这项工作得到国家杰出青年学者基金会 (81625002)、中国国家自然科学基金 (81470389、81270282、81601238) 的支持, 上海市学术研究领导 (18XD1402400) 项目教育统筹委员会高峰临床医学资助 (20152209), 上海中药肾康医院发展中心 (16CR3034A), 上海交通大学 (YG2013MS42), 上海交通大学医学院 (15ZH1003 和 14XJ10019),上海航海项目 (18YF1413000) 和蚌埠医学院研究生创新项目 (Byycx1722)。我们感谢乌尔博士以前在我们实验室的帮助。

所有动物实验都是根据国家卫生研究院关于使用实验动物的指导方针进行的, 并得到了研究所动物伦理委员会的批准。所有的实验都使用雄性 C57BL/6J 小鼠 (8-10 周龄)。小鼠被安置了在无病原体条件下24°c 4 °c, 在 12 h 光/黑暗周期, 与自由获得水和食物。
1. 腺病毒溶液的制备
<ol><li>纯化腺病毒溶液到达后, 将溶液贮存在-80 摄氏度的冷冻机中。 注: 腺病毒注射液是在存活的小鼠体内进行的。为确保腺病毒载体的不育, 制备并纯化了9、10的腺病毒 (荧光素酶、vdr 或 shRNA 靶向 vdr)。</li>
	<li>在手术当天, 从-80 °c 冰箱取出纯化的腺病毒溶液 (3 x 1010斑块形成单元 [pfu]/毫升), 并在冰上解冻腺病毒载体溶液。</li>
	<li>戴上面罩、消毒手套和无菌长袍。</li>
	<li>准备一支50µL 的哈密尔顿注射器, 在手术前一天就被消毒了。 注: 为杀菌, 哈密尔顿注射器包裹在纱布和放置在高温/高压灭菌器。选择 "固体灭菌" 模式, 按 "启动" 按钮开始灭菌。</li>
	<li>纯化腺病毒溶液完全解冻后, 用75% 乙醇喷洒含有腺病毒溶液的管, 将腺病毒溶液移入层流无菌罩中。</li>
	<li>在层流无菌罩, 吸入50µL 腺病毒溶液使用哈密尔顿注射器没有针, 其次是附着30克针的汉密尔顿注射器。</li>
	<li>按住注射器与30克针向上, 并轻轻推注射器的柱塞, 直到针充满腺病毒溶液 (确认由第一溶液的外观下降从针尖)。 注: 用大约45µL 溶液填充30克针, 所以现在几乎没有解决方案是可见的注射器。</li>
	<li>使用上述准备好的注射器, 仔细吸入另30µL 腺病毒溶液, 并将松散的上限注射器放在冰上供以后使用。</li>
</ol>2. 麻醉和手术准备
<ol><li>在异氟醚蒸发器中加入20毫升异氟醚, 并将异氟醚蒸发器与氧气罐连接。</li>
	<li>打开阀门, 让氧气从氧气罐流向异氟醚蒸发器。通过在异氟醚蒸发器中的氧气流量监测器, 保持2升/分的氧气流量。</li>
	<li>用4% 异氟醚在100% 氧 (2 升/分) 中诱导麻醉, 在与异氟醚蒸发器相连的塑料笼中2分钟。</li>
	<li>从塑料笼中取出麻醉鼠标, 并将其固定在层流消毒罩中俯卧位置的塑料平台上, 并通过鼻锥在100% 氧 (2 升/分) 内保持麻醉, 2% 异氟醚。</li>
	<li>确认足够的麻醉, 没有脚趾捏反应。</li>
	<li>将无菌眼霜应用于每只眼睛, 以保护角膜不受干燥。</li>
	<li>剃掉胸部和上腹部。将商业上可用的脱毛膏涂抹在剃须的地方1分钟. 去除脱毛霜和剩余的毛皮与湿纱布。</li>
	<li>用3的碘洗必泰消毒 (如 entoiodine) 消毒手术部位 (左下半部分)。用不育的褶皱覆盖手术部位。</li>
</ol>3. 心肌注射腺病毒在小鼠心脏中的研究
<ol><li>脱下手套, 戴上一双新的无菌手套。</li>
	<li>消毒钳, 微型蚊子止血, 一对手术剪刀, 和一个针持有人在运行前的高温/高压灭菌器 (见步骤1.4 注意)。</li>
	<li>沿着连接剑突和腋窝的线做一个0.5 厘米的皮肤切口。用镊子和微蚊止血直接解剖胸主和胸肌小肌肉。</li>
	<li>通过缩回胸主和胸小肌肉来暴露肋间空间。刺穿并打开第四肋间空间 (或观察最宽的肋骨空间) 与微蚊子止血。</li>
	<li>将心脏推向切口, 用非显性手的食指轻轻按压胸壁右侧, 将心脏外侧。</li>
	<li>用非显性手的食指和拇指轻轻地保护外在的心脏。</li>
	<li>通过用占主导地位的腺病毒 (图 1C) 填充的哈密尔顿注射器, 将30µL 腺病毒溶液注入三个部位 (腹侧、背部和左心室侧壁) 的左心室心肌。</li>
	<li>注射完成后, 立即将心脏放回胸腔内。</li>
	<li>通过轻轻按压胸壁向皮肤切口部位, 手动疏散胸腔内的空气。 注: 成功的空气疏散可以通过拍打的胸部少校和胸小肌肉造成的驱逐空气证明。</li>
	<li>用5-0 丝缝线缝合的水平床垫缝合皮肤。 注: 胸部少校和胸小肌不应缝合, 因为他们只是坦率地解剖和他们的解剖结构是完整的整个手术。</li>
</ol>4. 术后管理
<ol><li>每日两次通过皮下注射来管理丁丙诺啡 (3 毫克/千克), 以减少手术后48小时的术后疼痛11。</li>
	<li>手术后, 立即将小鼠保持在热垫上 (37 摄氏度), 直到完全恢复。将鼠标放回笼子后, 它完全恢复。</li>
	<li>在高温/高压灭菌器中杀菌使用的哈密尔顿注射器和30克注射器 (见步骤1.4 注)。在锋利的容器里收集消毒的30克注射器针。</li>
</ol>5.体内成像测量心脏荧光素酶表达的方法
<ol><li>在5天的腺病毒注射后, 准备一个新的库存溶液的荧光素在15毫克/毫升在 Dulbecco 的磷酸盐缓冲盐水 (DPBS)。通过0.2 µm 过滤器筛选解决方案。</li>
	<li>peritoneally 内注射荧光素溶液150毫克/千克体重的醒鼠。</li>
	<li>打开成像系统。</li>
	<li>成像系统自动自检后, 选择成像模式为 "发光", 并进行以下设置: 曝光: 自动;Binning: 8;FStop: 1;励磁: 块;发射: 打开。</li>
	<li>在荧光素注射液后10分钟, 用水合氯醛注射液麻醉小鼠 (300 毫克/千克体重)。确认足够的麻醉, 没有脚趾捏反应。</li>
	<li>将麻醉小鼠固定在俯卧部位的热成像台上, 将胸部对准摄像机。</li>
	<li>收集图像 (每只鼠标1图像), 并通过绘制相同的圆形测量区域 (ROI) 来量化信号的强度。</li>
	<li>在采集图像后, 牺牲小鼠并收集以下部分所述的组织。</li>
</ol>6. 收获组织
<ol><li>在病毒注射后的不同时间点 (图 1B), 麻醉小鼠与4% 异氟醚, 并通过鼻锥维持麻醉与2% 异氟醚, 并确保它在一个塑料平台上的俯卧位。</li>
	<li>在前颈部进行中线腹侧切口。通过缩回肩胛舌骨和胸锁乳突肌来暴露右颈总动脉。</li>
	<li>通过 transecting 正确的颈总动脉和排泄血液来牺牲老鼠。</li>
	<li>在腹部做一个中线腹侧切口。沿锁骨中线线切开肋笼两侧的肋骨, 并横断面膜片。</li>
	<li>举起剑突, 露出心脏。找到升主动脉, 轻轻地去除周围脂肪组织。</li>
	<li>Cannulate 主动脉和 retrogradely 灌注心脏与一个30克针连接到1毫升注射器填充0.5 毫升4°c 磷酸盐缓冲液 (PBS)。</li>
	<li>灌注后, 将心脏切除, 并将其分为左、右心室。切除肝脏、肺和脾脏。</li>
	<li>两次洗涤后, 冷 PBS (4 °c), 存储所有收集的组织分别在低温瓶中液氮。</li>
</ol>7. 蛋白质表达的测定
<ol><li>将蛋白酶抑制剂鸡尾酒加入到商业上可用的裂解缓冲液中, 以1:100 的比例制备均匀化缓冲剂。根据要处理的样本数计算总体积。将准备好的均匀缓冲保持在4摄氏度, 以供后续使用。</li>
	<li>从液氮罐中取出组织。在高精度鳞片上对组织进行称量 (每片组织大约60毫克)。</li>
	<li>将组织转移到一个新的1.5 毫升离心管。立即加入200µL 均匀缓冲和预换热器1.5 毫米钢球 (4 °c) 到离心管。</li>
	<li>将预换热器 (4 °c) 金属夹中的离心管固定在自动组织磨床上, 并以下列设置启动机器, 开始均匀化: 频率:70 赫兹;时间: 120s。</li>
	<li>均匀化后, 离心匀浆 (1.6万 x 克, 4 °c), 并小心转移到上清到一个新的1.5 毫升管与100µL 吸管。</li>
	<li>添加负载缓冲液 (5x) 到蛋白上清的比例为 1:4, 并变性蛋白质在100°c 5 分钟。</li>
	<li>在心脏和其他器官组织的蛋白质表达水平进一步监测的西方污点分析 (代表性的结果显示在图 2C和图 3) 根据协议, 先前描述的12。</li>
</ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramyocardial+cell+delivery:+observations+in+murine+hearts.">Intramyocardial cell delivery: observations in murine hearts.</a> J Vis Exp. (83), e851064 (2014).</li></ol>

作者没有什么可透露的。

本报告展示了一种改进的方法, 心肌注射病毒载体的心脏基因操作, 这是修改了从心肌梗死的方法诱导的高等。13当前,在体内对特定基因功能的刻画最常涉及产生挖空或转基因小鼠3,14,15,16,17, 这是昂贵的、耗时的和劳力密集型的。或者, 通过系统或局部注射来传递基因载体或 siRNA 也被广泛应用于心血管研究的基因操作4,5,7。特别是, 在某些情况下, 心肌注射液不能替代心脏基因的操作: 当需要定点注射 (例如, 心肌梗死模型中的边界区注射)11;当需要持续时间限制基因操作时 (例如, 腺病毒注射)。结果表明, 改进后的心肌注射方法简单、省时、效率高。
协议中的关键步骤和疑难解答:
为了成功地执行本议定书, 应注意到几个关键步骤。在吸病毒之前, 汉密尔顿注射器内的空气和附针必须疏散, 否则注入心肌的空气可能引起局部心脏损伤甚至死亡。为了进一步避免这一问题, 准备就绪使用的哈密尔顿注射器填充适当的病毒体积不应放置在柱塞末端向下, 因为这可能自发吸入空气的严重性, 金属柱塞。麻醉应仔细监测, 因为深度麻醉可能会延迟手术后的恢复, 严重的深度麻醉可能导致死亡。经过充分的麻醉后, 心脏不应该被外力外化, 因为这可能导致严重的肺损伤。事实上, 适当的心外化只需要温柔的心, 任何抵抗都可能表明向不正确的方向前进。
技术的局限性:
这项技术的取消插管, 减少了程序所需的时间, 建议心肌注射程序应在一个相对有限的时间窗口内完成, 以避免死亡。根据我们的经验, 心脏不应该被外部化超过三十年代, 因为这增加了死亡率和减慢手术后恢复。因此, 建议使用插管在这里描述的协议的第一次尝试。另一个限制是这种方法提供的病毒载体的不均匀分布, 这也存在于传统的心肌注射方法7中。此外, 这里描述的方法更有用的心脏特异基因操作在未受伤的心脏, 可以随后建立不同的心脏病模型18,19,20, 21,22,23。然而, 使用目前的方法来向受伤的心脏, 如梗死心脏提供药物可能是有限的, 因为这些老鼠可能不容忍的程序。
对于现有方法的意义:
常规的心肌注射需要插管和机械通气24, 并使得很难找到注射部位由于快速的鼠标心跳。这些问题大大延长了操作时间13, 从而增加了时间延迟带来的变化。这里提出的改进技术是快速的, 并允许精确的注射地点, 手动保护外部心脏;总的来说, 显著 potentiating 随后的研究。此外, 消除插管和机械通气使修改的方法几乎可以进入任何实验室。

心脏病是全球发病率和死亡率的主要原因1,2。缺乏有效的治疗策略, 包括心肌梗死和心力衰竭等危及生命的心脏疾病, 吸引了对基础 pathomechanisms 的深入探索, 并确定了新的治疗方案3。对于这些科学探索, 心脏特异基因操作被广泛使用4,5。心脏基因操纵可以通过基因组编辑使用强大的转录激活器样效应核酸酶 (塔伦) 和聚集定期 interspaced 短回文重复 (CRISPR)/CRISPR 相关蛋白 9 (Cas9) 工具, 或通过交付异位基因材料 (例如, 携带感兴趣的蛋白质编码基因的病毒载体)6。虽然基因组编辑允许精确和时空基因组修改的活鼠, 它仍然是一个耗时和劳动密集型的做法6。或者, 由病毒载体或小干扰 RNA (siRNA) 复杂传递的心脏特异基因操作通常执行6。
病毒载体传递到成年鼠心脏是通过大致两个策略: 系统性或局部注射。系统性注射 cardiotropic 血清自动增值服务如 AAV9 是无创性的心脏特异基因操作7。然而, 这种方法需要一个相对较高的载体, 以有效的传导和基因表达, 并可能导致显着转导的 nontarget 器官, 如肌肉和肝脏的7。局部病毒注射是通过心肌注射或冠状动脉内传递7实现的。与心肌注射液相比, 冠状动脉内分娩可导致心脏内更均匀的病毒分布。然而, 这一技术的缺点是快速洗出的病毒载体的系统循环和转导的 nontarget 器官8, 以及它的要求设备的压力测量在操作过程中。相比之下, 心肌注射液能使心肌中的病毒保留更好, 以及特定部位的分娩, 但它无法均匀分布病毒载体7。对小动物来说, 冠状动脉内分娩在技术上很难执行, 而系统性 AAV9 注射和心肌注射则更常用4,5,7。虽然全身注射是容易执行, 常规心肌注射需要机械通气和开胸, 造成广泛的组织损伤, 是费时。
在本报告中, 我们描述了一种简单、节省时间和高效的心肌注射方法。注射荧光素酶和 vdr 的腺病毒, 或 shRNA 靶向 vdr, 被注入以操纵心脏基因表达。一旦掌握, 这种方法可以用于基因操作, 以及注射细胞或其他材料的老鼠心脏。

<table border="0" cellpadding="0" cellspacing="0" width="914">
	<tbody>
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			<td colspan="4" height="40" style="height:40px;width:916px;">Equipments</td>
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			<td height="40" style="height:40px;width:351px;">Laminar flow sterile hood</td>
			<td style="width:267px;">Fengshi Animal Experimental&#160; Equipment Techonology Co., Ltd. (Soochow, China)</td>
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			<td style="width:156px;"></td>
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			<td style="width:143px;">75005282</td>
			<td style="width:156px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Tissue grinding machine</td>
			<td style="width:267px;">Scientz Biotechnology Co., Ltd. (Ningbo, China)</td>
			<td style="width:143px;">Scientz-48</td>
			<td style="width:156px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">High temperature/high pressure sterilizer</td>
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			<td height="40" style="height:40px;width:351px;">IVIS&#160; Lumina III imaging system</td>
			<td style="width:267px;">PerkinElmer (Waltham, USA)</td>
			<td style="width:143px;">CLS136334</td>
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			<td height="40" style="height:40px;width:351px;">Precision&#160;balance</td>
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			<td height="40" style="height:40px;width:351px;">Eppendorf pipette (100 &#181;L)</td>
			<td style="width:267px;">Eppendorf (Westbury, USA)</td>
			<td style="width:143px;">&#160;4920000059</td>
			<td style="width:156px;"></td>
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			<td height="40" style="height:40px;width:351px;">Eppendorf pipette (10 &#181;L)</td>
			<td style="width:267px;">Eppendorf (Westbury, USA)</td>
			<td style="width:143px;">&#160;4920000113</td>
			<td style="width:156px;"></td>
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			<td height="40" style="height:40px;width:351px;">Forceps</td>
			<td style="width:267px;">Shanghai Medical Instruments (Group) Ltd., Corp.&#160;</td>
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		<tr height="40">
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			<td style="width:267px;">Hamilton (Nevada, USA)</td>
			<td style="width:143px;">80501</td>
			<td style="width:156px;">Volume 50 &#956;L</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Micro-mosquito hemostat</td>
			<td style="width:267px;">F.S.T (Foster City, USA)</td>
			<td style="width:143px;">13011-12</td>
			<td style="width:156px;">Curved, tip width 1.3mm</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Needle holder&#160;</td>
			<td style="width:267px;">Shanghai Medical Instruments (Group) Ltd., Corp. (Shanghai, China)</td>
			<td style="width:143px;">J32110</td>
			<td style="width:156px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Surgical scissors</td>
			<td style="width:267px;">F.S.T (Foster City, USA)</td>
			<td style="width:143px;">14002-12</td>
			<td style="width:156px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">1-mL Syringe</td>
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			<td style="width:143px;"></td>
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			<td style="width:143px;">#2118</td>
			<td style="width:156px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Anti-Luciferase antibody</td>
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			<td style="width:143px;">ab187340</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Anti-rabbit IgG</td>
			<td style="width:267px;">CST (Danvers,&#160; USA)</td>
			<td style="width:143px;">&#160;#7074</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Anti-VDR antibody</td>
			<td style="width:267px;">Abcam (Cambridge, UK)</td>
			<td style="width:143px;">&#160;ab109234</td>
			<td style="width:156px;"></td>
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		<tr height="40">
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			<td style="width:267px;">Thermo Scientific (Waltham, USA)</td>
			<td style="width:143px;">PA175056</td>
			<td style="width:156px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Chloralic hydras</td>
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			<td style="width:156px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Cryogenic Vials</td>
			<td style="width:267px;">Thermo Scientific (Waltham, USA)</td>
			<td style="width:143px;">375418</td>
			<td style="width:156px;">1.8 mL&#160;</td>
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			<td height="40" style="height:40px;width:351px;">Depilatory cream</td>
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			<td style="width:156px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Dulbecco&#39;s phosphate buffered saline&#160;</td>
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			<td style="width:143px;">14040133</td>
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			<td height="40" style="height:40px;width:351px;">Entoiodine</td>
			<td style="width:267px;">LiKang (Shanghai, China)</td>
			<td style="width:143px;">310132</td>
			<td style="width:156px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">EP tube</td>
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			<td style="width:143px;">PCR001</td>
			<td style="width:156px;"></td>
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			<td height="40" style="height:40px;width:351px;">Filter</td>
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			<td style="width:143px;"></td>
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		<tr height="40">
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			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Luciferin</td>
			<td style="width:267px;">Promega (Madison, USA)</td>
			<td style="width:143px;">P1041</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Lysis buffer for western&#160; blot</td>
			<td style="width:267px;">Beyotime (Shanghai, China)</td>
			<td style="width:143px;">P0013J</td>
			<td style="width:156px;">Without inhibitors</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Ophthalmic cream</td>
			<td style="width:267px;">Apex Laboratories ( Melbourne, Australia))</td>
			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">PBS</td>
			<td style="width:267px;">Gibco (Grand Island,&#160; USA)</td>
			<td style="width:143px;">10010023</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Protease inhibitor cocktail</td>
			<td style="width:267px;">Thermo Scientific (Waltham, USA)</td>
			<td style="width:143px;">78438</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">5-0 silk suture</td>
			<td style="width:267px;">Shanghai Medical Instruments (Group) Ltd., Corp. (Shanghai, China)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Steel ball</td>
			<td style="width:267px;">Scientz Biotechnology Co., Ltd. (Ningbo, China)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;">Width 1.5 mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Syringe needle</td>
			<td style="width:267px;">Kindly Medical Devices Co., Ltd. (Zhejiang, China)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;">30 gauge&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Warm mat</td>
			<td style="width:267px;">Warmtact Electrical Heating Technology Co., Ltd. (Guangdong, China )</td>
			<td style="width:143px;">NF-GNCW</td>
			<td style="width:156px;"></td>
		</tr>
	</tbody>
</table>

a simple and efficient method for in vivo cardiac-specific gene manipulation by intramyocardial injection in mice

特别是在心脏的基因操作明显事实心脏疾病 pathomechanisms 的调查和他们的治疗潜力。体内心脏特异基因的传递通常是通过系统性或局部分娩来实现的。应用重组腺相关病毒 9 (AAV9), 采用尾静脉全身注射治疗心脏基因表达简便、有效。然而, 这种方法需要较高的载体, 有效传导, 并可能导致 nontarget 器官基因转导。在这里, 我们描述了一个简单的, 有效的, 节省时间的方法, 心肌注射液的体内心脏特异基因操作的小鼠。在麻醉 (没有通气) 下, 胸部主要和次要肌肉被坦率地解剖, 并且小鼠心脏通过手工外在化快速地暴露通过小切口在第四肋间空间。随后, 腺病毒的编码荧光素酶 (卢克) 和维生素 D 受体 (vdr), 或短发夹 RNA (shRNA) 靶向 VDR, 注射了汉密尔顿注射器进入心肌。随后的体内成像表明, 荧光素酶成功地抗原心脏。此外, 西方印迹分析证实了成功的过度表达或沉默的 VDR 在小鼠心脏。一旦掌握, 这种技术可以用于基因操作, 以及注射细胞或其他材料, 如磁性在鼠标心脏。

实验协议和报告方法的一些关键步骤显示在图 1中。5天后, 在心肌注射腺病毒荧光素酶 (), 在体内成像在对卢克注射小鼠表明, 显着的强力表达的荧光素酶特别在心脏 (图2A, B), 这是由西方印迹分析 (图 2C) 证实, 表明没有 nontarget 器官转导。相比之下, 在控制小鼠中没有发现荧光素酶表达。根据荧光素酶的成功过度表达, 西方印迹分析表明, 在注射腺病毒的小鼠左心室中, vdr 的表达明显增加 (如 vdr) (图 3A)。此外, shVDR 注射液显著减少左心室的 VDR 表达 (图 3B)。与此相反, 在右心室, shVDR 注射小鼠或注射小鼠 (图 3C, D) 中, vdr 表达没有明显改变, 因为腺病毒只注入左心室心肌。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57074/57074fig1.jpg"> 图 1.小鼠心肌注射和基因表达检测协议的模式.(A) 图示显示左心室心肌中有三个注射点。(B) 提示病毒注射后小鼠心脏基因表达检测的协议。荧光素酶: 腺病毒荧光素编码;AdVDR: 腺病毒编码 VDR;AdshVDR, 腺病毒编码 shRNA 靶向 VDR。(C) 代表图像, 显示修改后的鼠标心肌注射方法的多个步骤。a.通过商业上可用的脱毛膏去除毛皮。b. 3 度聚维酮碘磨砂手术部位的灭菌。c.用无菌悬垂覆盖手术部位。d. 0.5 厘米皮肤切口沿线连接剑突和腋窝。e.用镊子和微蚊子止血钝解剖胸大肌和胸小肌肉。f.心脏的外化。g.通过哈密尔顿注射器将30µL 腺病毒溶液注入左心室心肌。h-i.用5-0 丝缝线缝合的钱包线缝合皮肤。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57074/57074fig1large.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/57074/57074fig2.jpg"> 图 2.检测心脏中的荧光素酶表达式.(A) 影像系统采集的图像, 显示在注射后5天内, 在控制小鼠和注射腺病毒荧光素酶的小鼠体内, 心脏的发光信号。(B) 不同组的发光信号强度 (n = 3) 受t测试的统计分析。**p < 0.01 与控制鼠标。(C) 西方印迹分析结果显示, 在腺病毒注射后5天内, 表明组的心、肺、肝和脾中的荧光素酶蛋白水平 (n = 3)。由于荧光素酶的表达仅在对卢克注射小鼠心脏的检测, 没有进行统计分析。编码荧光素酶的腺病毒。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57074/57074fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57074/57074fig3.jpg"> 图 3.检测心脏中的 VDR 表达式.(A) 顶部面板: 在0天、5天、7天和在注射后14天, 显示在左心室 (腺病毒注射) 的 VDR 水平的西方印迹带。底部板: 对 VDR 表达水平的半定量分析在不同的小组 (n = 4 每时间点)。结果被规范化对 GAPDH 并且转换了到折叠变动相对于0天。对方差 (方差分析) 后的 Bonferroni 后检验 (等方差假设) 或 Tamhane 后检验 (不假设的等方差) 进行了统计分析。*p < 0.05 或 **p < 0.01 vs 0 天。(B) 顶部面板: 在0天、3天、5天和 shVDR 注射液后7天, 在左心室显示 VDR 水平的西方印迹带。底部板: 对 VDR 表达水平的半定量分析在不同的小组 (n = 4 每时间点)。*p < 0.05 vs 0 天。(C) 顶部面板: 在0天、5天、7天和在注射后14天内, 显示在右心室 (腺病毒未注射) 的 VDR 水平的西方印迹带。底部板: 对 VDR 表达水平的半定量分析在不同的小组 (n = 4 每时间点)。(D) 顶部面板: 在0天、3天、5天和 shVDR 注射液后7天, 在右心室显示 VDR 水平的西方印迹带。底部板: 对 VDR 表达水平的半定量分析在不同的小组 (n = 4 每时间点)。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57074/57074fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice at JoVE.com

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

在这里, 我们提出了一个小鼠心脏特异基因操作的协议。在麻醉下, 小鼠心脏通过第四肋间空间被外部化。随后, 将特定基因的腺病毒注射进心肌, 然后通过体内成像和西方印迹分析对蛋白质表达进行测量。

一种简便、有效的心肌注射小鼠心脏特异基因操作方法--体内

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

a-simple-efficient-method-for-vivo-cardiac-specific-gene-manipulation

Nanyang Technological University

Research

JoVE Journal

Biology

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

15.7K 次观看.  The First Affiliated Hospital of Bengbu Medical College. 在这里, 我们提出了一个小鼠心脏特异基因操作的协议。在麻醉下, 小鼠心脏通过第四肋间空间被外部化。随后, 将特定基因的腺病毒注射进心肌, 然后通过体内成像和西方印迹分析对蛋白质表达进行测量。

视频: A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

焦磷酸测序：一个准确的基因分型的简单方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

科研

生物学

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

简单和高效的睾丸细胞悬液的制备技术

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter implantation. The technical limitation of this surgical technique lies in the finesse of the hands. The injection is rapid, especially for a trained experimenter, and since tissue disruption with this technique is minimal, repeated injections are possible; moreover immune reaction to foreign tools (e.g. catheter) does not occur, thereby giving a better and more specific read out of spinal cord modulation. Since the application of the substance is largely limited to the target region of the spinal cord, drugs do not need to be applied in large dosages, and more importantly unwanted effects on other tissue, as observed with a systemic delivery, could be circumvented1,2. Moreover, we combine this technique with in vivo transfection of nucleic acid with the help of polyethylenimine (PEI) reagent3, which provides tremendous versatility for studying spinal functions via delivery of pharmacological agents as well as gene, RNA, and protein modulators.

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

体内转染和基因敲除在脊髓通过快速无创腰椎鞘内注射小鼠

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

从人类体细胞与仙台病毒高效的新一代人类诱导多能干细胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

在新生小鼠静脉注射

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤:一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.