由宁授予R01MH097949的自闭症试点补助7359到BWL和诺里斯棉花癌症中心光学成像仪器共享格兰特P30CA023108这项工作的支持和。

伦理学声明:所有协议是由达特茅斯生物安全制度和体制的动物护理和使用委员会审查委员会批准。 1.协议设计一个引导链（因组）的CRISPR / Cas9逆转录病毒注意:有许多可以用来生成sgRNAs非营利性网站为目标的目的基因（https://benchling.com/和http://crispr.mit.edu/）。这个协议的目标是设计并从该退火到彼此商业供应商订货单链寡聚物。此退火寡将连接入PXL转移载体。见补充视频1用于使用Benchling设计sgRNAs的一个例子。 <ol><li>使用致力于设计sgRNAs网站。 注意:例如，从目的基因的附近开始输入的编码/外显子序列插入该网站将产生一个20核苷酸因组。使用20核苷酸因组序列来设计有义和反义寡核苷酸S中的将被责令创建因组。 </li><li>产生因组后，复制此序列到一个字处理器。添加一个G（鸟嘌呤）的20个核苷酸因组序列的开始文档中，如果它不已经与一个（ 即，G-20核苷酸因组序列）开始。这是必要的，以确保良好的转录关闭U6启动子。 </li><li>包含本发现在20-21的核苷酸序列的反向互补。对于有义寡核苷酸中，添加"CACC"到文档（ 即，CACC-G-20核苷酸因组序列）中的序列的5&#39;端。这个突出端将用于连接序列插入PXL载体。 </li><li>对反义寡核苷酸中，添加AAAC到5&#39;端。使用这个悬垂到序列结扎到PXL载体。 </li><li>获得有义和反义寡核苷酸。接到寡核苷酸后，用DNA酶自由水使100μM股票。混合10微升各100μM的有义和反义寡聚物连同4μ微升10x NEB缓冲液2，和16微升水。 注:他们不需要页面纯化或5&#39;phosphorylation（作为BBSI悬是非相容性粘性末端）。 <ol><li>带来200ml水到沸腾的500毫升烧杯中，然后漂浮含有以泡沫"浮动"持有人此混合物的管。使水从95℃缓慢冷却，在室温下2小时。 </li><li>在无菌水中1000和下面的结扎立即使用或储存于-20℃下剩余的反应:稀释现在退火寡混合料1。 </li></ol></li><li>消化PXL，一个PX330矢量衍生物，在37℃下的BBSI限制性酶2小时。使用含有2微克为PXL的，4微升10X NEB缓冲2,1 BBS1和平衡水的微升40微升的反应。经受使用商业试剂盒将消化-PXL常规凝胶纯化。确保产量〜8.5 kb的产物。 </li><li>使用商业连接试剂盒，结扎1微升的1:1000退火，寡用50ng消化和凝胶纯化-PXL的。变换连接产物成缺陷型大肠杆菌主管重组大肠杆菌 （NEB 5-α，亚克隆效率）。筛选质粒DNA测序存在的正确导向的转化体。 </li><li>消化用BstB1和PAC1限制酶U6启动，引导链，和RNA脚手架元件（因组）出为PXL的和结扎到用相同的限制性酶消化所述荧光蛋白-T2A-Cas9病毒骨架。变换连接产物（NEB 5-α最大效率）。荧光蛋白-T2A-Cas9病毒骨架质粒是低拷贝质粒并因此低拷贝协议必须被使用。 注意:第二个因组可以由其它PXL引导链质粒的有PacI消化类似地引入并连接入PAC1消化和小牛肠磷酸酶处理已经包含第一引导链病毒骨架。病毒转移质粒的磷酸酶治疗将有助于减少来自不包含第二引导质粒的自身连接的转化体数目。 </li><li>顺序使用Nucleobond的Xtra马克西制备试剂盒验证最终的质粒和马克西准备并将其打包到使用下面的"复古/慢病毒生产协议 - CaPO4法"的病毒。 </li></ol> 2.准备293FT / 293GP细胞转染（复古/慢病毒生产 - CaPO4法） <ol><li> （第1天）快速解冻1小瓶每10cm细胞培养板的细胞在37℃水浴中。对于慢病毒包装，用293FT细胞。对于逆转录病毒包装，使用293GP（GAG / POL）细胞。 </li><li>吸取全部来自冷冻管解冻细胞的入15ml锥形管中，并加2ml预温热的 CO 2平衡-完整的Iscove氏改良的Dulbecco氏培养基。 </li><li>在500 XG离心机细胞5分钟以沉淀。吸出上清液，重悬细胞沉淀在10ml完全是海湾的改良Dulbecco培养基。板上的细胞到10cm的细胞培养皿。孵育细胞过夜，在37℃在5％二氧化碳培养箱。 </li><li> （第2天）电镀后24小时，通过吸现有媒体和加入10ml预热的Iscove氏改良的Dulbecco培养基到板改变上盘介质。 <ol><li> （天3-4）后24-48小时的媒体变化，并且一旦细胞变得汇合，细胞分裂，2.5-3.0×10 6个细胞/板（10厘米板）的汇合。 </li><li>分裂的细胞，吸出介质并用5毫升的PBS洗涤板。 1毫升0.25％胰蛋白酶的添加到板并于37℃孵育直至细胞抬离板。将0.5ml的Iscove氏改良的Dulbecco培养基，以中和0.25％胰蛋白酶反应和吸取细胞至1.5 ml管中。 </li><li>旋转细胞在500×g离心5分钟。重悬细胞于1ml的Iscove氏改良的Dulbecco氏培养基。 10稀81;在90微升PBS中的细胞的湖计数使用一个血球或自动细胞计数细胞。重板2.5-3.0×10 6个细胞/板具有完整的Iscove氏改良的Dulbecco氏培养基。 注:细胞就会〜50％汇合电镀后24-34小时。转染的细胞时，他们〜50％汇合。 </li></ol></li></ol> 3.（第5天）CaPO4转染和病毒颗粒集合<ol><li>通过吸现有媒体和加入10ml预热的Iscove氏改良的Dulbecco氏培养基的介质2小时改变转染前。确保这正是存在的10ml媒体在10cm平板。 </li><li>制备转染试剂用2,5毫升圆底聚苯乙烯试管2的菜肴。标记在第一管的"DNA"和第二管"2X HBS"。调节DNA的浓度在pH 7.4至1微克在Tris-EDTA /微升。 <ol><li>对于慢病毒，慢慢加入转移载体的20微升（维拉感兴趣升构建体），13微升CMVdelta8.9的，9微升VSV-G，860微升分子生物学等级H 2 O和100微升的2.5M 的 CaCl 2，以在第一管（"DNA"），同时连续攻丝对管进行混合。 </li><li>对于逆转录病毒，省略CMVdelta8.9质粒。加入20μl转移载体，15微升VSV-G，860微升分子生物学级的 H 2 O和100微升的2.5M的CaCl 2到标记为"DNA"的管。 </li><li>加入1ml 2×HEPES-缓冲盐水中（pH为7.0;该pH是绝对关键的）标有"2X HBS"管。 </li><li>缓慢的"DNA"管的1ml的内容添加到"2X HBS"管，每次一滴。连续点击用食指或中指的"2X HBS"管而添加的"DNA"管中的内容。每一滴后视观察投诉警察课4囊泡。孵育在黑暗中管在室温下30分钟。 </li></ol></li><li>在缓慢滴加入1ml转染的每10 1cm池板，然后孵育过夜，在37℃。 </li><li> （第6天）用8ml的Iscove氏改良的Dulbecco氏培养基+ 0.5％FBS和40毫克咖啡因/100毫升媒体更换介质。如果转移载体含有荧光标记物，那么荧光团表达式表示成功转染。 </li><li> （第7天），通过使用10ml的血清吸管收集含有病毒颗粒的介质，分装到50毫升锥形管中。储存在4℃的50ml锥形管中。 8毫升0.5％FBS的培养基添加到每个板。 <ol><li> （8日）再次，收集含有病毒颗粒的介质，并与前一天的收获在50毫升的锥形管相结合。 </li></ol></li></ol> 4.浓度与病毒的纯化<ol><li>通过加入40％的聚乙二醇6000和1.5M NaCl至DDH 2 O作5倍的聚乙二醇6000溶液高压灭菌的溶液在121℃下45分钟的液体循环。冷却时，慢慢地混合的解决方案。 注:该解决方案将开始浑浊，然后变得清晰，因为它冷却。 </li><li>离心含有病毒上清的两个集合的50ml锥形管中，在2000×g离心10分钟以沉淀不溶物质。由通过0.45μm低蛋白结合的注射器过滤器（PES或PVDF）过滤纯化的病毒媒体。 </li><li>添加5倍的聚乙二醇6000溶液媒体（终浓度应为8％的聚乙二醇6000和0.3 M氯化钠）。颠倒离心管数次（不涡）混合。孵育在4℃下在病毒含有聚乙二醇溶液为12个或更多小时，偶尔重新混合。 </li><li> （9日）离心2,500 xg离心45分钟病毒含有聚乙二醇溶液。取出并弃去上清液和2分钟再次旋转。再次，捞出弃去上清液。 </li><li>加入320微升悬浮颗粒无菌磷酸盐缓冲盐水（320微升是1/100收集病毒含有介质的原体积的），并孵育过夜，在4℃。任选地，悬浮在室温下颗粒上的摇杆30分钟。 </li><li>重新悬浮沉淀后，等分试样的病毒（5-10每0.5ml微升管）并在-80℃（冷冻在4℃解冻或存储将大大减少滴度）冷冻等分试样。 </li><li>滴定使用标准协议， 即病毒，对HEK293T细胞的6孔平板进行稀释系列和48小时后手动计数荧光菌落。 </li></ol> 5. CRISPR病毒测试功效注意:使用以下步骤顺序克隆测试对于生产用鼠标Neuro2A细胞NHEJ修复双链断裂的。这具有的优点超过测量员测定的，因为它可被用来确定已修改的细胞的百分比和自然从NHEJ造成的插入缺失。 <ol><li>涂层用凝胶状蛋白质混合物3.5公分细胞培养皿如基质胶稀释在Iscove氏改良的Dulbecco氏培养基1:50，于37℃孵育30分钟。吸出凝胶状蛋白混合物镀Neuro2A细胞。 </li><li>在Neuro2A细胞达到50％汇合后，添加CRISPR慢病毒或逆转录病毒以10的感染复数（即每个细胞10病毒颗粒）。 注:Neuro2A细胞不作为友好感染因为是HEK293细胞，从而，单个感染不会导致被感染了慢病毒的细胞的100％。另外，逆转录病毒只感染分裂的细胞，因此，也不会导致100％的感染。为了实现感染的接近100％的速度，除病毒的可重复上多天，可以根据需要分裂的细胞。备选地，荧光阳性细胞可使用荧光激活细胞分选进行分离。钍E要实现具有慢病毒100％的感染率最有效的方法是进行一个单一感染，随后通过FACS隔离。对于逆转录病毒，进行4轮感染相隔24小时，并用流式细胞仪分离遵循保证100％的感染。 </li><li>感染细胞1周后，展开细胞接近汇合于10cm板，吸出介质，并用5毫升磷酸盐缓冲盐水洗该板。加入1ml 0.25％胰蛋白酶并在37℃下孵育直至细胞抬离板。将0.5ml的Iscove氏改良的Dulbecco培养基，以中和0.25％胰蛋白酶反应和吸取细胞到1.5毫升管和沉淀细胞，在500×g离心5分钟。 </li><li>以分离的DNA，加入100微升的50mM氢氧化钾，重悬浮细胞，并在95孵育 °C为5分钟。中和，用10微升的1M Tris，pH值= 8.0的溶液中。 </li><li> PCR扩增该区域的侧翼由CRISPR因组定位的基因组区使用〜该DNA的300纳克分离在步骤5.5使用高保真PCR主混合物4。 </li><li>运行上的2.5％的琼脂糖凝胶和凝胶纯化使用凝胶纯化试剂盒的适当大小的片段的PCR反应如凝胶回收DNA试剂盒。结扎成为PGEM-T-容易相处的PCR克隆载体和等转化为感受态细胞9。 </li><li> 24小时后，加2ml LB肉汤中入15ml圆底管以及合适的抗生素（氨苄青霉素，新霉素等 ）。通过用无菌移液管尖端或循环选择来自LB板单菌落接种与单个菌落的管。扩大和在250RPM和37℃，24小时惊天管生长的殖民地。根据需要重复许多殖民地。 <ol><li>使用微型制备试剂盒，从大肠杆菌分离DNA 大肠杆菌 ，并与设计成放大由因组靶向的基因的区域，以分析小鼠基因组的Cas9目标区域的引物序列。 </li></ol></li></ol> 6.成年小鼠注射立体定位协议<ol><li>准备手术 注:为了保持在生存手术无菌条件是很重要的。这在此协议通过加热来完成灭菌的外科手术工具，使用优碘消毒注射部位，并且它被关闭之后加入抗生素软膏到切口部位。同样重要的是使用无菌手套，以及一个彻底消毒，专用手术区域。 <ol><li>热杀菌手术工具，通过高压灭菌两种或使用热珠灭菌后才能使用。通过接通加热垫手术和恢复期间保持体温准备回收室和手术区域。组装用于创建头骨注射孔钻。 </li><li>负载4微升病毒进入注射针直接从无菌PCR管吸病毒。简单地说，从冰删除病毒等分。保持病毒在室温下的注射器用于注射之前不超过1小时。 </li></ol></li></ol> 7.立体定位注射<ol><li>确认通风，手术套件是开放的，以确保适当的空气流通。通过在感应腔室，用4％的异氟醚麻醉准备鼠标进行手术。以下麻醉，剃的头部以制备注射部位。 <ol><li>通过使用镊子移动舌头向下和侧面放置在立体定位仪鼠标。将咬杆插入口中，直至牙齿放入插槽，然后固定耳酒吧。确保鼠标的主体是在加热垫与鼻位于鼻锥体。直接4％异氟醚到鼻锥。按捏用镊子脚，并确保没有惊跳反射确认麻醉。 </li><li>应用人工泪液润滑眼睛。完成与交替轮POV的擦拭光头准备注射部位吡咯烷酮碘和利多卡因。 </li></ol></li><li>使用手术刀，切开小切口沿着头皮中心。干后用棉签和过氧化氢头骨在必要时帮助可视化前囟门。 <ol><li>使用解剖范围，定位在头骨前囟门和地方钻尖的前囟门。零（或记录）在x，y和z平面的数字立体定向坐标。 </li><li>为了确保该头在喙尾鳍y轴水平，放置在λ钻头和使得z坐标是在两个前囟和lambda大致等于平头。 </li><li>为了确保该头是在x轴的水平，钻头放置到y = 1/2的λ。确保的z坐标是在上矢状缝的每一侧（X = +/- 1毫米）1毫米相等。在1mm至左和拉姆达的右调整颅骨如果有在差z坐标。 </li></ol></li><li>放置在钻在所需坐标。对于齿状回，使用的坐标SY从囟并且x =±1.1 = -1.9。钻孔之前，降低异氟烷至2％，然后缓慢并小心地通过颅骨钻孔。 <ol><li>钻所有的洞后，贴上填充注射器到立体定位仪。视觉中心维修过孔注射器和零头骨z坐标。 </li><li>慢慢放下注射器最深的Z深度。对于齿状回，在Z深度是-2.5，-2.4，-2.3和。在采用立体喷射器为0.25 L / min的速率开始注入。 <ol><li>注射是最低的Z深度完成后，等待1分钟，然后提升到下个坐标，重新开始注入。直到所有的z坐标注射注射继续这种模式。在最后一次注射后除去注射器之前等待2分钟。 注:无不良影响已在小鼠大脑注射到2微升每半球病毒对组织指出。 </li></ol></li><li>其他钻孔重复进样。 </li></ol></li><li>注射后，从立体定位仪中删除的小鼠和用6-0丝缝线缝合头皮。应用利多卡因和抗菌膏到伤口。 <ol><li>注射0.8-1.0毫升生理盐水+酮洛芬（3-5毫克/千克），通过IP路由管理的疼痛。然后将鼠标放到加热回收室。不要离开无人看管的动物，直到它已经恢复了足够的意识，以保持胸骨斜卧。不要将动物返回到公司的其他动物，直到它已完全恢复。 </li></ol></li><li>在跟随手术的日子里，每天权衡鼠标和提供软的食物和对待。检查伤口及注意全身情况/风范。 </li><li>以下立体定位注射时，小鼠可以安乐死用于切片制备电1或灌注和他们的大脑取出，切片，并通过免疫组织化学染色分析10。 </li></ol>

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<li>Sander, J. D., Joung, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR-Cas+systems+for+editing%2C+regulating+and+targeting+genomes%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">CRISPR-Cas systems for editing, regulating and targeting genomes.</a> Nat Biotechnol. 32 (4), 347-355 (2014).</li>
<li>Luikart, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((miR-132+mediates+the+integration+of+newborn+neurons+into+the+adult+dentate+gyrus%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">miR-132 mediates the integration of newborn neurons into the adult dentate gyrus.</a> PLoS One. 6 (5), e19077(2011).</li>
<li>Nasri, M., Karimi, A., Allahbakhshian Farsani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production%2C+purification+and+titration+of+a+lentivirus-based+vector+for+gene+delivery+purposes%5BTitle%5D)+AND+%22Cytotechnology%22%5BJournal%5D)">Production, purification and titration of a lentivirus-based vector for gene delivery purposes.</a> Cytotechnology. 66 (6), 1031-1038 (2014).</li>
<li>Ellis, B. L., Potts, P. R., Porteus, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Creating+higher+titer+lentivirus+with+caffeine%5BTitle%5D)+AND+%22Hum+Gene+Ther%22%5BJournal%5D)">Creating higher titer lentivirus with caffeine.</a> Hum Gene Ther. 22 (1), 93-100 (2011).</li>
<li>Bacterial Transformation: The Heat Shock Method. JoVE Science Education Database. Basic Methods in Cellular and Molecular Biology. , JoVE. Cambridge, MA. Available from:
 <a target="_blank" href="http://www.jove.com/science-education/5059/bacterial-transformation-the-heat-shock-method">http://www.jove.com/science-education/5059/bacterial-transformation-the-heat-shock-method</a> (2015).</li>
<li>Fricano, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fatty+acids+increase+neuronal+hypertrophy+of+Pten+knockdown+neurons%5BTitle%5D)+AND+%22Front+Mol+Neurosci%22%5BJournal%5D)">Fatty acids increase neuronal hypertrophy of Pten knockdown neurons.</a> Front Mol Neurosci. 7, 30(2014).</li>
<li>Park, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lentiviral+vectors%3A+are+they+the+future+of+animal+transgenesis%3F%5BTitle%5D)+AND+%22Physiol+Genomics%22%5BJournal%5D)">Lentiviral vectors: are they the future of animal transgenesis?</a> Physiol Genomics. 31 (2), 159-173 (2007).</li>
</ol>

作者什么都没有透露。

但是也有一些成功的病毒包装重要的几个关键步骤。细胞的健康至关重要之前和转染期间，为不健康细胞会大大降低病毒的产生量。如果转染和包装是成功的，然后将细胞的100％应该表达的荧光团和细胞应形成功能合胞体。在步骤3.2.4，轻敲管是必要的高滴度的转染效率，以及在HEPES缓冲的盐水的pH值必须准确。产生所需的病毒包装质粒的马克西棉片必须非常纯净。到这一点，是有帮助的乙醇沉淀DNA终的洗脱和在Tris-EDTA缓冲液重新悬浮。这也是非常重要的血清的量减少到2％或更少，所述咖啡因被添加到对病毒采集前6天（步骤3.4）的介质。如果血清不降低，那么最终纯化病毒包含血清PROT的不希望的量EIN。利用聚乙二醇沉淀病毒颗粒时6000的排除超速离心的必要性。同样重要的是要注意，Cas9含有CRISPR病毒通常具有约10滴度倍比单独含有荧光团病毒少。 对于立体定向手术，使用吸入麻醉允许快速和精确的控制或动物的意识相比，注射麻醉剂和麻醉可以在更大的年龄范围。它是保持外科手术器械的清洁和无菌非常重要，并且可再现的定位需要头的精确定位。确保没有在立体定位仪和头骨牢牢握在地方头部的俯仰或滚动。它可以是有用的，以允许颅骨中以便找到缝线来确定立体坐标干燥。此外，对于各立体坐标的速率和体积应根据经验确定。 这种技术限制，相对于腺相关病毒（自动增值服务）。因此当一个lenti-或反转录病毒的传播受到限制，尤其是，这些病毒感染的离散大脑区域时是有价值的，但不是对整体感染自动增值服务相关联用于动物行为分析。在这个协议中使用咖啡因的大大增加这些病毒的滴度，但它们仍然不那样高的AAV包装达到的效价。此外，稳定整合只有荧光团表达的优点，如CRISPR / Cas9形成甚至稳定的基因组编辑时，瞬时转染的和可能的是持续的Cas9和因组的表达可能最终产生脱靶效应。瞬时表达的CRISPR / Cas9系统自动增值服务足以产生在整个细胞分裂繁殖的基因组的改变，但是，荧光团表达不会被维持。 LEN创作抗-IGF和利用CRISPR / Cas9系统逆转录病毒将赋予针对任何新的基因在多种生物体的能力。基因编辑效率似乎依赖于导的RNA靶向Cas9裂解的序列。经验已经确定，％和克隆的80％至10包含插入缺失后测序感染Neuro2A细胞。它是目前未知的Neuro2A细胞计算插入缺失的频率是否反映那些神经元。指导的RNA等设计软件Benchling现在包括一个"上的目标"得分可能能够预测给定靶序列的效率。到什么程度，例如"关于目标"分数是可靠的需求中的神经元和其他细胞类型作为CRISPR-Cas9系统根据经验确定变得更加广泛实施。 基于慢病毒的转基因动物生产已经成功可变处理报表的慢病毒载体转基因交付SI成为lenced 11的DNA CRISPR介导的基因编辑可通过种系传递产生整个动物模型。因此，稳定的基因组编辑可以是尽管病毒递送的荧光团和Cas9转基因的沉默可以实现的。这可用于靶向基因组改变提供一种有效的平台。在病毒递送的CRISPR / Cas9系统，同时不需要的转基因生物，是这些技术的补充。例如，注射这种病毒颗粒成化合物转基因动物诱导性地表达的Cre和Cre的依赖性opto-或化学 - 基因的转基因应促进复杂的研究成遗传操作和神经元活性之间的关系。第二个例子是将这些Cas9 /因组病毒颗粒递送到一个条件性敲除，企图以筛选基因 - 基因相互作用。最后，这项研究的另一个令人兴奋的途径是患者的细胞表型与治疗化合物的筛选，从而可以用于验证和发现，在各种疾病打乱基因网络。

学习正常生理和疾病病理的基础上，有必要精确地操纵在模式生物的基因表达。用于哺乳动物的模式生物，这在很大程度上是在建立和转基因小鼠的发展，其中感兴趣的遗传元件是由重组酶识别位点两侧的中心。这会导致这些侧翼的基因的位点特异性操纵。虽然这是一个成功的策略，这是时间和资源密集型;例如，创建一个三转基因动物，将表达基因两侧装接loxP，Cre重组酶和酶Cre报告基因需要多次交配和验证。相反，复制的立体定位注射编码荧光蛋白和重组到两侧装接loxP基因动物缺陷病毒颗粒不需要复杂的基因分型或育种策略1。另外，如果荧光蛋白和Cre表达病毒是共注射与第二病毒ENCO鼎不同的荧光蛋白，那么这提供了一个内部​​组织控制目标遗传操作。虽然这个策略仍然需要使用敲入动物，病毒介导的基于RNA的策略规避转基因动物的需要。例如，编码荧光蛋白和短发夹RNA（shRNA）的复制缺陷型病毒立体定向注射可以使用细胞的内源RNAi机制导致一种有效减少所关注的基因的转录物。然而，shRNA的策略产生微妙的基因敲除的下调通常会导致适度的细胞表型2。而击倒可能更生理学相关的杂合基因功能障碍，其降低的鲁棒性相比，敲除是不理想的新基因的表型的发现。 第三种技术是最近出现的CRISPR（聚集定期相互间隔短回文重复）/ Cas9（CRISPR相关蛋白9）系统，依靠这两个小外源RNA和DNA的切割酶的表达。的CRISPR / Cas9系统适于从该演进识别外国，侵入从病毒DNA和经由Cas9酶3,4-定位使其降解的方法，该原核免疫系统。这种强大的基因组编辑技术可用于创建有针对性的缺失，插入和突变;和下面的协议将概述如何使缺失感兴趣的基因以敲除其在体内的表达。该Cas9酶必须以引导RNA的同源感兴趣区域和邻接的支架的RNA来表达。使用这种技术的基因的敲除需要使用合成导的RNA（因组）在基因组中靶向Cas9到特定的区域，并诱导在感兴趣的位点双链断裂（DSB的）。这些双链断裂然后由内源性细胞修复机制通过非同源末端连接（NHEJ）的文件修复广告可能产生错义或无义突变，因此可以创建功能蛋白表达5的损失插入缺失。因为这个系统产生的基因组的改变，只需要在Cas9和因组的瞬时表达。然而，希望有一个稳定的荧光指示剂，以确定在这样的方式操作的细胞和它们的后代。 Lenti-和逆转录病毒具有稳定地整合目的DNA在其中保持长期的表达和有丝分裂过程中下降传递到子代细胞的宿主细胞的优势。这个协议描述了设计和生产两种类型的复制缺陷型，高滴度逆转录病毒的:人类免疫缺陷病毒衍生的慢病毒颗粒（慢病毒）和基于鼠马洛尼白血病病毒（反转录病毒）。尽管这两种病毒是能够支持大的转基因的稳定表达，反转录病毒颗粒可以仅整合入基因组杜与核膜的降解，和因此环细胞分裂可以用作标记的工具和出生日期的细胞6。尽管慢病毒有是相对低滴度7，这种方法，包括病毒收集期间使用咖啡因8的名声，通常会产生10 9和10 10颗粒/ ml的滴度。 lenti-和逆转录病毒的另一个优点是对于非常大的插入的公差。协议的下列集合概述设计伦蒂或逆转录病毒编码荧光记者，sgRNAs和Cas9地利用CRISPR / Cas9系统修改DNA以及表达荧光蛋白的过程。 小鼠立体定位神经外科是用于体内注射病毒研究形态学，功能和感染的神经元的连接性的有价值的方法。神经细胞病毒感染可反复使用的添长时间操纵表达水平即，如在整个开发和表达可通过使用各种不同的药物可诱导系统和特定的Cre驱动的表达的精确控制。这种特定的协议说明如何注入表达一种因组和Cas9敲除目标基因在成年小鼠脑中的病毒。小鼠恢复很快从病毒转基因的这个过程，表达可注射后48小时之内可见。然而，荧光表达出现了3个星期感染后，增加了产生接近最大水平星期的课程。该病毒进行注射立体小鼠可用于行为，电生理学或形态学研究。总体而言，这些程序的目的是展示如何敲除使用立体定位手术并表达特定因组和Cas9病毒在成年小鼠大脑的基因。

<table><tbody><tr><td colspan="4">&lt;strong&gt;List of Cell Culture Reagents&lt;/strong&gt;</td></tr><tr><td>293FT cell line</td><td>Life Technologies</td><td>R700-07</td><td>For Lentivirus</td></tr><tr><td>293GP cell line</td><td>Clontech</td><td>631458</td><td>For Retrovirus</td></tr><tr><td>Iscove&#039;s Modification of DMEM (IMDM)</td><td>Corning&amp;nbsp;</td><td>10-016-CV</td><td>Complete IMDM with 10% FBS, 1% NEAA, 1% L-Gln, and&amp;nbsp; 1% P/S</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Corning</td><td>35-011-CV</td><td></td></tr><tr><td>MEM Nonessential Amino Acids (NEAA)</td><td>Corning</td><td>25-025-CI</td><td></td></tr><tr><td>L-Glutamine solution, 100x (L-Gln)</td><td>Corning</td><td>25-005-CI</td><td></td></tr><tr><td>Pennicillin/Streptomycin solution, 100x (P/S)</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>Polystyrene 10&amp;nbsp;cm plate</td><td>USA Scientific</td><td>CC7682-3394</td><td></td></tr><tr><td>Trypsin EDTA 1x</td><td>Corning</td><td>25-053-CI</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;List of Transfection Reagents&lt;/strong&gt;</td></tr><tr><td>5&amp;nbsp;ml polystyrene tubes</td><td>Fisher Scientific</td><td>352054</td><td></td></tr><tr><td>Calcium Chloride Dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Fisher Scientific&amp;nbsp;</td><td>C69-500</td><td>Make a 2.5 M solution in ddH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Fisher Scientific&amp;nbsp;</td><td>S271-3</td><td></td></tr><tr><td>HEPES</td><td>Fisher Scientific&amp;nbsp;</td><td>BP2939-100</td><td></td></tr><tr><td>Sodium phosphate dibasic (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher Scientific&amp;nbsp;</td><td>S369-500</td><td></td></tr><tr><td>2x HEPES Buffered Saline (HBS)</td><td></td><td></td><td>500 ml: 8.2 g NaCl, 5.95&amp;nbsp;g HEPES, 0.106 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, pH 7.01 (exact!)</td></tr><tr><td>Caffeine</td><td>Sigma-Aldrich</td><td>C0750-5G</td><td></td></tr><tr><td>0.22 &amp;micro;M syringe filter unit</td><td>EMD Millipore&amp;nbsp;</td><td>SLGV033RS</td><td></td></tr><tr><td>0.45 &amp;micro;M syringe filter unit</td><td>EMD Millipore&amp;nbsp;</td><td>SLHP033RS</td><td></td></tr><tr><td>60 cc L/L Syringe</td><td>Med-Vet International</td><td>MV60CCLL</td><td></td></tr><tr><td>50 ml Conical Tube</td><td>Corning</td><td>352098</td><td></td></tr><tr><td>polyethylene glycol&amp;nbsp;&amp;nbsp; 6000 (PEG 6000)</td><td>Millipore</td><td>528877</td><td></td></tr><tr><td>(10x) Phosphate Buffered Saline (PBS)</td><td>National Diagnostics</td><td>CL-253</td><td></td></tr><tr><td>0.5 ml microcentrifuge tubes</td><td>USA Scientific</td><td>1605-0000</td><td></td></tr><tr><td>Matrigel</td><td>Fisher Scientific</td><td>CB-40230A</td><td></td></tr><tr><td>6-well plate</td><td>Fisher Scientific</td><td>353046</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Fisher Scientific</td><td>AC41678-5000</td><td></td></tr><tr><td>Donor Horse Serum</td><td>Cellgro</td><td>35-030-CV</td><td></td></tr><tr><td>TritonX-100</td><td>Sigma-Aldrich</td><td>X100-500ML</td><td></td></tr><tr><td>10 ml serological pipette</td><td>Fisher Scientific</td><td>357551</td><td></td></tr><tr><td>anti-GFP, rabbit, 488 conjugate</td><td>Invitrogen</td><td>A21311</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Stereotaxic Surgery Reagents&lt;/strong&gt;</td></tr><tr><td>Vet-Syringe vet use T.B. Syringe only 1 cc Luer slip T.B. 100/bx</td><td>Med-Vet International</td><td>1CCVLS</td><td></td></tr><tr><td>Isoflurane</td><td></td><td></td><td></td></tr><tr><td>Stainless Steel Scalpel Blades, #10, 100-pk</td><td>Med-Vet International</td><td>JOR580S</td><td></td></tr><tr><td>artificial tear ointment</td><td>Med-Vet International</td><td>RXPARALUBE-O</td><td></td></tr><tr><td>PVP PrepSolution&amp;nbsp;</td><td>Med-Vet International</td><td>HPIV108208H</td><td></td></tr><tr><td>normal saline&amp;nbsp;</td><td>Med-Vet International</td><td>DYND500MLSH</td><td></td></tr><tr><td>cotton tipped applicators</td><td>Med-Vet International</td><td>CTA6</td><td></td></tr><tr><td>Triple antibiotic ointment&amp;nbsp;</td><td>Med-Vet International</td><td>RXTRIP-OI15</td><td></td></tr><tr><td>MONOJECT&amp;reg; Needles Soft Pack 25 g x 5/8&quot;</td><td>Med-Vet International</td><td>25058</td><td></td></tr><tr><td>6-0 silk sutures</td><td>Med-Vet International</td><td>MV-711</td><td></td></tr></tbody></table>

designing, packaging, and delivery of high titer crispr retro and lentiviruses via stereotaxic injection

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto- or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.

继"协议设计一个引导链（因组）为CRISPR / Cas9逆转录病毒"，寡核苷酸靶向特定序列被插入HU6启动子下游的PXL克隆载体，并使用BBSI克隆位点导向的RNA支架的下游（ 图1，步骤1）。此因组然后从PXL切除并插入到使用BstBI和有PacI位点（ 图1，步骤2）的pRubi骨干。最后，另一个因组克隆到PXL可以下游放置在pRubi-Guide1（ 图1，步骤3）第一导向的，靶向的基因的另一个区域，增加通过NHEJ淘汰赛的机会。正确的结构验证应通过序列分析来确定。一旦该构建而成，它可以被包装成继"协议复古/慢病毒产品 - CaPO4方法"的病毒。成功的包装是由病毒感染确诊为HEK293细胞，以滴定在V病毒属。如果不存在荧光团表达然后有可能的病毒的包装过程中的错误。 图2是2逆转录病毒，人们表达GFP和其他表达mCherry一个代表性的结果，共注射到新生小鼠（7日龄）的齿状回和成像21天注射后。与mCherry或GFP的神经元的标记允许在相同的组织的各种遗传操作，其中一个病毒可表达CRISPR的形态学评估/ Cas9介导KO和另一个对照病毒，表达仅一个荧光团。立体定向注入允许就证明了预期的坐标，齿状回的离散感染精确的解剖选择性。当分析脑切片感染，它保持周围的组织，直到它被确定是正确的解剖区域感染是重要的。如果没有感染的迹象，则有可能使喷射发生在邻近的区域中的D可在周围部分被识别。它也可以是有帮助的定位针轨道找到准确注入区域。 VSVG假病毒很少摊开齿状回的边缘的体内注射时，并趋向于沿着整个齿状回延髓/尾轴感染细胞扩散，由三维重建（ 图3），为进行分析。 <img alt="图1" src="/files/ftp_upload/53783/53783fig1.jpg" /> 图1:在逆转录病毒pRubi-Guide1-Guide2-CRISPR质粒克隆策略该策略是基于FU-慢病毒质粒相同。因组的寡核苷酸被退火并用BSBI克隆位点插入PXL。后测序，以确保因组被成功插入PXL，消化与BstBI和PacI位的质粒。然后该被丢弃了（黑盒子）的插入物克隆入病毒背部B一（黑色虚线）pRubi-Guide1 CRISPR。第二因组也可以被插入到PXL消化出使用有PacI酶。这是克隆到使用PacI位的pRubi-Guide1 CRISPR载体（红色虚线）。得到的质粒，然后同时包含导链，以及必要的病毒元件，推动者和荧光。 <a href="https://www.jove.com/files/ftp_upload/53783/53783fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53783/53783fig2.jpg" /> 图2:表达mCherry（红色）或绿色荧光蛋白（绿色） 鼠标齿状回的逆转录病毒注射逆转录病毒注射到一个P7小鼠的齿状回。在21天后将小鼠灌注和脑切片并染色GFP和mCherry。 （A）5X宽视野荧光图像显示PR齿状回注射ecision和标签齿状回颗粒神经元的特异性。海马的形态可以通过DAPI（蓝色）染色可见。比例尺测量200微米。 （B）10X宽视野荧光图像表明，这些高滴度慢病毒感染的大量的细胞，其形态可通过荧光表达进行访问。比例尺测量100微米。表达GFP或mCherry（C）的病毒进行共注入齿状回。使用逆转录病毒的系统时，可以使用一个病毒，使由GFP和由mCherry标记的另一个操纵打上一架遗传操作，然后评估由于每一病毒单或添加剂的变化。比例尺测10微米。 <img alt="图3" src="/files/ftp_upload/53783/53783fig3.jpg" /> 图3:慢病毒注射的解剖立体定向传播共注射<html>一个GFP-shPten病毒和mCherry对照病毒为成人的脑的Pten loxP序列/ +小鼠导致沿着海马的齿状回的整个延髓/尾轴的病毒传播。这显示在注射的，其中关闭所述病毒蔓延的轮廓被追踪比使用重建软件21连续切片（Z = 50微米/部分）的程度的3D重建。然后轮廓描被对准以产生用于音量量化的三维图像。总病毒式传播显示为绿色（体积= 54730800微米3）和齿状本地化传播在紫色显示（体积= 27275200微米3）。病毒沿针轨道和在针轨道的除了填充所述齿状回的延髓/尾轴的交点胼胝体传播。比例尺测量200微米。 LES / ftp_upload / 53783 / 53783video1frame.jpg"/&gt; 补充视频1. sgRNAs设计克隆到逆转录病毒和慢病毒的骨干。 在该合成导的RNA（因组）设计例如小鼠CHD8的基因组序列是从NCBI下载。起始密码子和外显子结构，然后在载体NTI可视化。这让我们的基因组区域复制围绕第一外显子编码，并输入该序列为Benchling。 Benchling使我们能够想象的所有潜在sgRNAs在该地区。此外，说明我们输入了基因组区域后，Benchling将向我们展示上的目标和脱靶得分每个导的RNA。然后，用户可以选择具有最高在线和脱靶得分指导RNA。 <a href="https://www.jove.com/files/ftp_upload/53783/Benchling_GuideRNA_Design.mp4" target="_blank">请点击此处观看该视频。 （右键点击下载）。</a>

Watch this Scientific Journal Video about Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection at JoVE.com

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

设计，包装，并通过立体定向注射高效价CRISPR复古和慢交付

该CRISPR / Cas9系统提供作出有针对性的基因组编辑获得和负担得起科学界的潜力。该协议旨在演示如何创建病毒将基因敲除使用CRISPR / Cas9系统感兴趣的基因，然后立体定位将它们注入成年小鼠大脑。

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique ...

designing-packaging-delivery-high-titer-crispr-retro-lentiviruses-via

Research

JoVE Journal

Neuroscience

17.5K 次观看.  Geisel School of Medicine at Dartmouth College. 该CRISPR / Cas9系统提供作出有针对性的基因组编辑获得和负担得起科学界的潜力。该协议旨在演示如何创建病毒将基因敲除使用CRISPR / Cas9系统感兴趣的基因，然后立体定位将它们注入成年小鼠大脑。 

视频: Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

可伸缩流控喷油器阵列病毒完整的3 - D的大脑回路为目标

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

科研

神经科学

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with na&iuml;ve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

立体定向注射的microRNA表达慢病毒的小鼠海马CA1区和行为结果的评估

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

行为学

Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or knockdown gene expression in a relatively focused region without significant damage to the brain tissue. After recovery, the injected mouse can be tested on various behavioral tasks such as the Open Field Test (OFT) and the Forced Swim Test (FST). The OFT is designed to assess locomotion and the anxious phenotype in mice by measuring the amount of time that a mouse spends in the center of a novel open field. A more anxious mouse will spend significantly less time in the center of the novel field compared to controls. The FST assesses the anti-depressive phenotype by quantifying the amount of time that mice spend immobile when placed into a bucket of water. A mouse with an anti-depressive phenotype will spend significantly less time immobile compared to control animals. The goal of this protocol is to use the stereotactic injection of a lentivirus in conjunction with behavioral tests to assess how genetic factors modulate animal behaviors.

Stereotactic injection of lentiviruses expressing cDNAs or shRNAs can modulate gene expression in specific brain areas of mice. Here, we present a protocol to combine stereotactic injections with behavioral tasks, such as the Open Field Test (OFT) and the Forced Swim Test (FST).

应用立体定向注射技术在研究动物行为的遗传效应

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or ...

Packaging HIV- or FIV-based Lentivector Expression Constructs &amp; Transduction of VSV-G Pseudotyped Viral Particles

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient expression of effectors. Packaging lentiviral expression constructs into pseudoviral particles, however, enables up to 100% transduction, even with difficult-to-transfect cells, such as primary, stem, and differentiated cells. Moreover, the lentiviral delivery does not produce the specific cellular responses typically associated with chemical transfections, such as cell death resulting from toxicity of the transfection reagent 1, 2. When transduced into target cells, the lentiviral construct integrates into genomic DNA and provides stable expression of the small hairpin RNA (shRNA), cDNA, microRNA or reporter gene 3, 4. Target cells stably expressing the effector molecule can be isolated using a selectable marker contained in the expression vector construct such as puromycin or GFP. After pseudoviral particles infect target cells, they cannot replicate within target cells because the viral structural genes are absent and the long terminal repeats (LTRs) are designed to be self-inactivating upon transduction 5, 6.
There are three main components necessary for efficient lentiviral packaging 1, 5, 6, 7. 
1. The lentiviral expression vector that contains some of the genetic elements required for packaging, stable integration of the viral expression construct into genomic DNA, and expression of the effector or reporter. 
2. The lentiviral packaging plasmids that provide the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant pseudoviral particles. This protocol uses the pPACK plasmids (SBI) that encode for gag, pol, and rev from the HIV or FIV genome and Vesicular Stomatitis Virus g protein (VSV-G) for the viral coat protein. 
3. 293TN producer cells (derived from HEK293 cells) that express the SV40 large T antigen, which is required for high-titer lentiviral production and a neomycin resistance gene, useful for reselecting the cells for maintenance.
An overview of the viral production protocol can be seen in Figure 1. Viral production starts by co-transfecting 293TN producer cells with the lentiviral expression vector and the packaging plasmids. Viral particles are secreted into the media. After 48-72 hours the cell culture media is harvested. Cellular debris is removed from the cell culture media, and the viral particles are precipitated by centrifugation with PEG-it for concentration. Produced lentiviral particles are then titered and can be used to transduce target cells. Details of viral titering are not included in this protocol, but can be found at:
<a href="http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf" target="_blank">http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf</a> 8.
This protocol has been optimized using the specific products indicated. Other reagents may be substituted, but the same results cannot be guaranteed.

Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles

Lentiviral expression vectors are the most effective vehicles for stably expressing different effector molecules or reporter constructs in dividing and non-dividing mammalian cells and whole organisms. Here we provide a protocol on how to package lentivector expression constructs in pseudoviral particles and to transduce target cells using the pseudoviral particles.

包装艾滋病毒或FIV的基于Lentivector表达结构与VSV-G假型病毒颗粒转导

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient ...

免疫与感染

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

扩展胚胎及成体神经干细胞<em&gt;在子宫内</em电穿孔或病毒立体定向注射

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

单个锥体神经元的高保真光遗传学控制方法<em&gt;在体内</em

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

立体定向注射病毒载体的基因操作在小鼠脊髓条件

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

使用球后注射技术对小鼠进行有效的全身递送

Systemic Treatment for Postnatal, Juvenile, and Runted Adult Mice by Retrobulbar Sinus Injection

While tail vein injections are frequently used as a systemic route of delivery in adult mice, retrobulbar injections are an alternative method for systemic delivery with fewer limitations. First, tail vein injections (TVIs) are limited to adult mice where the size of the tail vein is suitable for access. Being restricted to treating adult mice can be problematic when dealing with mouse models that do not survive to adulthood. Second, TVIs are not feasible for mouse models with growth retardation phenotypes where the mice never achieve the size of adult wildtype mice. Therefore, retrobulbar injections can be successfully used to treat both young and small adult mice. Lastly, retrobulbar injections are performed under anesthesia, which is less stressful on the mice than TVIs that are commonly performed without anesthesia. This article presents a protocol and detailed instructions for retrobulbar injections that can be used for systemic delivery to small and young mice.

作者聚焦:通过在小鼠模型中进行球后注射克服有机酸血症基因治疗的挑战

This article provides a protocol and an accompanying video for the retrobulbar sinus injection of up to a total volume of 150 &#181;L for postnatal, juvenile, and runted adult mice. This procedure is particularly well suited for the injection of small mice (15 g) when tail vein injection is not feasible.

设计，包装，并通过立体定向注射高效价CRISPR复古和慢交付

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