throughput purposes. Here, a homogenous fluorescence-based &#8220;mix and measure&#8221; hammerhead ribozyme in vitro reporter assay to monitor RNA editing, is presented. Only as a consequence of RNA editing of the hammerhead ribozyme a fluorescence resonance energy transfer (FRET) oligoribonucleotide substrate undergoes cleavage. This in turn results in separation of the fluorophore from the quencher thereby producing a signal. In contrast, when the editosome function is inhibited, the fluorescence signal will be quenched. This is a highly sensitive and simple assay that should be generally applicable to monitor in vitro RNA editing or high throughput screening of chemicals that can inhibit the editosome function.

A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

RNA催化剂作为记者为筛选药物对RNA编辑在锥虫

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been ...

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle&#8217;s body cavity.
In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

RNA interference (RNAi)-based gene knockdown techniques are at the core of Tribolium research. Here, we provide an overview of our larval RNAi technique in Tribolium castaneum. Larval RNAi is a simple, but powerful technique that provides quick access to loss-of-function phenotypes, allowing researchers to study gene functions in diverse contexts.

幼虫RNA干扰在赤拟谷盗，<em&gt;赤拟谷盗</em

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully ...

Production of Double-stranded DNA Ministrings

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ministrings possess a heightened safety profile and also efficiently deliver DNA cargo to targeted cells. Conventional DNA vectors carry undesirable prokaryotic sequences, including antibiotic resistance genes, CpG motifs, and bacterial origins of replication, which may lead to the stimulation of host immunological responses. The bioavailability of conventional DNA vectors is also compromised due to their larger molecular size. Their circular nature may also impart chromosomal integration, leading to insertional mutagenesis.
Bacterial sequences are excised from DNA minivectors, leaving only the gene of interest (GOI) and necessary eukaryotic expression elements. Our LCC DNA minivectors, or DNA ministrings, are devoid of immunogenic bacterial sequences; therefore improving their bioavailability and GOI expression. In the event of vector integration into the chromosome, the LCC DNA ministring will lethally disrupt the host chromosome, thereby removing the potentially dangerous mutant from the proliferating cell population. Consequently, DNA ministrings offer the benefits of &#39;minicircle&#39; DNA while eliminating the potential for undesirable vector integration events. In comparison to conventional plasmids and their isogenic circular covalently closed (CCC) counterparts, DNA ministrings demonstrate superior bioavailability, transfection efficiency, and cytoplasmic kinetics - they thus require lower amounts of cationic surfactants for effective transfection of target cells.
We have constructed a one-step inducible in vivo system for the production of DNA ministrings in Escherichia coli that is simple to use, rapid, and scalable.

The linear covalently closed (LCC) DNA minivector (DNA ministring) is a non-viral gene delivery vector offering high transfection efficiency and is relevant to any DNA delivery application. The production system is simple, rapid, and scalable in vivo. The following protocol provides visual step-by-step instructions for the production of DNA ministrings.

双链DNA Ministrings的生产

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

RNA结合蛋白的检测通过<em&gt;体外</em在脂肪细胞培养&gt; RNA下拉

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

tchip-seq: 细胞专用表观体分析

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...