Name: preparation of raav9 to overexpress or knockdown genes in mouse hearts
Uploaded: 2016-12-17T13:00:00.000+00:00
Duration: 11 min 11 s
Description: Watch this Scientific Journal Video about Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts at JoVE.com

We thank Dr. Zaffar Haque for careful reading of the manuscript. We thank Drs. Masaharu Kataoka and Gengze Wu for discussions and help. Work in the Wang lab is supported by the American Heart Association, Muscular Dystrophy Association, and NIH (HL085635, HL116919, HL125925).

在由生物安全委员会和波士顿儿童医院的机构动物护理和使用委员会批准的规程进行所有描述的步骤。波士顿儿童医院与监管的光/暗循环和气候控制无病原鼠标设施。兽医和动物关爱员工的变化笼，保证了老鼠的健康。该设施是AAALAC认证，并有活跃的动物福利保障认证（AAALAC认证在1992年2月24日动物福利保障授予数量:A3303-01）。小鼠通过CO 2从压缩气体源输送安乐死。组织样品证实心脏速率，运动和动物的呼吸停止后收集。新生儿啮齿动物抗CO 2安乐死，并通过使用锋利的剪刀斩首被实施安乐死。这些方法都与小组的美国兽医本草安乐死的建议相一致社团出版社。 1.由克隆的cDNA或shRNA表达盒插入质粒骨架rAAV9构建的代注:rAAV9质粒含有用于基因表达的末端反向重复序列AAV2的（ITRS），已被修改为怀有鸡TNNT2 子（rAAV9.cTNT），使转基因25,26,29的特定心肌表达。独特NheⅠ和KpnI位点已被引入到质粒中，启动子的下游。编码感兴趣的基因的cDNA片段克隆到用这两种限制性位点25,26,29的rAAV9骨干。这里，作为一个例子，产生在小鼠心脏GFP基因的过表达的rAAV9载体。所得质粒含有由两个ITR位点（ 图1）侧翼的肌钙蛋白:: GFP盒。 rAAV9.U6 ::的shRNA构建体用于基因敲除25。设计使用的shRNA在线shRNA的设计服务器。 rAAV9.U6 :: shRNA的可产生或者通过退火和结扎的shRNA序列插入的限制酶消化的rAAV9载体携有U6启动子的DNA含有-寡核苷酸，或者通过远距离PCR和分子内吉布森组件基于"无缝"建设30。所得质粒中应包含的U6-shRNA的盒由两个ITR位点（ 图2）侧接。在此，作为一个例子，rAAV9.U6 :: shRNA的载体构建拦截TRBP的mRNA（TRBP shRNA的序列:GCAGTGATGGATATGCATCTTCTCGAGAAGATGCATATCCATCACTCG）。一个争夺的shRNA作为阴性对照（CCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGG）。 <ol><li>克隆的基因或shRNA表达盒到rAAV9质粒骨架。变换DNA导入感受态大肠杆菌细胞25。 注:使用stbl2或stbl3 大肠杆菌细胞rAAV9 DNA转化，以尽量减少意外ITR重组。 </li><li>拿起从转化的大肠杆菌的细胞阳性克隆。放大500毫升莉莉-巴尼特介质的文化和提取细菌细胞25-30 rAAV9质粒。 注意:MIDI /马克西预习的rAAV9质粒得到的DNA（&gt; 100微克）的高含量。产生病毒之前，总是通过限制性消化和琼脂糖凝胶电泳分析AAV质粒的序列完整性，如先前所述（http://www.vvf.uzh.ch/cloningservice/11bpdeletion/itrintegrity.html）。 </li></ol> 2. HEK293细胞rAAV9质粒转染<ol><li>准备的线性聚乙烯亚胺（PEI）的解决方案1微克/微升。溶解PEI粉末在无内毒素的dh已经加热到70-80℃2 O。后冷却至RT，中和该溶液至pH 7.0，用1M HCl中。过滤消毒（0.22微米）的溶液中。分装1微克/微升PEI原液（1,400微升/管）和存储SOLUT离子在-20℃。 </li><li>培养HEK293细胞在Dulbecco改进的Eagle培养基（DMEM）中，用10％胎牛血清（FBS）和1％青霉素/链霉素。培养的细胞在37℃培养箱用5±0.5％的二氧化碳（CO 2）。 </li><li>在第0天，板块HEK293细胞十150毫米的菜肴在1分裂&gt; 90％汇合细胞转染前18-20小时:2的稀释。 注:在第1天，将细胞要达到90％汇合。 </li><li>第1天，转染HEK293细胞与rAAV9质粒（ 例如，rAAV9.cTNT :: GFP或rAAV6.U6 :: shRNA的构建），广告辅助质粒，并采用PEI 25,26,29 AAV-REP /帽质粒。 <ol><li>在90％汇合10菜肴的细胞，在一个50毫升的离心管中混合70微克的AAV-REP /盖质粒，70微克粉煤rAAV9质粒，和200微克的Ad-辅助质粒的。 </li><li>如果细胞较少汇合时，按比例调整的DNA量。例如，如果细胞是在75％汇合时，减少DNA金额比例（步骤2.4.1所示的金额75/90）:混合70×75/90 = 58.3微克AAV-REP /帽质粒，70×75/90 = 58.3 rAAV9质粒微克和200× 75/90 = 166.7的Ad-辅助质粒的在50毫升离心管微克。 </li><li>加入49毫升RT DMEM（无FBS）到50毫升试管中拌匀。 </li><li>添加1,360微升的PEI溶液，使PEI:DNA比值（体积/重量）是4:1。拌匀。孵育在室温下进行15 - 30分钟。 </li><li>添加在步骤2.4.4制备每150毫米培养皿（50毫升混合物的十150-mm培养皿中）的混合物的5毫升</li></ol></li><li>培养的细胞在37℃培养箱用5±0.5％CO 2的60-72小时。 </li></ol> 3.转染的HEK293收获细胞与rAAV9载体纯化<ol><li>收获细胞转染后60-72小时。去除并通过上下抽吸与培养基暂停在餐具的细胞。转移所有的细胞悬液无菌50毫升管。 </li><li>离心细胞，在500×g离心5分钟。悬浮用5ml的PBS将细胞沉淀在每个管和所有的细胞悬浮液合并成一个50ml管中。 </li><li>离心细胞，在500×g离心5分钟。弃去上清液。在此步骤中，存储所述细胞沉淀在-80℃下或立即从粒料净化AAV，如在步骤3.4-3.15说明。 </li><li>制备裂解缓冲液:150mM的氯化钠和20mM的Tris-HCl，pH值8.0。过滤消毒（0.22微米）。储存在4℃的缓冲液中。 </li><li>悬浮用10ml裂解缓冲液的沉淀。 </li><li>冷冻裂解物在-80℃下或在干冰/乙醇浴中，然后解冻它在37℃。涡旋1分钟。冻结和解冻的裂解物3次。 </li><li>添加MgCl 2溶液的解冻溶胞产物（使的 MgCl 2中的裂解物中的最终浓度为1mM）。核酸酶添加到250单位/毫升的最终浓度。孵育在37℃下15分钟以溶解该DNA /蛋白质AGGRegation。 注:如果DNA /蛋白聚集没有得到核酸内切酶或治疗后溶解，均匀DOUNCE的裂解液的20倍。 </li><li>离心样品以4800rpm×g离心在4℃下20分钟。收集上清液。 </li><li>同时，准备碘克沙醇梯度的解决方案: <ol><li>通过混合5毫升10×PBS中，0.05毫升的1摩尔的 MgCl 2，0.125mmol毫升的1摩尔的KCl，10毫升5M的NaCl，和12.5毫升ofdensity梯度介质制备梯度溶液的17％。调整总体积为用H 2 O50毫升</li><li>通过混合5毫升10×PBS中，0.05毫升的1摩尔的 MgCl 2，0.125mmol毫升的1摩尔的KCl，20毫升的密度梯度介质和0.2毫升0.5％（重量/体积）酚红制备25％的溶液。调整总体积为用H 2 O50毫升</li><li>通过混合5毫升10×PBS中，0.05毫升的1摩尔的 MgCl 2，0.125mmol毫升的1摩尔的KCl，和33.3毫升密度梯度介质的制备40％的溶液。调整用总体积至50mlH 2 O </li><li>通过混合制备60％溶液0.05毫升的1M MgCl 2的，0.125mmol毫升的1摩尔的KCl，50毫升的密度梯度介质的，和0.1ml 0.5％（重量/体积）酚红。 </li></ol></li><li>用针和注射器，加载碘克沙醇梯度溶液进聚丙烯管以5毫升17％，5毫升25％，5毫升40％和5毫升60％的数量级，从底部开始。加载从渐变的最高领奖台3.8（14-16毫升）中获得的所有裂解液。梯度从底部列出上方，是60％，40％，25％，17％，和所述裂解物层。填用裂解缓冲液的管中，用软木塞覆盖。 </li><li>离心在16℃185000 XG 90分钟。 </li><li>收获病毒级分（40％层）用注射器。插入针（21号）插入40％和60％的级分之间的交叉点，只吸移40％层。 注:避免吸ANY 25％的层。 </li><li>混合病毒的部分用消毒聚氧乙烯 - polyoxypropylene嵌段共聚物的PBS溶液（10％聚氧乙烯 - 聚氧丙烯嵌段共聚物库存1:10,000稀释在PBS中）至15ml的总体积。加载混合物进入过滤管（截止MW = 100,000道尔顿）。离心机在2000×g离心在4℃下30分钟。 </li><li>丢弃在底部的溶液。重新填充聚氧乙烯 - 聚氧丙烯嵌段共聚物PBS溶液过滤管的15毫升总体积。离心机在2000×g离心在4℃下20分钟。重复此步骤两次。收集纯化rAAV9病毒（过滤器上面的分数）。 </li><li>转移纯化rAAV9在过滤器管1.7毫升管。分装纯化rAAV9（100 - 400微升/管，这取决于所述AAV的体积和滴度）和病毒储存在-80℃。 注:避免重复冷冻解冻。 </li></ol> 4. rAAV9的效价测定<ol><li> 制备标准DNA样本。 <ol><li>具体的设计和高效的PCR引物rAAV9矢量和优化的PCR条件。 注:在本研究中使用的引物是"转发:TCGGGATAAAAGCAGTCTGG;反向:TCGGACGGAGATACGTGAGT"。在95℃下3分钟初始变性;:PCR反应以下列条件下进行95℃35个循环持续20秒，60℃15秒，72℃，10秒;和在72℃最终延伸10分钟。然而，优化引物和PCR条件是质粒特异性的，如在rAAV9矢量的插图序列可影响的PCR 31的特异性和效率。 </li><li>执行与步骤4.1.1所示的条件进行PCR反应。净化用凝胶提取试剂盒将PCR产物。 </li><li>测量使用分光光度计纯化的DNA的浓度。计算基于所述PCR产物的分子量/长度的DNA分子数的浓度。 <ol><li>使用下列公式计算的分子浓度:莫lecular浓度（DNA分子或片段/毫升）= 6.23×10 23摩尔1×体质。 ×10 -6 /兆瓦。注:（6.23×10 23摩尔-1是Avagadro的数量;刀豆:DNA浓度微克/毫升; MW:以g / mol的分子量）。例如，如果获得的PCR产物的浓度为100微克/毫升，其长度为200碱基，该双链DNA的分子量为2×200×310 = 124000（平均在每个核苷酸的分子量单链DNA是约310克/摩尔）。分子浓度（DNA分子/毫升）= 6.23×10 23摩尔-1×100微克/毫升×10 -6 /124000克/摩尔= 5.18×10 14的DNA分子/毫升。 </li><li>执行稀释系列的DNA片段，并制备标准样品，以10 13个分子/毫升，10 12个分子/毫升，10 11个分子/毫升，10 10个分子/毫升，10 9个分子/毫升，10个浓度<suP&gt; 8分子/ ml，并将10 7分子/毫升。使用1微升解决方案的定量PCR每个标准样品（定量PCR，在步骤4.6）。 </li></ol></li></ol></li><li>混合5微升纯化的rAAV9溶液与5微升10×DNA酶缓冲液，1μl的DNA酶的（10,000单位/毫升），和的DDH 2 O的39微升的总体积应为50微升。 </li><li>孵育在37℃下的小瓶中30分钟以去除残留的未封装质粒DNA。 </li><li>灭活DNA酶在95℃下10分钟。冷静下来的溶液，在加入44微升H 2 0，5微升10X DNA酶缓冲液，1微升蛋白酶K的股票（10毫克/毫升）。 </li><li>孵育在50℃下该溶液2小时。终止反应，灭活蛋白酶K在95℃下10分钟。 </li><li>使用1微升样本的定量PCR（定量PCR）检测的。计算滴度。 <ol><li>用样品FR步骤4.1.1设计引物，运行定量PCR（定量PCR）嗡步骤4.1.4（标准样品），并从步骤4.5（样品被测量）。 <ol><li>对于每个反应，混合2倍青主混合物的10微升（含有Taq聚合酶，dNTP混合物，缓冲液，MgCl 2的，与绿色染料），0.5微升正向引物（5微米），0.5微升反向引物（5微米）的， 8微升的H 2 O和1μl的样品进行测量。在以下条件下进行的qPCR:握住样品在50℃下2分钟和95℃，10分钟;在95℃下进行40个循环15秒，在60℃，1分钟;对于熔融阶段，孵育样品在95℃下30秒和60℃持续15秒。基于所述标准样品（ 图3）的（C T）的数字标准曲线。 </li></ol></li><li>计算相对于标准曲线的AAV样品的分子浓度/效价。的rAAV9具有单链DNA基因组，因此，分子的浓度将是2倍比更高计算值（2×功率（10，y）时， 图3B）。此外，纯化的rAAV9的滴度将是20倍比什么是从计算由于1:20稀释在DNA酶和蛋白酶K反应的病毒（5微升在100微升总）的获得更高。 </li></ol></li></ol> 5. rAAV9注射液在心脏新生小鼠与基因表达分析<ol><li>制备在聚氧乙烯 - 聚氧丙烯嵌段共聚物的PBS溶液rAAV9工作溶液。使病毒库存以1-7×10 12个粒子/ ml的效价。 注:提供50-70微升rAAV9溶液到每个日龄0.5-1.5小鼠皮下注射。实现高效的基因过表达或敲除，建议为每个研究以优化注入的AAV的量进行试验性测试。使用rAAV9.cTNT ::吕克或rAAV9.U6 ::扰频控制相同数量的每个研究，以减少偏差。 注:我们使用1-1.5×10 11粒/小狗的过度表达与2.5 - 5×10 11粒/为击倒在出生后0.5-1.5英里CE）的小狗。 </li><li> 通过皮下注射治疗在P0.5-P2.5与rAAV9新生小鼠。 <ol><li>预填充29G1 / 2，0.33点¯x12.7毫米胰岛素与rAAV9液的注射器。小心去除气泡。 </li><li>握住一只手低温麻醉的小狗用拇指和食指。在注射前，滑动用70％异丙醇饱和维持无菌状态的拭子棒小狗的背部皮肤。将注射器针头插入动物的前侧 - 背侧皮下组织以5到10°的角度。注射50-70微升使用胰岛素注射器rAAV9解决方案。 注:rAAV9也可通过腹膜内或静脉内注射26,27递送到小鼠。能够得到在心脏的递送基因的有效表达。然而，腹腔注射离子有时可导致在肝脏渗漏表达。注射后，幼崽的条件每天监测。 </li></ol></li><li>在心脏基因表达的水平可以与定量PCR，免疫荧光或蛋白印迹分析进行监测（代表性的结果示于图4和5）25,26。 注:小鼠用CO 2从压缩气体源输送安乐死。组织样品证实心脏速率，运动和动物的呼吸停止后收集。新生儿啮齿动物抗CO 2安乐死，并通过使用锋利的剪刀斩首被实施安乐死。该方法是在美国兽医协会的安乐死小组的建议相一致。 </li></ol>

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The authors have nothing to disclose.

质粒构建中，以尽量减少不希望的ITR重组是重要的。产生病毒之前，必须始终监视用限制性消化和琼脂糖凝胶电泳所述AAV质粒的ITR完整性。这是不可能的，获得100％完好的质粒，但重组比值应该被最小化，尽可能。小于20％是成功rAAV9包装上可接受的。值得注意的是，在与低级振荡速度（180-200转）低的温度（30℃）培养所述细菌可减少ITR重组的机会。 重要的是要确保HEK293细胞?...

A correction was made to the Authors section in: <a href="http://www.jove.com/video/54787">Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts</a>.
One of the authors names and affiliation was corrected from:
Jian-Ming Jiang3,4 
3 Department of Genetics, Harvard Medical School 
4 Howard Hughes Medical Institute
to:
Jianming Jiang5 
5 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore

A correction was made to the Authors section in: <a href="http://www.jove.com/video/54787">Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts</a>.

Erratum: Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

控制各种生物系统的特定基因的表达或活性已经成为在基因功能1的研究的有价值的策略。实现这一目标的一个直接的方法是对操纵核苷酸序列，并产生突变等位基因。虽然进行精确的，有针对性的变化到活细胞的基因组中仍 一个耗时耗力的做法，强大的TALEN和CRISPR / Cas9工具的发展开辟基因组编辑2-5的新时代。对于基因操作更常规的实验室方法重点推出遗传物质（DNA的含有编码序列或siRNA的/的shRNA的RNA）进入细胞表达或击倒感兴趣1,6基因（S）的。 在许多情况下，用于基因操作的主要瓶颈是递送DNA，RNA或蛋白的导入细胞。对于体外研究，高效transfecti上系统已经建立了许多培养的细胞系。然而，在特定的小鼠模型中， 体内基因递送是更具挑战性。有一系列的需要，以实现外源试剂的高效细胞摄取被绕过细胞内外的障碍。其他障碍包括快速清除和交付的材料7,8的持续时间短。规避这些问题的一个策略是使用病毒载体的"载体"或"车辆" 用于体内基因递送。病毒的自然进化的传导属性允许感兴趣进入细胞7,9,10基因的有效提供。许多类型的病毒载体，已经开发和实现在不同类型的细胞和器官的小鼠体内基因操作灵活。 最常用的病毒系统包括逆转录病毒，慢病毒，腺病毒，和腺相关病毒（AAV） <s达&gt; 11。逆转录病毒是单链RNA病毒，并且可以有丝分裂过程中引入遗传物质，以稳定的方式在宿主细胞基因组中，提供用于在靶细胞和器官12-14转导的基因的终身表达的潜力。但是，许多类型的逆转录病毒只感染分裂的细胞，以及它们在非分裂细胞中的功效是非常低的15。这限制了其效用的基因传递。慢病毒是逆转录病毒科的一属。从其他逆转录病毒不同，慢病毒可以感染两个分割和非分裂细胞，并已被广泛用于基因转移到有丝分裂后和高度分化的细胞16。慢病毒的生命周期也涉及整合载体DNA导入宿主基因组中。因此，慢病毒介导的基因递送使转导的遗传元件16-18的稳定和长持续时间的表达。但是，此功能可能代表一个双-E在使用这些病毒dged剑操纵基因表达，作为载体DNA的整合可能导致插入诱变 在宿主细胞中，并可能导致假象的影响。腺病毒是另一种广泛使用的基因递送系统。不像逆转录病毒和慢病毒，腺病毒是非集成，并且不与宿主细胞8,10,11,19的基因组完整性干扰。此外，腺病毒可以转染DNA引入许多细胞类型和感染是不依赖于活性细胞分裂19。腺病毒的另一个重要特性是易于矢量纯化的，因为病毒载体有能力要复制19,20。然而，这种系统的主要告诫是腺病毒感染可触发靶细胞和器官19强烈的免疫反应，限制在许多调查其使用，特别是在基因治疗研究。 这些不同类型的比较病毒载体的S，重组腺相关病毒（腺相关病毒）似乎是理想的基因递送系统21,22。它具有最小的免疫原性和致病性23,24。此外，腺相关病毒感染广泛的细胞类型，包括分割和非分裂细胞。在大多数情况下，重组腺相关病毒不整合到宿主基因组中;因此，在靶细胞不希望的基因或基因组的改变的风险是低的22。 最近，重组腺相关病毒系统已被成功地用于DNA编码的蛋白质中，miRNA，shRNA的，和CRISPR-gRNAs的体内递送到小鼠心肌23,25-29。这种方法在心血管领域的研究促进了基础研究和基因治疗的研究。这里，详细过程生成rAAV9载体能够有效地过表达或敲除的小鼠心脏目的基因进行了说明。该协议提供了一个简单而有效的方法在小鼠实验模型中操纵心脏基因表达。

<table><tbody><tr><td>Polyethylenimine, Linear (MW 25,000)</td><td>Polysciences, Inc.&amp;nbsp;</td><td>#23966-2</td><td></td></tr><tr><td>Tube, Polypropylene, 36.2 ml, 25 x 87 mm, (qty. 56)</td><td>Beckman Coulter, Inc</td><td># 362183</td><td></td></tr><tr><td>Nuclease, ultrapure</td><td>SIGMA</td><td>#E8263-25KU</td><td></td></tr><tr><td>Density Gradient Medium(Iodixanol)</td><td>SIGMA</td><td>#D1556-250ML</td><td></td></tr><tr><td>Centrifugal Filter Unit with Ultracel-100 membrane</td><td>EMD Millipore Corporation</td><td>#UFC910008</td><td></td></tr><tr><td>Laboratory pipetting needle with 90&amp;deg; blunt ends,gauge 14, L 6 in., nickel plated hub</td><td>SIGMA</td><td>#CAD7942-12EA</td><td></td></tr><tr><td>Poloxamer 188 solution (Pluronic&amp;reg; F-68 solution)</td><td>SIGMA</td><td>P5556-100ML</td><td></td></tr><tr><td>Proteinase K</td><td>SIGMA</td><td>3115828001</td><td></td></tr><tr><td>DNase I</td><td>Roche</td><td>10104159001</td><td></td></tr><tr><td>Centrifuge machine</td><td>Thermo Scientific</td><td>75004260</td><td></td></tr><tr><td>Centrifuge System</td><td>Beckman Coulter</td><td>363118</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>DMEM medium</td><td>Fisher Scientific</td><td>SH30243FS</td><td></td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Atlanta Biologicals&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>S11150</td><td></td></tr><tr><td>rAAV9 vector</td><td>Penn Vector Core</td><td>P1967</td><td></td></tr></tbody></table>

preparation of raav9 to overexpress or knockdown genes in mouse hearts

控制通过在鼠模型的心肌递送遗传物质的表达或特定基因的活性使得基因功能的调查。它们在心脏治疗潜力也可确定。有在小鼠心脏体内分子介入有限的方法。重组腺相关病毒（腺相关病毒）系的基因组工程已被利用作为用于体内心脏基因操作的基本工具。这项技术的具体优点包括高效率，高特异性，低基因组整合率，最小 免疫原性，和最小的致病性。这里，构造，封装，和纯化rAAV9矢量的详细过程进行说明。皮下注射rAAV9成新生幼仔导致鲁棒表达或在小鼠心脏感兴趣的基因（多个）的有效敲低，但不是在肝脏和其他组织。使用心脏 - 技术规格获得ÇTNNT2子，在心脏GFP基因的高表达。此外，靶mRNA是在心脏抑制被利用的rAAV9-U6-shRNA的时候。工作rAAV9技术方面的知识可能对心血管有益的调查。

为rAAV9施工rAAV9.cTNT :: GFP或rAAV9.U6 :: shRNA的质粒的策略示于图1和2，分别为。作为例子，在生成rAAV9矢量过表达GFP基因在小鼠心脏。所得质粒含有由两个ITR位点（ 图1）侧翼的肌钙蛋白:: GFP盒。所述rAAV9.U6 :: shRNA的载体构建拦截TRBP的mRNA（ 图2） 用通过线性回归的qPCR数据生成rAAV9滴...

Watch this Scientific Journal Video about Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts at JoVE.com

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

在小鼠心脏rAAV9的制备过表达或敲除基因

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

preparation-of-raav9-to-overexpress-or-knockdown-genes-in-mouse-hearts

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12.7K 次观看.  Boston Children's Hospital. In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described. 

视频: Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Delivery of Modified mRNA in a Myocardial Infarction Mouse Model

Myocardial infarction (MI) is a leading cause of morbidity and mortality in the Western world. In the past decade, gene therapy has become a promising treatment option for heart disease, owing to its efficiency and exceptional therapeutic effects. In an effort to repair the damaged tissue post-MI, various studies have employed DNA-based or viral gene therapy but have faced considerable hurdles due to the poor and uncontrolled expression of the delivered genes, edema, arrhythmia, and cardiac hypertrophy. Synthetic modified mRNA (modRNA) presents a novel gene therapy approach that offers high, transient, safe, nonimmunogenic, and controlled mRNA delivery to the heart tissue without any risk of genomic integration. Due to these remarkable characteristics combined with its bell-shaped pharmacokinetics in the heart, modRNA has become an attractive approach for the treatment of heart disease. However, to increase its effectiveness in vivo, a consistent and reliable delivery method needs to be followed. Hence, to maximize modRNA delivery efficiency and yield consistency in modRNA use for in vivo applications, an optimized method of preparation and delivery of modRNA intracardiac injection in a mouse MI model is presented. This protocol will make modRNA delivery more accessible for basic and translational research.

This protocol presents a simple and coherent way to transiently upregulate a gene of interest using modRNA after myocardial infarction in mice.

在心肌梗死小鼠模型中传递修正的mRNA

Myocardial infarction (MI) is a leading cause of morbidity and mortality in the Western world. In the past decade, gene therapy has become a promising ...

科研

遗传学

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

成人的心肌细胞的分离和遗传操作共聚焦成像

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

生物学

小鼠心脏中基于电穿孔的心肌细胞转基因

An Efficient Transgenesis Approach for Gene Delivery in the Mouse Embryonic Heart

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, and fate of these progenitors are vital for the proper development of this organ. The molecular mechanisms that govern the morphogenesis of the heart are essential for understanding the pathogenesis of congenital heart diseases and embryonic cardiac regeneration. Classical approaches to investigate these mechanisms employed the generation of transgenic mice to assess the function of specific genes during cardiac development. However, mouse transgenesis is a complex, time-consuming process that often cannot be performed to assess the role of specific genes during heart development. To address this, we have developed a protocol for efficient electroporation and culture of mouse embryonic hearts, enabling transient transgenesis to rapidly assess the effect of gain- or loss-of-function of genes involved in cardiac development. Using this methodology, we successfully overexpressed Meis1 in the embryonic heart, with a preference for epicardial cell transfection, demonstrating the capabilities of the technique.

作者聚焦:用于快速基因筛选的心肌细胞转基因

This protocol presents a detailed methodological framework for electroporation-based transgenesis of cardiac cells in developing mouse hearts. The video assets provided here will facilitate learning of this versatile technique.

一种在小鼠胚胎心脏中进行基因递送的高效转基因方法

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, ...

发育生物学

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

腺相关病毒介导的 CRISPR 在小鼠心脏基因编辑中的传递

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

医学

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

细胞标记和注射胚胎发展小鼠心脏

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including Mef2c, Gata4, Tbx5 (MGT), fibroblasts can be reprogrammed into induced cardiomyocytes (iCMs). These iCMs, when generated in situ in an infarcted heart, integrate electrically and mechanically with the surrounding myocardium, leading to a reduction in scar size and an improvement in heart function. Because of the relatively low reprogramming efficiency, purity, and quality of the iCMs, characterization of iCMs remains a challenge. The currently used methods in this field, including flow cytometry, immunocytochemistry, and qPCR, mainly focus on cardiac-specific gene and protein expression but not on the functional maturation of iCMs. Triggered by action potentials, the opening of voltage-gated calcium channels in cardiomyocytes leads to a rapid influx of calcium into the cell. Therefore, quantifying the rate of calcium influx is a promising method to evaluate cardiomyocyte function. Here, the protocol introduces a method to evaluate iCM function by calcium (Ca2+) flux. An &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mouse strain was established by crossing Tg(Myh6-cre)1Jmk/J (referred to as Myh6-Cre below) with Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as Rosa26A-Flox-Stop-Flox-GCaMP3 below) mice. Neonatal cardiac fibroblasts (NCFs) from P0-P2 neonatal mice were isolated and cultured in vitro, and a polycistronic construction of MGT was introduced to NCFs, which led to their reprogramming to iCMs. Because only successfully reprogrammed iCMs will express GCaMP3 reporter, the functional maturation of iCMs can be visually assessed by Ca2+ flux with fluorescence microscopy. Compared with un-reprogrammed NCFs, NCF-iCMs showed significant calcium transient flux and spontaneous contraction, similar to CMs. This protocol describes in detail the mouse strain establishment, isolation and selection of neonatal mice hearts, NCF isolation, production of retrovirus for cardiac reprogramming, iCM induction, the evaluation of iCM Ca2+ flux using our reporter line, and related statistical analysis and data presentation. It is expected that the methods described here will provide a valuable platform to assess the functional maturation of iCMs for cardiac reprogramming studies.

We describe here, the establishment and application of an Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 below) mouse reporter line for cardiac reprogramming assessment. Neonatal cardiac fibroblasts (NCFs) isolated from the mouse strain are converted into induced cardiomyocytes (iCMs), allowing for convenient and efficient evaluation of reprogramming efficiency and functional maturation of iCMs via calcium (Ca2+) flux.

使用GCaMP3报告器测量心脏特异性钙通量，对心脏重编程进行体外评估

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including ...

脉络丛中的选择性基因沉默

Targeted Knockdown of Genes in the Choroid Plexus

The choroid plexus (ChP) serves as a critical gateway for immune cell infiltration into the central nervous system (CNS) under both physiological and pathological conditions. Recent research has shown that regulating ChP activity may offer protection against CNS disorders. However, studying the biological function of the ChP without affecting other brain regions is challenging due to its delicate structure. This study presents a novel method for gene knockdown in ChP tissue using adeno-associated viruses (AAVs) or cyclization recombination enzyme (Cre) recombinase protein consisting of TAT sequence (CRE-TAT). The results demonstrate that after injecting AAV or CRE-TAT into the lateral ventricle, the fluorescence was exclusively concentrated in the ChP. Using this approach, the study successfully knocked down the adenosine A2A receptor (A2AR) in the ChP using RNA interference (RNAi) or Cre/locus of X-overP1 (Cre/LoxP) systems, and showed that this knockdown could alleviate the pathology of experimental autoimmune encephalomyelitis (EAE). This technique may have important implications for future research on the ChP's role in CNS disorders.

作者聚焦：脉络丛研究中基因表达操作技术的见解和创新 

Here, we describe a method to selectively alter gene expressions in the choroid plexus while avoiding any impact in other brain areas.

脊索丛中基因的靶向敲除

The choroid plexus (ChP) serves as a critical gateway for immune cell infiltration into the central nervous system (CNS) under both physiological and ...

在小鼠心脏rAAV9的制备过表达或敲除基因

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此视频中的章节

在心肌梗死小鼠模型中传递修正的mRNA

成人的心肌细胞的分离和遗传操作共聚焦成像

一种在小鼠胚胎心脏中进行基因递送的高效转基因方法

一种在小鼠胚胎心脏中进行基因递送的高效转基因方法

一种在小鼠胚胎心脏中进行基因递送的高效转基因方法

腺相关病毒介导的 CRISPR 在小鼠心脏基因编辑中的传递

细胞标记和注射胚胎发展小鼠心脏

使用GCaMP3报告器测量心脏特异性钙通量，对心脏重编程进行体外评估

脊索丛中基因的靶向敲除

脊索丛中基因的靶向敲除