我们感谢彭博公共卫生学院生物化学与分子生物学系的安东尼 k.l. 梁, 他在设计 miR-181-sponge 结构方面的技术帮助。我们还感谢分子和比较病理部的宝琳娜 Sysa 和凯瑟琳 Gabrielson, 约翰霍普金斯医疗机构的技术援助, 通过体内成像的 miRNA 海绵分娩。
这项工作得到了 NIH、HL39752 (查尔斯 Steenbergen) 的赠款和美国心脏协会 14SDG18890049 (Samarjit Das) 的科学家发展补助金的支持。在辛辛那提儿童医院, 杰弗里 d. Molkentin 慷慨地提供了鼠心特异性启动子。

所有实验程序均经约翰霍普金斯大学机构动物护理和使用委员会批准。
1. 海绵设计
<ol><li>
MicroRNA 绑定 3 ' UTR 注: miRNA 函数通过特定的基配对与部分互补点在其目标基因的 3 ' 未翻译区域 (UTR) 中的相互作用 (为全面审查, 见巴特尔)15。<ol>
<li>设计 miRNA 表达抑制剂作为寡核苷酸, 对 miRNA 序列具有显著的互补性。通过广泛的基配对将 miRNA 在寡核苷酸复合体中, 以阻止 miRNA 功能。</li>
	</ol>
</li>
	<li>
miRNA 海绵的设计
	<ol>
<li>设计 tandemly 阵列部分互补 miRNA 序列, 包括一个凸起的位置通常由 Argonaute 2。凸起位于成熟 miRNA 序列的核苷酸 9-12, 以防止任何 RNA 干扰类型的分裂和海绵的降解。</li>
		<li>如果需要, 设计诱饵目标序列绑定到所有 miR-181 家庭成员。在检查和比较 miR-181 家庭 miRNA 序列, 发现一个共同的序列在 5 ' 边的凸起包含8个连续核苷酸, 或者发现一个序列共同的4个 miR-181 家庭成员在 3 ' 边膨胀的另外8连续核苷酸。这2个序列分别表示左和右半绑定站点。 注: 诱饵序列是由 GGA 间隔分隔的左右半绑定站点的串联数组。将 5 '-端序列和 3 ' 端序列 (在步骤1.2.2 中描述) 连接在一起, 其设计为在退火到 miRNA 时创建凸起 (如步骤1.2.1 中所述)。重复这 1 miRNA 绑定站点10x 在最后的和平号-海绵结构。</li>
	</ol>
</li>
	<li>
其他 miRNA 海绵的注意事项 注意: 为了将海绵序列复制到接收者表达式向量中, 必须在序列的每一端包含限制核酸酶识别站点。在硅消化分析中, 应使用所选择的限制酶和在下游克隆应用中使用的其他限制酶来确定海绵序列的非特异裂解。<ol>
<li>在合成 3 ' UTR 的 5 ' 端添加一个双停止密码子 (TAA TAA), 这样 miR-181-sponge 序列不是增强的绿色荧光蛋白 (EGFP) 编码序列的一部分。</li>
	</ol>
</li>
	<li>
克隆海绵 DNA 的合成
	<ol>
<li>使用 DNA 合成器或利用商业可用的来源, 以合成最终 miRNA 海绵序列与限制核酸酶识别网站到位, 克隆。海绵被运送到一种质粒上, 可以包含可选择的标记, 用于放大细菌。此外, 海绵可以通过聚合酶链反应 (PCR) 来进行克隆, 或者使用步骤1.3 中描述的限制点 (也见步骤 3.1) 来简单地丢弃捐赠向量。</li>
	</ol>
</li>
</ol>2. 克隆受体载体, 包括组织特定的启动子
注: 使用 pEGFP-C1 向量作为 miRNA 海绵表达盒的接收向量。pEGFP-C1 向量包含一个 EGFP 由巨细胞病毒 (CMV) 启动子驱动的阅读框架。巨细胞病毒启动子是组成性活跃在哺乳类。如果需要组织特定的启动子, 则可以通过消化 AseI 和 NheI 酶来去除巨细胞病毒启动子 (见步骤 2.1)。该质粒包含一个广泛的多克隆站点 (MCS) 和卡那霉素 (菅直人) 和新霉素 (G418) 标记的细菌选择和一个稳定的表达在哺乳动物细胞, 分别。
<ol><li>
从 pEGFP-C1 中去除巨细胞病毒启动子
	<ol>
<li>制作50的消化反应 ul, 其中含有1x 商业上可用的消化缓冲剂, 所用的限制酶的制造, 5 微克的质粒 DNA (在水中), 5 ul 每 AseI 和 NheI 酶。将所有组件添加到离心管中, 然后轻轻地将其混合。十年代在离心中旋转管子以收集底部的反应。在37摄氏度的水浴中孵化反应15分钟。</li>
		<li>纯化经消化的线性化质粒。添加5x 列绑定缓冲区的反应量 (用于制造 dna 纯化柱的适当绑定缓冲区的专有组合), 并按照制造商描述的 dna 纯化柱纯化反应。洗脱与 25 ul 洗脱缓冲 (水) 添加仔细到中心的专栏。</li>
		<li>凝胶纯化0.5% 琼脂糖凝胶上的消化质粒, 如下所示: 在洗脱质粒上添加5的负载缓冲 (50% glycerol-3x 加载染料) ul。在0.5% 琼脂糖凝胶上将整个样品装入1井。包括一个单独的车道与500吴未切割的载体。运行 30-40 分钟, 直到达到适当的分离切割和未切割的质粒。</li>
		<li>对线性化质粒进行纯化: 将琼脂糖凝胶中的 DNA 可视化为 UV 光盒。用干净的剃刀刀片, 仔细地使用与线性质粒对应的带。 注: 线性质粒应低于未切割的质粒, 在大约 4.2 kb 时将显示为单波段。该切除的巨细胞病毒启动子将出现作为一个光带在大约500核苷酸 (nt)。</li>
		<li>利用商业上可用的试剂盒从凝胶切片中提取 dna, 并洗脱 25 ul 的水柱上的 dna。</li>
	</ol>
</li>
	<li>
将心脏特定的启动子克隆到 pEGFP-C1 中 注: 阿尔法肌球蛋白重链 (α MHC) 启动子以前的特点和证明, 以直接的强健水平的心脏限制表达, 如以下 Molkentin, 乔布, 和锦马16。接下来的部分描述了如何放大克隆的向量的启动子。<ol>
<li>克隆的α MHC 启动子之间的 +420 至-2934 之间的转录起始点到 p40 质粒16首先设计 pcr 底漆, 侧面这个区域, 然后质粒作为 pcr 模板。使用 PCR 引物放大启动子区域以前描述的5。正向和反向 PCR 引物包含重叠序列的 pEGFP-C1 (步骤 2.1) 的消化结束, 以允许融合定向克隆。请参阅融合手册, 以全面解释融合克隆的底漆设计。底漆序列在表 1中找到。</li>
		<li>准备一个 50 ul PCR 反应, 其中包含1微米的每一个底漆和 10 ng 的模板 DNA。根据所选 pcr 试剂的制作, 添加适当数量的 pcr 酶和 dNTPs。在标准 DNA 循环块上进行 PCR。执行3分钟98°c 变性步骤, 其次为5、30和120s 的35个变性、退火和延伸周期分别。包括最后延长10分钟在72°c。</li>
		<li>清理 PCR 和凝胶萃取。在 pcr 循环完成后, 将柱状结合缓冲器的反应体积加5x 到 pcr 反应中, 并按照制造商描述的 DNA 纯化柱纯化。洗脱与 25 ul 的洗脱缓冲 (水) 的混合物, 小心地添加到柱的中心。<ol>
<li>添加5的负载缓冲 ul [50% 甘油 (3x) 加载染料] 到洗脱质粒。在1% 琼脂糖凝胶上将整个样品装入1井。运行 30-40 分钟;如上文所述, 可视化和消费 PCR 产品 (步骤 2.1.5)。</li>
		</ol>
</li>
		<li>结扎α MHC 启动子与 pEGFP-C1 如下: 添加 10 ul 结扎反应, 包括1x 商用结扎缓冲, 2 ul 的纯化 PCR, 1 ul 的线性化向量。执行结扎在50°c 15 分钟, 然后冷却它在冰上。</li>
		<li>执行细菌转化如下: 染 50 ul 的合格细胞与 2.5 ul 的融合结扎混合通过添加成分。把离心管放在冰上20分钟, 放在42摄氏度的水浴中, 四十年代, 然后把管子放在冰上2分钟。<ol>
<li>转换后, 添加 350 ul 的 SOC 介质和生长细胞在37°c 45 分钟. 板 200 ul 在 LB 板上与菅直人. 执行菌落 PCR 鉴定含有α MHC 启动子结扎的抗菅细胞。 注: 筛选底漆的设计, 以 PCR 横跨结扎连接。</li>
		</ol>
</li>
		<li>用标准的微型预制备质粒纯化协议, 以制造商描述的方法, 在 LB-坎汤中扩增 PCR 阳性克隆。</li>
	</ol>
</li>
</ol>3. 海绵的克隆
<ol><li>
摘要收件人向量
	<ol>
<li>用α-MHC-pEGFP 质粒线性克隆 EcoRI 和 KpnI, 并纯化上述凝胶 (步骤 2.1)。</li>
	</ol>
</li>
	<li>
PCR 放大和消化海绵 DNA
	<ol>
<li>使用 miR-181-sponge 和相应的 284 nt 长的争夺航运载体作为 PCR 模板。使用先前描述的 PCR 引物5放大海绵区域。注意, 正向和反向 PCR 引物含有限制酶序列, 允许结扎成α-MHC-pEGFP-C1。执行 PCR 如上文所述 (步骤 2.2.2)。在消化前纯化 PCR 产物 (步骤 2.2.3), 并洗脱水进行消化。对于底漆序列, 见表 1.
</li>
		<li>消化海绵 DNA 进行结扎。50 ul 消化反应包括1x 消化缓冲, 凝胶纯化 PCR 反应从步3.2.1 和 5 ul EcoRI 和 KpnI 酵素。将所有组件添加到离心管中, 然后轻轻地将其混合。十年代在离心中旋转管子以收集底部的反应。在37摄氏度的水浴中孵化反应15分钟。</li>
		<li>使用 pcr 清理柱纯化消化后的 pcr 海绵 (步骤 2.2.1)。</li>
	</ol>
</li>
	<li>
结扎 miR-181-sponge 成α-MHC-pEGFP C1 向量
	<ol>
<li>建立 21 ul 结扎反应, 其中包含1x 结扎缓冲, 3 ul 的消化和纯化 miR-181-sponge PCR, 1 ul 的线性α MHC-pEGFP C1 向量, 1x dna 缓冲, 1 ul 的 dna 连接酶。在室温下进行结扎5分钟, 然后放在冰上。</li>
		<li>50 ul 的 XL1-blue 主管细胞与 2 ul 的结扎组合。使用以上所述的转换和电镀协议 (步骤 2.2.5)。进行菌落 PCR 鉴定含有正确的α-MHC-pEGFP-181 海绵序列的耐药性细菌。设计筛选引物的 PCR 横跨结扎连接。见表 1为底漆序列。</li>
		<li>放大和序列的向量。用标准的微型预制备质粒纯化协议, 在 LB 汤中扩增 PCR 阳性克隆, 并将其纯化成粒。进行 dna 测序以验证表达所需 dna 序列的质粒。</li>
	</ol>
</li>
</ol>4. 生成稳定的 H9c2 miR-181-sponge 表达细胞
<ol><li>
H9c2 细胞的生长与维持
	<ol>
<li>培养 H9c2 细胞在 Dulbecco 的改良鹰的培养基 (DMEM) 含有胎牛血清 (血清) 到最后浓度为10%。亚文化细胞使用标准的协议, 并在5% 二氧化碳37摄氏度增长。</li>
	</ol>
</li>
	<li>
H9c2 α-pEGFP-C1-181-海绵表达细胞的转染与选择
	<ol>
<li>对 H9c2 细胞进行电穿孔, 以取得最佳效果。染大鼠成肌细胞 (H9c2), 用一只爬行海绵 (一个 284 nt 长的争夺序列取代 miR-181-sponge-sequence) 或α-MHC-pEGFP 和平号-181 海绵结构与 electroporator。<ol>
<li>简单地, 染 4x10-5细胞与2微克的质粒 DNA (在5µL 的 H2O) 和100µL 的解决方案兼容的电穿孔装置。使用 DS-120 的电穿孔程序染 H9c2 细胞。</li>
			<li>室温下在试管中孵育转染细胞10分钟。将500µL DMEM 直接添加到试管。将总试管内容轻轻地转移到6井板的井中。</li>
		</ol>
</li>
		<li>选择荧光蛋白阳性细胞后, 48 小时后转染的外地资产管制。仅板绿色 GFP 表达的细胞入6井板材与增加新霉素 (G418) (400 ng/毫升) 到完全成长媒介。 注意: 在 G418 选择之前, 应建立一个终止曲线, 以确定选定内容所需的新霉素量。对于 H9c2 细胞, 400 G418 的80% 的非质粒转染 H9c2 细胞死亡率约为24小时后, 被认为是这些细胞所期望的 G418 工作浓度。在 G418 维持16天后, 细胞线被认为是稳定的。</li>
		<li>用 GFP 的表达作为海绵阳性选择的标记, 对 G418 稳定细胞进行分类。通过流动排序, 在96井板的每一个井中种 5-10 个细胞。将单克隆细胞从96井板扩展到24井板上的多个通道。使用冷冻储存的细胞从通道 3 (P3) 细胞作为后续实验的出发点。</li>
		<li>在 P5 和 P10 之间执行所有基于细胞线的低通道细胞实验。</li>
	</ol>
</li>
</ol>5. 海绵纳米微粒的合成和纯化 (miR-181-sponge 纳米粒子)
<ol><li>将阳离子 amphiphile (DOTAP) 和共脂、胆固醇和 DSPE 钉在 5:5: 0.1 毫米的比值中, 分别用一个玻璃瓶中的氯仿和甲醇混合物制备脂质体纳米颗粒。</li>
	<li>在真空下使用旋转蒸发器将有机溶剂去除 44-45 摄氏度, 其次是温和流动的无水分氮气。将剩余的脂质干膜保持在高真空下8小时。</li>
	<li>在真空干脂膜中加入5% 葡萄糖来制备混合物。让它过夜, 让这种混合物水合物的电影。在室温下将瓶子旋转2到3分钟, 以便在 45° C 水浴中偶尔摇动, 产生 multilamellar 泡。<ol>
<li>油脂实验 multilamellar 泡在冰浴中准备小 unilamellar 泡, 3-4 分钟, 直到可以观察到清晰度, 在100% 的工作周期和 25 W 输出功率。</li>
	</ol>
</li>
	<li>将 miR-181-sponge 序列或克隆成 pEGFP (α-MHC) 载体和脂质体的一组混合序列与1:3 电荷比基础, 形成 nanovector4。 注: 有正电荷的脂质体纳米粒子和带负电荷的粒 DNA 的静电复合物被称为 nanovector。</li>
</ol>6. 海绵纳米微粒的全身输送和体内效应的验证
<ol><li>
海绵纳米颗粒在大鼠体内的系统传递
	<ol>
<li>使用大 (SD) 大鼠。静脉注射 miR-181-sponge nanovector 的大鼠, 或通过尾静脉 nanovector。使用 SD 雄性大鼠, 其体重约为200克。</li>
		<li>使用4毫克 nanovector/千克体重的剂量进行尾静脉注射, 并在2周内使用6尾静脉注射方案进行重复。在重复 nanovector 交付后, 允许1周 (天 15-21) 在体内表达载体。</li>
		<li>通过在 nanovector 交付之前、期间和之后对 GFP 信号进行成像, 确认优化。利用层析成像软件对 GFP 信号进行分析, 生成半定量数据。</li>
	</ol>
</li>
	<li>
miR-181-sponge 对体内miR-181 抑制作用的验证
	<ol>
<li>执行西方印迹, 以证明在心脏组织的功能 miR-181-sponge 的表达。 注: 本研究对 mt-COX1 表达进行了检查。</li>
	</ol>
</li>
	<li>
心脏体内miR-181-sponge 表达的功能后果
	<ol>
<li>在 nanovector 分娩前后进行二维、M 型和多普勒超声心动图分析心脏功能和形态学改变。为了更好的扫描能力, 在啮齿动物的心脏, 使用一个超声系统配备 15 MHz 线性阵列传感器。</li>
		<li>麻醉可能影响心脏收缩力;因此, 在超声心动图过程中, 执行 miR-181-sponge nanovector 或争夺 nanovector 的动物, 并无任何麻醉试剂。请见 Das,等等。4进一步澄清。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Hammond, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=microRNA+detection+comes+of+age.">microRNA detection comes of age.</a> Nat Methods. 3 (1), 12-13 (2006).</li><li>van Rooij, E., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+powerful+new+regulators+of+heart+disease+and+provocative+therapeutic+targets.">MicroRNAs: powerful new regulators of heart disease and provocative therapeutic targets.</a> The Journal of Clinical Investigation. 117 (9), 2369-2376 (2007).</li><li>Das, S., 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=Nuclear+miRNA+regulates+the+mitochondrial+genome+in+the+heart.">Nuclear miRNA regulates the mitochondrial genome in the heart.</a> Circulation Research. 110 (12), 1596-1603 (2012).</li><li>Das, S., 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=miR-181c+regulates+the+mitochondrial+genome,+bioenergetics,+and+propensity+for+heart+failure+in+vivo.">miR-181c regulates the mitochondrial genome, bioenergetics, and propensity for heart failure in vivo.</a> PLoS One. 9 (5), e96820 (2014).</li><li>Das, S., 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=Divergent+effects+of+miR-181+family+members+on+myocardial+function+through+protective+cytosolic+and+detrimental+mitochondrial+microRNA+targets.">Divergent effects of miR-181 family members on myocardial function through protective cytosolic and detrimental mitochondrial microRNA targets.</a> Journal of the American Heart Association. 6 (3), e004694 (2017).</li><li>Sicard, F., Gayral, M., Lulka, H., Buscail, L., Cordelier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+miR-21+for+the+therapy+of+pancreatic+cancer.">Targeting miR-21 for the therapy of pancreatic cancer.</a> Molecular Therapy. 21 (5), 986-994 (2013).</li><li>Jopling, C. L., Yi, M., Lancaster, A. M., Lemon, S. M., Sarnow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+hepatitis+C+virus+RNA+abundance+by+a+liver-specific+microRNA.">Modulation of hepatitis C virus RNA abundance by a liver-specific microRNA.</a> Science. 309 (5740), 1577-1581 (2005).</li><li>Lanford, R. E., 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=Therapeutic+silencing+of+microRNA-122+in+primates+with+chronic+hepatitis+C+virus+infection.">Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection.</a> Science. 327 (5962), 198-201 (2010).</li><li>Ucar, A., 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=The+miRNA-212/132+family+regulates+both+cardiac+hypertrophy+and+cardiomyocyte+autophagy.">The miRNA-212/132 family regulates both cardiac hypertrophy and cardiomyocyte autophagy.</a> Nature Communications. 3, 1078 (2012).</li><li>Zhu, X., 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=Identification+of+micro-RNA+networks+in+end-stage+heart+failure+because+of+dilated+cardiomyopathy.">Identification of micro-RNA networks in end-stage heart failure because of dilated cardiomyopathy.</a> Journal of Cellular and Molecular Medicine. 17 (9), 1173-1187 (2013).</li><li>Bandiera, S., 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=Nuclear+outsourcing+of+RNA+interference+components+to+human+mitochondria.">Nuclear outsourcing of RNA interference components to human mitochondria.</a> PLoS One. 6 (6), e20746 (2011).</li><li>Barrey, E., 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=Pre-microRNA+and+mature+microRNA+in+human+mitochondria.">Pre-microRNA and mature microRNA in human mitochondria.</a> PLoS One. 6 (5), e20220 (2011).</li><li>Bian, Z., 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=Identification+of+mouse+liver+mitochondria-associated+miRNAs+and+their+potential+biological+functions.">Identification of mouse liver mitochondria-associated miRNAs and their potential biological functions.</a> Cell Research. 20 (9), 1076-1078 (2010).</li><li>Kren, B. T., 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=MicroRNAs+identified+in+highly+purified+liver-derived+mitochondria+may+play+a+role+in+apoptosis.">MicroRNAs identified in highly purified liver-derived mitochondria may play a role in apoptosis.</a> RNA Biology. 6 (1), 65-72 (2009).</li><li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+genomics,+biogenesis,+mechanism,+and+function.">MicroRNAs: genomics, biogenesis, mechanism, and function.</a> Cell. 116 (2), 281-297 (2004).</li><li>Molkentin, J. D., Jobe, S. M., Markham, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-myosin+heavy+chain+gene+regulation:+delineation+and+characterization+of+the+cardiac+muscle-specific+enhancer+and+muscle-specific+promoter.">Alpha-myosin heavy chain gene regulation: delineation and characterization of the cardiac muscle-specific enhancer and muscle-specific promoter.</a> Journal of Molecular and Cellular Cardiology. 28 (6), 1211-1225 (1996).</li><li>Poliseno, L., 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=A+coding-independent+function+of+gene+and+pseudogene+mRNAs+regulates+tumour+biology.">A coding-independent function of gene and pseudogene mRNAs regulates tumour biology.</a> Nature. 465 (7301), 1033-1038 (2010).</li><li>Henao-Mejia, J., 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=The+microRNA+miR-181+is+a+critical+cellular+metabolic+rheostat+essential+for+NKT+cell+ontogenesis+and+lymphocyte+development+and+homeostasis.">The microRNA miR-181 is a critical cellular metabolic rheostat essential for NKT cell ontogenesis and lymphocyte development and homeostasis.</a> Immunity. 38 (5), 984-997 (2013).</li><li>Williams, A., Henao-Mejia, J., Harman, C. C., Flavell, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-181+and+metabolic+regulation+in+the+immune+system.">miR-181 and metabolic regulation in the immune system.</a> Cold Spring Harbor Symposia on Quantitative Biology. 78, 223-230 (2013).</li><li>Hori, D., 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=miR-181b+regulates+vascular+stiffness+age+dependently+in+part+by+regulating+TGF-beta+signaling.">miR-181b regulates vascular stiffness age dependently in part by regulating TGF-beta signaling.</a> PLoS One. 12 (3), e0174108 (2017).</li><li>Ebert, M. S., Sharp, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+sponges:+progress+and+possibilities.">MicroRNA sponges: progress and possibilities.</a> RNA. 16 (11), 2043-2050 (2010).</li><li>Ma, L., 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=Therapeutic+silencing+of+miR-10b+inhibits+metastasis+in+a+mouse+mammary+tumor+model.">Therapeutic silencing of miR-10b inhibits metastasis in a mouse mammary tumor model.</a> Nature Biotechnology. 28 (4), 341-347 (2010).</li><li>Davis, S., Lollo, B., Freier, S., Esau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+targeting+of+miRNA+with+antisense+oligonucleotides.">Improved targeting of miRNA with antisense oligonucleotides.</a> Nucleic Acids Research. 34 (8), 2294-2304 (2006).</li><li>Davis, S., 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=Potent+inhibition+of+microRNA+in+vivo+without+degradation.">Potent inhibition of microRNA in vivo without degradation.</a> Nucleic Acids Research. 37 (1), 70-77 (2009).</li><li>Elmen, J., 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=LNA-mediated+microRNA+silencing+in+non-human+primates.">LNA-mediated microRNA silencing in non-human primates.</a> Nature. 452 (7189), 896-899 (2008).</li><li>Esau, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+microRNA+with+antisense+oligonucleotides.">Inhibition of microRNA with antisense oligonucleotides.</a> Methods. 44 (1), 55-60 (2008).</li><li>Stenvang, J., Kauppinen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+as+targets+for+antisense-based+therapeutics.">MicroRNAs as targets for antisense-based therapeutics.</a> Expert Opinion on Biological Therapy. 8 (1), 59-81 (2008).</li><li>Lennox, K. A., Behlke, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+direct+comparison+of+anti-microRNA+oligonucleotide+potency.">A direct comparison of anti-microRNA oligonucleotide potency.</a> Pharmaceutical Research. 27 (9), 1788-1799 (2010).</li><li>Lennox, K. A., Behlke, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+modification+and+design+of+anti-miRNA+oligonucleotides.">Chemical modification and design of anti-miRNA oligonucleotides.</a> Gene Therapy. 18 (12), 1111-1120 (2011).</li><li>van Rooij, E., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+therapeutics+for+cardiovascular+disease:+opportunities+and+obstacles.">MicroRNA therapeutics for cardiovascular disease: opportunities and obstacles.</a> Nature Reviews. Drug Discovery. 11 (11), 860-872 (2012).</li><li>Stenvang, J., Petri, A., Lindow, M., Obad, S., Kauppinen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+microRNA+function+by+antimiR+oligonucleotides.">Inhibition of microRNA function by antimiR oligonucleotides.</a> Silence. 3 (1), 1 (2012).</li><li>Levin, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+issues+in+the+pharmacokinetics+and+toxicology+of+phosphorothioate+antisense+oligonucleotides.">A review of the issues in the pharmacokinetics and toxicology of phosphorothioate antisense oligonucleotides.</a> Biochimica et Biophysica Acta. 1489 (1), 69-84 (1999).</li><li>Flynt, A. S., Li, N., Thatcher, E. J., Solnica-Krezel, L., Patton, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+miR-214+modulates+Hedgehog+signaling+to+specify+muscle+cell+fate.">Zebrafish miR-214 modulates Hedgehog signaling to specify muscle cell fate.</a> Nature Genetics. 39 (2), 259-263 (2007).</li><li>Kloosterman, W. P., Lagendijk, A. K., Ketting, R. F., Moulton, J. D., Plasterk, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+inhibition+of+miRNA+maturation+with+morpholinos+reveals+a+role+for+miR-375+in+pancreatic+islet+development.">Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development.</a> PLoS Biology. 5 (8), e203 (2007).</li><li>Martello, G., 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=MicroRNA+control+of+Nodal+signalling.">MicroRNA control of Nodal signalling.</a> Nature. 449 (7159), 183-188 (2007).</li><li>Fabani, M. M., Gait, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-122+targeting+with+LNA/2'-O-methyl+oligonucleotide+mixmers,+peptide+nucleic+acids+(PNA),+and+PNA-peptide+conjugates.">miR-122 targeting with LNA/2'-O-methyl oligonucleotide mixmers, peptide nucleic acids (PNA), and PNA-peptide conjugates.</a> RNA. 14 (2), 336-346 (2008).</li><li>Fabani, M. M., 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=Efficient+inhibition+of+miR-155+function+in+vivo+by+peptide+nucleic+acids.">Efficient inhibition of miR-155 function in vivo by peptide nucleic acids.</a> Nucleic Acids Research. 38 (13), 4466-4475 (2010).</li><li>Babar, I. A., 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=Nanoparticle-based+therapy+in+an+in+vivo+microRNA-155+(miR-155)-dependent+mouse+model+of+lymphoma.">Nanoparticle-based therapy in an in vivo microRNA-155 (miR-155)-dependent mouse model of lymphoma.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (26), E1695-E1704 (2012).</li><li>Torres, A. G., 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=Chemical+structure+requirements+and+cellular+targeting+of+microRNA-122+by+peptide+nucleic+acids+anti-miRs.">Chemical structure requirements and cellular targeting of microRNA-122 by peptide nucleic acids anti-miRs.</a> Nucleic Acids Research. 40 (5), 2152-2167 (2012).</li><li>Kent, O. A., McCall, M. N., Cornish, T. C., Halushka, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+from+miR-143/145:+the+importance+of+cell-type+localization+of+miRNAs.">Lessons from miR-143/145: the importance of cell-type localization of miRNAs.</a> Nucleic Acids Research. 42 (12), 7528-7538 (2014).</li></ol>

作者没有什么可透露的。

本文介绍了 miRNA 海绵的设计和合成, 并论证了海绵组织特异表达是抑制组织特异 miRNA 家族表达的有力工具。
我们已经证明, 一个 miR-181 的家庭靶向海绵可以克隆成表达质粒与心脏特定的启动子。该质粒可以有效地包装成 nanovector 颗粒, 用于在体外和体内使用电穿孔或尾静脉注射, 分别 (图 1)。miR-181 海绵可以抑制 miR-181 家族的心脏特异表达, ?...

在过去的两年里, 已经有许多研究指出 miRNAs 在人类疾病中的重要作用。大量文献表明, miRNAs 在疾病的病理生理学中具有不可否认的重要性, 如癌症1和心血管疾病2、3、4、5。例如, miR-21 是上调在许多癌症, 导致细胞周期增加和细胞增殖6。在丙型肝炎感染中, miR-122 在病毒7的复制中起着重要作用, 并表明 miR-122 抑制病毒负荷8。在心肌肥厚, miR-212/132 是上调在心脏和参与病理表型9。上调 miRNA 的下调或功能性抑制的明显重要性表明, 在几乎所有疾病中, 治疗利用 miRNA 生物学的机会。
在人类基因组的三个基因组中发现了四 miR-181 家庭成员, miR-181a/b/c/d。非编码 RNA 宿主基因 (MIR181A1-HG) 的内含子区域编码 miR-181-a/b-1 簇。NR6A1 基因的内含子区域对 miR-181-a/b-2 进行编码。miR-181-c/d 星团位于 uncharacterized 19 号染色体上。所有的 miR-181 家庭成员共有相同的 "种子" 序列, 所有四 miR-181 家庭成员可以有可能调节相同的 mRNA 目标。
我们3,4和其他10突出了 miR-181 家庭成员在末期心力衰竭的重要性。我们也认识到, miR-181c 上调发生在与心脏病风险增加有关的病理条件下, 如 II 型糖尿病、肥胖症和衰老3、4、5。据推测, miR-181c 的过度表达会导致氧化应激, 导致心脏功能障碍4。
有几个小组建议 miRNA 存在于线粒体11、12、13、14, 但我们是第一个证明 miR-181c 来源于核基因组、加工和随后移位到线粒体在 RISC3。此外, 我们检测到 miR-181a 和 miR-181b 的低表达在心脏线粒体室5。重要的是, 我们发现 miR-181c 压抑 mt-COX1 mRNA 表达, 从而证明 miRNAs 参与线粒体基因调控和改变线粒体功能3,4。
本文讨论了设计一个 miRNA 海绵, 以摧毁整个 miR-181 家庭在心肌细胞所需的方法。此外, 我们概述了在体内应用的 miR-181-sponge 的协议。

<table>
	<tbody>
		<tr>
			<td>pEGFP-C1 vector</td>
			<td>Addgene</td>
			<td>6084-1</td>
			<td></td>
		</tr>
		<tr>
			<td>In-fusion</td>
			<td>Clontech</td>
			<td>121416</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Miniprep</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>miR-181-sponge synthesis</td>
			<td>Introgen GeneArt</td>
			<td>custome made</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td>Integrated DNA Technologies</td>
			<td>custome</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRI enzymes</td>
			<td>New Endland Biolabs</td>
			<td>R0101S</td>
			<td></td>
		</tr>
		<tr>
			<td>KpnI enzymes</td>
			<td>New Endland Biolabs</td>
			<td>R0142S</td>
			<td></td>
		</tr>
		<tr>
			<td>Rapid DNA Ligation Kit</td>
			<td>Sigma-Aldrich</td>
			<td>11635379001</td>
			<td></td>
		</tr>
		<tr>
			<td>H9c2 cells</td>
			<td>ATCC</td>
			<td>CRL-1446</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Media</td>
			<td>Thermo Fisher Scientific</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>10082139</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleofector 2b Device</td>
			<td>Lonza</td>
			<td>AAB-1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleofector Kits for H9c2 (2-1)</td>
			<td>Lonza</td>
			<td>VCA-1005</td>
			<td></td>
		</tr>
		<tr>
			<td>G418, Geneticin</td>
			<td>Thermo Fisher Scientific</td>
			<td>11811023</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSAria II Flow cytometer</td>
			<td>BD Bioscience</td>
			<td>644832</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson 450 sonifier</td>
			<td>Marshall Scientific</td>
			<td>EDP 100-214-239</td>
			<td></td>
		</tr>
		<tr>
			<td>The Xenogen IVIS Spectrum optical imaging device</td>
			<td>Caliper Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-MTCO1 antibody</td>
			<td>Abcam</td>
			<td>ab14705</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-tubulin antibody</td>
			<td>Abcam</td>
			<td>ab7291</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequoia C256 ultrasound system</td>
			<td>Siemens</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo nanovector delivery of a heart-specific microrna-sponge

MicroRNA (miRNA) 是一种小的非编码 rna, 它抑制转录后信使 rna (mRNA) 的表达。人类疾病, 如癌症和心血管疾病, 已被证明激活组织和/或细胞特异的 miRNA 表达与疾病进展有关。miRNA 表达的抑制提供了治疗干预的潜力。然而, 传统的抑制 miRNAs 的方法, 使用 antagomir 寡核苷酸, 影响特定的 miRNA 功能, 在全球交付。本文提出了一种在大鼠模型中对 miR-181 家族进行心脏特异性抑制的协议。miRNA 海绵结构的设计包括10重复 anti-miR-181 绑定序列。心脏特异的α MHC 启动子被克隆成 pEGFP 骨干, 以驱动心脏特异的 miR-181 miRNA 海绵表达。为了创建一个稳定的细胞系表达 miR-181-sponge, 骨骼肌 H9c2 细胞被转染的α-MHC-EGFP 和平号-181 海绵结构, 并排序由荧光活化细胞分类 (资产管制署) 到 GFP 阳性 H9c2 细胞培养与新霉素(G418)。随着新霉素的稳定增长, 单克隆细胞的数量是由额外的和单细胞克隆建立的。由此产生的骨骼肌 H9c2-miR-181-sponge-GFP 细胞表现为 miR-181 家庭成员的功能丧失, 这是通过增加 miR-181 靶蛋白的表达来评估的, 而与表达非功能性海绵的 H9c2 细胞相比。此外, 我们还开发了一个 nanovector 的系统交付 miR-181-sponge 结构的络合正电荷脂质体纳米粒子和负电荷 miR-181-sponge 质粒。在活体成像的 GFP 显示, 多尾静脉注射的 nanovector 在三周的时间, 能够促进一个重要的表达 miR-181-sponge 在心脏特定的方式。重要的是, 在心脏组织, 而不是在肾脏或肝脏中观察到 miR-181 功能的丧失。miRNA 海绵是抑制组织特异 miRNA 表达的有力方法。驱动 miRNA 海绵表达从组织特定的启动器提供特异性的 miRNA 抑制, 这可以被限制在靶器官或组织。此外, 结合 nanovector 和 miRNA 海绵技术, 允许有效的分娩和组织特定的 miRNA 抑制在体内。

在稳定转染的 pEGFP-miR-181-sponge-expressing H9c2 细胞 (从步骤 4.2), 整个 miR-181 家族 (miR-181a, miR-181b, miR-181c 和 miR-181d) 的表达相对于 pEGFP--表达 H9c2 细胞有适度的减少。MiR-181-sponge 作为整个 miR-181 家族的一个竞争性抑制剂, 因此我们预计 miR-181c 线粒体靶基因的表达将会增加, mt-COX1。西方的印迹数据表明, pEGFP-miR-181-sponge-expressing H9c2 细胞中的 mt-COX1 表达比 pEGFP-爬行表达的 H9c2 细胞?...

Watch this Scientific Journal Video about In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge at JoVE.com

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

体内Nanovector 心脏特异性 MicroRNA 海绵的交付

组织特异性 microRNA 抑制是一种在 microRNA 领域发展不发达的技术。在这里, 我们描述了一个协议, 以成功地抑制 miR-181 microRNA 家族在骨骼肌细胞从心脏。Nanovector 技术是用来提供一个 microRNA 海绵, 表明重要的体内心脏特定 miR-181 家庭抑制。

MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and ...

in-vivo-nanovector-delivery-of-a-heart-specific-microrna-sponge

Research

JoVE Journal

Biology

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

7.3K 次观看.  University of Toronto. 组织特异性 microRNA 抑制是一种在 microRNA 领域发展不发达的技术。在这里, 我们描述了一个协议, 以成功地抑制 miR-181 microRNA 家族在骨骼肌细胞从心脏。Nanovector 技术是用来提供一个 microRNA 海绵, 表明重要的体内心脏特定 miR-181 家庭抑制。

视频: In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with high precision while minimizing localized trauma and inflammation. Also, the ability to customize the system to accommodate animals of different species and sizes is desirable. This video presentation demonstrates the construction of an infusion system that can be fitted to any particular research animal. The delivery microcannula diameter is approximately 10-fold smaller than most infusion cannulas presently used. This translates into enhanced accuracy and reduced trauma to the brain region under study. The delivery cannula can also be sculpted to fit the contour of the surface of the animal's skull, thereby allowing closure of the scalp incision neatly over the infusion system, precluding the need for a skull-mounted pedestal, reducing risk of infection, and ensuring a greater level of comfort to the animal. The system is assembled in an air-free environment and requires the researcher to fashion glass micropipettes with a heat source. These construction methods require special skills that are best acquired, if not in person, using video instruction.  (This article is based on work first reported in J Neurosci Methods. 2008 Jan 30;167(2):213-20. Epub 2007 Aug 28.).

As neuroscience inquiry becomes more sophisticated, investigation of brain structures and circuitry requires improved levels of accuracy and higher resolution. We have developed a method for the preparation and implantation of a chronic infusion system within the brain utilizing a borosilicate microcannula with a tip diameter of 50 microns.

为持续给药Neuroactive代理Microinfusion系统的建设和植入。

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with ...

科研

生物学

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

In vivo Imaging of Deep Cortical Layers using a Microprism

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size and have a reflective coating on the hypotenuse to allow internal reflection of excitation and emission light.  The microprism probe simultaneously images multiple cortical layers with a perspective typically seen only in slice preparations.  Images are collected with a large field-of-view (~900 &mu;m).  In addition, we provide details on the non-survival surgical procedure and microscope setup.  Representative results include images of layer V pyramidal neurons from Thy-1 YFP-H mice showing their apical dendrites extending through the superficial cortical layer and extending into tufts.  Resolution was sufficient to image dendritic spines near the soma of layer V neurons.  A tail-vein injection of fluorescent dye reveals the intricate network of blood vessels in the cortex.  Line-scanning of red blood cells (RBCs) flowing through the capillaries reveals RBC velocity and flux rates can be obtained. This novel microprism probe is an elegant, yet powerful new method of visualizing deep cellular structures and cortical function in vivo.

In vivo Imaging of Deep Cortical Layers using a Microprism

Right-angle microprisms inserted into the mouse neocortex allows for deep imaging of multiple cortical layers with a viewpoint typically found in slice. One-millimeter microprisms offer a wide field-of-view (~900 &mu;m) and spatial resolutions sufficient to resolve dendritic spines. We demonstrate layer V neuronal imaging and neocortical vascular imaging using microprisms.

在使用微深皮质层的活体成像

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

果蝇胚胎的体内离心

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

啮齿动物附睾精子磷酸分析

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal development.

RNAi的触发传送到<em&gt;冈比亚按蚊</em&gt;蛹

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases.

We describe a novel method for the delivery of DNA plasmid into the urothelial cells of mouse bladder in vivo through urethra catheterization and electroporation. It offers a fast and convenient way for generating autochthonous mouse models of bladder diseases.

电穿孔对小鼠膀胱 Urothelium 质粒 DNA 传递的新方法

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...

An Ex Vivo Tissue Culture Model of Cartilage Remodeling in Bovine Knee Explants

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique tissue important for proper function of the synovial joint and is constituted by a dense extracellular matrix (ECM), rich in proteoglycan and type II collagen. Chondrocytes are the only cell type present within cartilage and are widespread and relatively low in number. Altered external stimuli and cellular signalling can lead to changes in ECM composition and deterioration, which are important pathological hallmarks in diseases such as osteoarthritis (OA) and rheumatoid arthritis.
Ex vivo cartilage models allow 1) profiling of chondrocyte mediated alterations of cartilage tissue turnover, 2) visualizing the cartilage ECM composition, and 3) chondrocyte rearrangement directly in the tissue. Profiling these alterations in response to stimuli or treatments are of high importance in various aspects of cartilage biology, and complement in vitro experiments in isolated chondrocytes, or more complex models in live animals where experimental conditions are more difficult to control.
Cartilage explants present a translational and easily accessible method for assessing tissue remodeling in the cartilage ECM in controllable settings. Here, we describe a protocol for isolating and culturing live bovine cartilage explants. The method uses tissue from the bovine knee, which is easily accessible from the local butchery. Both explants and conditioned culture medium can be analyzed to investigate tissue turnover, ECM composition, and chondrocyte function, thus profiling ECM modulation.

Here, we present a protocol describing isolation and culturing of cartilage explants from bovine knees. This method provides an easy and accessible tool to describe tissue changes in response to biological stimuli or novel therapeutics targeting the joint.

牛膝外生软骨重塑的外骨组织培养模型

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique ...

In Vivo Surface Electrocardiography for Adult Zebrafish

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of zebrafish not only as a research model for human cardiac electrophysiology and myopathies but also as a surrogate model in high throughput pharmaceutical screening for potential cardiotoxicities to humans, such as QT prolongation. As such, in vivo electrocardiography for adult zebrafish is an electrical phenotyping tool that is necessary, if not indispensable, for cross-sectional or longitudinal in vivo electrophysiological characterizations. However, too often, the lack of a reliable, practical, and cost-effective recording method remains a major challenge preventing this in vivo diagnostic tool from becoming more readily accessible. Here, we describe a practical, straightforward approach to in vivo electrocardiography for adult zebrafish using a low-maintenance, cost-effective, and comprehensive system that yields consistent, reliable recordings. We illustrate our protocol using healthy adult male zebrafish of 12-18 months of age. We also introduce a rapid real-time interpretation strategy for quality validation to ensure data accuracy and robustness early in the electrocardiogram recording process.

Here, we present a reliable, minimally invasive, and cost-effective method to record and interpret electrocardiograms in live anesthetized adult zebrafish.

在 Vivo 表面心电图中成人斑马鱼

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of ...

体内Nanovector 心脏特异性 MicroRNA 海绵的交付

摘要

探索更多视频

此视频中的章节