This work was supported by the Australian National Health and Medical Research Council (NHMRC fellowship #454330 to JX, project grant #628761 to SM and APP1058647 to JX), Multiple Sclerosis Research Australia (MSRA #12070 to JX), the University of Melbourne Research Grant Support Scheme and Melbourne Research CI Fellowship to JX as well as Australia Postgraduate Scholarships to HP and AF.&#160;We would like to acknowledge the Operational Infrastructure Scheme of the Department of Innovation, Industry and Regional Development, Victoria Australia.

注:用于此研究中的所有动物的混合性别和繁殖的解剖及病理科的动物设施和神经科学与心理健康研究的弗洛里学院在墨尔本大学。所有动物的程序进行，在墨尔本大学经动物实验伦理委员会。 1.克隆2K7慢病毒的<ol><li>前克隆感兴趣的基因插入2K7慢病毒载体，亚克隆的基因导入用标准分子技术11的载体pENTR载体（3637 bp的，卡那霉素抗性）。使用EcoRI和SacⅡ位限制位点亚克隆。 </li><li>在2K7慢病毒扩增<ol><li>变换2K7慢病毒的DNA，轻轻混合100ng的质粒DNA的感受态细胞，并在冰上孵育30分钟。 </li><li>热休克的DNA /感受态细胞混合，在42℃下90秒和板它们含有ampici的LB琼脂平板LLIN（100微克/毫升）和氯霉素（15微克/毫升），在37℃进行16-18小时。 注:氯霉素用于防止所述长末端重复之间的重组。 </li><li>第二天，生长在含有氨苄青霉素（100微克/毫升）和氯霉素（15微克/毫升），在37℃下进行16-18小时的LB培养基中选择的细菌克隆。提取使用市售的质粒DNA的大量制备试剂盒按照制造商的说明将DNA。 </li></ol></li><li>子克隆从载体pENTR载体2K7慢病毒（ 图1） <img alt="图1" src="/files/ftp_upload/52179/52179fig1highres.jpg" /> 图1:该Gateway重组过程的示意图感兴趣的基因，这里表示为标示-ERK1，被克隆入载体pENTR L1-L2矢量。这被加入到含有CMV启动子和骨干2K7矢量的载体pENTR L4-R 1向量。这三个矢量由在LR克隆酶II正酶重组插入的感兴趣的基因和启动子插入病毒就绪2K7矢量。 <ol><li>以下内容添加到1.5毫升管，轻轻混匀: L4-R1载体pENTR-pDNOR-CMV（子）（60 BNG）1-2.5微升 L1-L2 pENTR4IRES2GFP（与感兴趣的基因）（80 BNG）1-2.5微升 K7慢病毒（80纳克/微升）1微升 的TE缓冲液，pH值82-5微升 共有8微升</li><li>从-80℃取出克隆酶酶混合物和解冻冰上2分钟。确保这种酶是从-80℃的新鲜等分试样作为酶大大减少了克隆效率与反复冻融</li><li>加入2微升每反应的酶，并通过涡旋拌匀，孵育23-25​​℃下克隆酶反应混合物6-24小时。 </li><li>加入1微升蛋白酶K溶液（用酶试剂盒提供）到克隆酶反应混合物中，并孵育10分钟，在37℃。 </li><li>变换克隆酶反应混合物注入感受态细胞和成长的选择菌落在含有氨苄青霉素（100微克/毫升），在37℃下进行16-18小时的LB培养基。 </li><li>提取和使用商业质粒DNA的小量制备试剂盒按照制造商的说明纯化DNA。 </li><li>确认通过使用限制性消化酶SpeⅠ和SacI II和合适的缓冲液根据制造商的指示，以除去含有感兴趣的启动子和基因的片段的DNA。 注意:此步骤是关键的，以检查是否该亚克隆的DNA具有与右载体骨架的兴趣正确插入片段的基因。该载体本身没有插入片段的大小为6.7 kb的。所释放的插入片段的大小既包括启动子和感兴趣的插入物的基因。质粒编辑器-通过DNA序列编辑程序， 例如 ，APE计算预期的片段大小从消化的特异基因。 </li><li>检查两个DNA载体主链，并通过运行在1％琼脂糖凝胶中释放的片段的大小在1×TAE缓冲液（见表储备液）在100伏</li><li>通过在37℃下生长的细菌在500ml含有氨苄青霉素（100微克/毫升）的LB培养基中16-18小时，扩增的DNA证实。 </li><li>提取和使用商业质粒DNA的大量制备试剂盒按照制造商的说明纯化DNA。 注:大量制备通常产生的DNA所需的病毒产生足够量。 </li></ol></li><li>重复步骤1.3.9-1.3.10以扩增为慢病毒制剂以下的DNA:包络线矢量（PMD2.G，6.1 kb的），封装向量（pBR8.91，12.5 kb的），以及一个空的慢病毒载体作为对照（GFP -CMV-2K7，8.7 KB）。 </li></ol> 2. 2K7病毒生产注:第1天: <ol><li>在转染的当天，板32000000 HEK293 T细胞中有25ml的HEK293 T细胞培养基T175烧瓶（见储备溶液的表）。另外，板16万个细胞的一天在染前如果时间紧运行转染的一天。 注:转染可通过转染的当天或前一天电镀细胞同样成功。使用这两种替代的，这里的关键点是确保细胞被卡住向下培养皿表面之前转染。 注意: 第2天: </li><li>转染<ol><li>在转染之前，稀释的DNA至1微克/微升TE缓冲液中包含10毫摩尔Tris pH值8，在去离子水中1毫摩尔EDTA pH为8。 </li><li>在50ml管中，制备主混合物（ 表1），用于转染在T175烧瓶中。加入DNA，以预热贝科的改良Eagle培养基（DMEM）和涡拌匀，再加入无菌聚乙烯亚胺（PEI）（请参见库存解决方案的表），以避免过早沉淀。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr height="21"><td height="21" style="height:21px;width:344px;"> 向量 </s<html>仲&gt; </td><td style="width:120px;"> 浓度 </td><td style="width:105px;"> 音量 </td></tr><tr height="21"><td height="21" style="height:21px;width:344px;"> pMDG.2 </td><td style="width:120px;"> 1微克/微升</td><td style="width:105px;"> 5微升</td></tr><tr height="21"><td height="21" style="height:21px;width:344px;"> pBR8.91 </td><td style="width:120px;"> 1微克/微升</td><td style="width:105px;"> 15微升</td></tr><tr height="21"><td height="21" style="height:21px;width:344px;">与GFP +感兴趣的基因2K7矢量</td><td style="width:120px;"> 1微克/微升</td><td style="width:105px;"> 22微升</td></tr><tr height="21"><td height="21" style="height:21px;width:344px;">无菌聚乙烯亚胺（PEI） </td><td style="width:120px;"> 1g / L的</td><td style="width:105px;"> 500微升</td></tr>"&gt; <td height="21" style="height:21px;width:344px;"> DMEM </td><td style="width:120px;"></td><td style="width:105px;"> 2100微升</td></tr></tbody></table> 表1:准备转染混合物为2K7病毒。 </li><li>通过倒转试管3-4倍拌匀孵育在室温下15分钟（RT）。 </li><li>执行对HEK293T细胞全媒体转变。抽吸掉从细胞完全培养基和饲料用预温的HEK293T细胞培养基（每T175烧瓶25ml）中。 </li><li>添加转染混合物（DNA / PEI混合物）逐滴于单层细胞。移动轻轻拌匀，并培育转染的细胞在37℃，5％CO 2过夜。 注:第3天: </li><li>以确保转染是成功的，通过荧光显微镜检查GFP表达24小时后转染。 注:超过50％的细胞表达GFP的通常表示一个很好的转染。 注:第4天: </LI&gt; </ol></li><li>在48小时后转染，收集的病毒上清，并替换为新鲜的HEK293Ť培养基（每T175烧瓶25ml）中。 <ol><li>离心病毒上清在1140×g离心10分钟，在4℃下清理从上清液细胞碎片。转印清零上清液至50ml管中并储存于4℃。 注:第5天: </li></ol></li><li>在72小时后转染，收集第二批病毒上清液。重复步骤2.3.1和游泳池48和72小时清除上清液。 </li><li>使用30毫升超速离心管在4℃下浓缩病毒，离心机病毒上清在170000×g离心90分钟。 </li><li>弃去上清液，并重复步骤2.6，直到所有被清除上清液已经离开离心病毒的（不可见的）粒料加在管的底部沉淀的PEI。 </li><li>重悬病毒，加入500μlSATO媒体（见表股票方案）的超速离心管。涡旋30秒第二刮管的底部用一个移液管尖，以机械地松开该病毒。以松病毒颗粒重复此步骤6倍。 </li><li>池中的悬浮病毒进入离心管和旋转很简单，除去不溶PEI。通过0.45微米的过滤器过滤上清液，以除去蛋白质。 </li><li>分装成病毒20微升，50微升，100微升等分试样并储存在-80℃。 </li></ol> 3.病毒滴度测定HEK293T细胞<ol><li>检查感兴趣的蛋白质的表达，并确定最佳的病毒浓度为实验中，添加了一系列病毒原料的系列稀释（ 例如 ，0，5，10，20，40，80微升）以转导的HEK293 T细胞接种于6孔板中，并培养24小时，在37℃，5％的CO 2。这个协议典型地产生病毒，可以在浓度范围为1:50至1被使用:200实现的基因的表达鲁棒兴趣。 </li><li> 48小时后的病毒转导，裂解HEK293T细胞在TNE缓冲液（见表原液）与蛋白酶抑制剂。 <ol><li>漂洗孔两次用冷的DPBS，然后添加150微升TNE缓冲液到每个孔中。 </li><li>裂解细胞通过上下吹打5到10倍。传送全细胞裂解物，以1.5 ml离心管，并在冰上孵育15-30分钟。 </li><li>离心溶胞产物在4℃下进行30分钟，在最大速度（20,000 xg离心），并转移清零上清液至新管用于通过Bradford蛋白测定和随后的免疫印迹分析。 </li></ol></li><li>确定通过标准蛋白质印迹分析目的蛋白质的表达水平，而探测的抗体对蛋白质本身和熔合标记（ 即 ，标记）。使用的病毒稀释能产生&gt; 95％的GFP +细胞和用于实验目的蛋白的稳健表达。 </li></ol> 4.分离培养的DRG和（Figure 2步骤1＆2） <img alt="图2" src="/files/ftp_upload/52179/52179fig2highres.jpg" /> 图2: 在体外试验髓鞘 DRG神经元的示意图 ，从P2-3幼鼠，然后纯化和培养两周（1-2）解剖。 OPCs的是从P7-9大鼠脑使用immunopanning纯化（3）。 OPCs的随后感染慢病毒和48小时（4）中培养。的OPCs然后接种到的DRG，和感兴趣的任何生长因子，如BDNF加入（5）。共培养物，然后培养2周，以允许OPCs的分化和髓鞘的轴突（6）。最后，共培养物或者裂解为蛋白印迹或固定为免疫细胞化学（7）。 注:1天前2天清扫: &lt;OL&gt; <li>外套蒸压22毫米X22毫米盖玻片用聚-L-鸟氨酸（0.5毫克/毫升）在6孔板中，并孵育在37℃下过夜。 </li><li>第二天，再次涂覆盖玻片与层粘连蛋白（在MEM 20微克/毫升）在37℃下过夜，吸去多余和干燥20分钟，在组织培养罩。 注:第2天:按病种付费的解剖和隔离: </li><li>牺牲P2-P3幼鼠12倍颈椎横断。 </li><li>去除皮肤覆盖从该动物的背部的肌肉。使用vannas剪刀剪开椎孔，然后使用镊子轻轻舀出脊髓。 </li><li>摘下指出在于椎骨列之间的DRG和切断附脊神经纤维，然后发生的DRG中装有3毫升的L-15介质33毫米的培养皿。它通常是可以从脊髓的每一侧8和12之间的DRG收集</li><li>传递收集的DRG随着L-15介质装入一个15毫升的管中，离心，在180×g离心5分钟。 </li><li>吸气上清，加2ml的0.25％胰蛋白酶至DRG粒料和孵育在37℃下30分钟。 </li><li>加入5 ml M1介质（见表原液）到DRG粒料以停止胰蛋白酶，和离心机在180×g离心3分钟，在RT。 </li><li>吸去上清液，重悬浮沉淀在2ml预热M1介质（未用生长因子）。吹打50倍，或直至神经节被分散磨碎的节粒料。离心细胞悬浮液在180×g离心3分钟。 </li><li>重悬解离的神经元在M1的介质，并在6孔板板下来每孔100μl5节。 </li><li>以除去增殖非神经元细胞，最少4小时培养后，以方便附着，除去M1介质和进料的神经元与M2的介质（见表原液）。文化DRG神经元NGF（100毫微克/毫升）的存在，净化NGF依赖TrkA的表达病种付费的文化。另外，使用的BDNF（100毫微克/毫升）以纯化的BDNF依赖的TrkB-EXPRES星病种付费。 </li><li>维持神经元M1的媒体（+ NGF或BDNF的在100纳克/毫升）与抗有丝分裂的M2介质交替为如下2周。 </li><li>维持介质M1（+ NGF或BDNF的在100纳克/毫升）天:4-6，8-10，12-14和M2培养基（+ NGF或BDNF的在100纳克/毫升）与FDU和尿苷天1至4，6-8，以及10-12。最少的M2培养基纯化3个循环是必需的。 </li><li>保持货币供应量M1病种付费，仅一个星期再完成2周抗有丝分裂周期之后。改变M1媒体每2-3天。 </li></ol> 5.分离的OPCs文化（图2步骤3） 注:1天前2天清扫: <ol><li>外套10厘米组织培养板用聚-D-赖氨酸（PDL，在无菌去离子水10微克/毫升）在4℃下过夜。 注:第2天:4天前清扫: </li><li>用无菌去离子水进行3次洗PDL板。让干燥〜6小时在组织培养罩。如果不使用直线距离，包装和储存最多至4周，在4℃。 </li><li>准备二次抗体板为immunopanning。为1脑解剖，准备2个IgG的板（用于RAN2抗体以除去星形细胞和01抗体以除去premyelinating少突），以每10cm的培养皿中的DPBS 45微升山羊α小鼠IgG（15毫升）; 1倍的IgM板（O4抗体用于选择少突胶质细胞前体细胞），45微升山羊α小鼠IgM在DPBS中每10cm培养皿（15毫升）中。 注:第3天:清扫日: </li><li>添加200单位木瓜蛋白酶在10毫升的木瓜蛋白酶缓冲液（ 表2），并升温于37℃，直到缓冲区变清。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr height="21"><td height="21" style="height:21px;width:259px;"></td><td style="width:104px;"> 浓度 </td><td style="width:73px;"> 对于250毫升 </td><td style="width:83px;"> 终浓度 </td></tr><tr height="21"><td height="21" style="height:21px;"> EBSS股票</td><td> 10倍</td><td> 25毫升</td><td> 1X </td></tr><tr height="25"><td height="25" style="height:25px;"> 硫酸镁 </td><td> 100毫米</td><td> 2.5毫升</td><td> 1毫米</td></tr><tr height="21"><td height="21" style="height:21px;">葡萄糖</td><td> 30％ </td><td> 3毫升</td><td> 0.46％ </td></tr><tr height="21"><td height="21" style="height:21px;"> EGTA </td><td> 0.5M的</td><td> 1毫升</td><td> 2毫米</td></tr><tr height="25"><td height="25" style="height:25px;"> 碳酸氢钠 </td><td> 1M的</td><td> 6.5毫升</td><td> 26毫米</td></tr><tr height="21"><td colspan="4" height="21" style="height:21px;">带体积达250毫升去离子水和过滤杀菌</td></tr></tbody></table> 表2:木瓜蛋白酶缓冲准备。 </li><li>清洗所有二抗板的DPBS的3倍。 </li><li>倒入RAN2和O1抗体上的IgG板和O4杂交到IgM的板。培育这些初级抗体板超过2小时，在RT。 </li><li>从P7老鼠解剖大脑的一个。斩首用削尖剪刀小狗，并消除皮肤上覆用剪刀头骨。 <ol><li>从枕叶，颞叶和额叶切断围绕颅骨。除去使用镊子从颅骨脑并轻轻它用1ml DPBS转移到35毫米培养皿中。 </li><li>骰子大脑切成小块大致用剪刀或刀片消毒。 </li></ol></li><li>滤波器预温热木瓜蛋白酶缓冲液（10毫升）到一个新的15ml试管含有L-半胱氨酸的几粒，然后加入200μl的DNA酶（12500 U / ml的）与过滤木瓜蛋白酶缓冲液中。 </li><li>倒入木瓜蛋白酶缓冲到切块脑组织;孵育在37℃下90分钟。 </li><li> Gentlly转移解离脑组织到50ml管中，用25毫升吸管和允许定居。 </li><li>除去木瓜蛋白酶缓冲液，加2ml螺大毛（卵类粘蛋白），以脑组织和titurate 5到10倍，通过移液打碎脑组织块，允许沉降，除去顶部2毫升上清液到新的管中。 </li><li>添加另一个2毫升罗大毛到脑组织，然后重复步骤5.11，直到没有大块的组织仍然存在。 Tituration能得到越来越咄咄逼人。 </li><li>离心分离细胞悬浮液15分钟，在200×g下。抽吸掉上清，在10毫升您好大毛重悬细胞沉淀，并离心15分钟，在200×g下。 </li><li>与DPBS的3倍洗第一immunopanning板（RAN 2板）。吸去上清液，重悬细胞在10ml淘洗缓冲液（ 表2）和倾到第一immunopanning板（冉2片）。孵育细胞15分钟，在室温。 </li><li>洗第二immunopanning板（01片）与DPBS的3倍。 </li><li>在第一immunopanning板培养后，小费的细胞悬浮到第二immunopanning板（01片），冲洗任何松散细胞离该板的表面用1-3毫升平移缓冲器和转移用移液管向01板。孵育细胞15分钟，在室温。 </li><li>洗净第三immunopanning板（O4板）。该板是正选择板，其中所述的OPCs结合其表面上。与DPBS的3倍洗O4板。 </li><li>在第二immunopanning板培养后，在RT下将细胞转移到最终immunopanning板（O4板），孵育细胞45分钟。这一步将选择O4 + OPCs的。 </li><li>吸从上immunopanning板（O4版）上清液，冲洗板EBSS的6倍。 </li><li>以除去OPCs的断板，孵育细胞用5毫升温暖的0.05％胰蛋白酶-EDTA的1:10稀释用EBSS在37℃下8分钟。 </li><li>加入5 ml的30％的FBS（在EBSS制造），以中和胰蛋白酶。去除细胞脱盘吹打表面约50倍。 </li><li>转移所有的细胞悬浮液到一个新的管中，离心15分钟，在200×g下。 </li><li>弃上清，重悬细胞沉淀在1ml预热佐藤介质，随后通过细胞计数。 1大脑的解剖可以产生1.5-2万元的OPC。 </li><li>板单元上干燥的PDL包被的板在1×10 5和5×10 5个每10cm平板的密度与佐藤介质（10毫升）中含有睫状神经营养因子（CNTF，10纳克/毫升），血小板衍生的生长因子（PDGF，10纳克/毫升，），神经营养因子3（NT3，1毫微克/毫升），和毛喉素（4.2微克/毫升）2,12。培养的OPCs在37℃，8％的CO 2。 </li></ol> 6.转导的OPC <ol><li>在佐藤介质（10毫升/ 10厘米板）配有CNTF培养初级OPCs的（10毫微克/毫升），PDGF（10毫微克/毫升），NT3（1纳克/毫升），和毛喉素（4.2微克/毫升），在37℃， 8％CO 2的解剖后24小时。 </li><li>完全吸出的OPC文化传媒，饲料细胞和新鲜的SATO媒体（10毫升）用生长因子（见上面在步骤6.1）。 </li><li>添加病毒到OPCs的由步骤3（ 图2，步骤4），培养的OPCs要进一步48小时确定的最佳浓度。 </li></ol> 7. OPC播种的髓鞘形成共同的文化（图2，步骤5和6） <ol><li>以除去离表面的OPCs，第一冲洗的OPC板用8ml EBSS两次，然后孵育细胞用5毫升温暖的0.05％胰蛋白酶-EDTA的1:10稀释用EBSS在37℃下2分钟。 </li><li>中和，用30％的FBS的胰蛋白酶中EBSS（5毫升）中，并通过移液除去从板的细胞。转移细胞悬液至15ml管中，离心机在180×g离心15分钟，在RT。 </li><li>抽吸掉在1ml预热佐藤媒体随后细胞计数上清液和重悬细胞沉淀。 </li><li>前OPC播种，从DRG培养板完全吸媒体。轻轻种子20万的OPC逐滴到DRG神经元如前所述4-6。 注意:总的OPC播种体积必须每22毫米盖玻片小于200微升。 </li><li>离开细胞不移动板在组织培养罩10分钟沉降，然后轻轻地补足用1ml预热佐藤媒体每孔。 </li><li> Replate其余姐妹的OPC与SATO媒体与生长因子（见步骤6.1）。使用这些姐姐OPCs的验证感兴趣的蛋白质的表达。 </li><li> 24小时后，用共培养培养基（2毫升/孔）含有佐藤培养基（无因子）和的Neurobasal（V / V），用1％B27的更换佐藤介质。保持共培养物14天与媒体变化每2-3天。 </li><li>通过Western印迹分析评估的共培养物为髓鞘蛋白表达和可视化用于通过双免疫染色用抗髓鞘碱性蛋白标记物和神经元标记物4-6的形成有髓轴突的段。 </li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendroglial+expression+of+TrkB+independently+regulates+myelination+and+progenitor+cell+proliferation.">Oligodendroglial expression of TrkB independently regulates myelination and progenitor cell proliferation.</a> The Journal of Neuroscience. 33 (11), 4947-4957 (2013).</li><li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning,+Characterization+and+Expression+of+miR-15a-3p+and+miR-15b-3p+in+Dairy+Cattle.">Molecular cloning, Characterization and Expression of miR-15a-3p and miR-15b-3p in Dairy Cattle.</a> Molecular and Cellular Probes. , (2014).</li><li>Emery, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+gene+regulatory+factor+is+a+critical+transcriptional+regulator+required+for+CNS+myelination.">Myelin gene regulatory factor is a critical transcriptional regulator required for CNS myelination.</a> Cell. 138 (1), 172-185 (2009).</li><li>Murai, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+receptor+TLX+stimulates+hippocampal+neurogenesis+and+enhances+learning+and+memory+in+a+transgenic+mouse+model.">Nuclear receptor TLX stimulates hippocampal neurogenesis and enhances learning and memory in a transgenic mouse model.</a> Proc Natl Acad Sci U S A. 111 (25), 9115-9120 (2014).</li></ol>

The authors declare that there is no conflict of interest regarding this research.

轴突的髓鞘为脊椎动物的中央和外周神经系统的最佳功能的关键过程。髓轴突的生成和维护是一项涉及神经元之间的分子相互作用，胶质（来自雪旺氏细胞或少突胶质细胞）和细胞外基质蛋白的复杂和协调的过程。这个协议的意义和适用性是它允许操纵蛋白的内混合共培养设置一个特定的细胞类型。作为多种细胞类型中涉及髓鞘在体内和在体外髓鞘测定中，使用钝的药理学工具，以?...

轴突的髓鞘是用于在中央和周围神经系统的快速和有效的传输动作电位是至关重要的。特化的细胞，雪旺氏细胞中的外周神经系统和少突胶质细胞在中枢神经系统中，环绕并ensheathe轴突在髓鞘，有效隔热的神经和推动跳跃式传导1。髓鞘形成的方法，可使用的视网膜神经节神经元2中 ，工程化的纳米纤维3，或背根神经节神经元共培养要么雪旺氏细胞4或少突5-7来研究在体外 。 在体外测定髓鞘形成是一个建立的模型用于研究神经系统的髓鞘和它复制许多髓鞘体内 5-8期间发生的基本过程。该法涉及背根神经节（DRG）神经元的纯人口的共培养，用的OPC（对于CNS髓鞘）或雪旺氏细胞（PNS髓鞘）。在特定条件下，这些髓鞘细胞ensheathe DRG轴突在有序，超结构验证，多层状绝缘表达髓鞘本体内特异性蛋白质的相同补质膜片。 学习的CNS髓鞘体外的最常用的细胞模型是在共培养物的DRG神经元和OPCs的，并已成功地用于研究的影响，外源因素，如神经营养蛋白产生关于CNS髓鞘体外 5,6。外源因子如生长因子或小分子的药理学抑制剂已被广泛用于研究用DRG-OPC共培养模型7,9的信号通路中髓鞘形成的作用。然而，在包含两个神经元和少突胶质细胞的混合共培养的设置，但仍可能正式，无论是生长因子或所述的医药macological抑制剂可以发挥经DRG神经元和少突胶质细胞（OL）两种效果。这确实提供了具体剖析该蛋白的病种付费仅明示或少突胶质细胞在发挥髓鞘使用这种双电池系统中的作用的能力。明确地确认该信号通路在少突胶质细胞直接调节髓鞘，OPCs的慢病毒转导，对接种到DRG神经元的体外髓鞘测定之前，已被证明是一种优雅的方式来过表达野生型和突变体蛋白质，以及作为组成型表达的蛋白质由少突胶质细胞敲低表达。因此，这种方法提供了一条专门询问和操作信号少突胶质细胞内途径研究髓鞘9,10。 在本文中，我们报告，我们已经发展到过度表达的目的蛋白选择性地少突胶质细胞通过慢病毒的方法方法在体外研究髓鞘形成。该技术始于含有感兴趣的基因的表达载体的产生，无论是在野生型，组成型活性或显性负形式被随后克隆到载体pENTR载体（载体pENTR L1-L2 pENTR4IRES2GFP）。该载体（含有目的基因）中，巨细胞病毒启动子给体（载体pENTR L4-R 1载体pENTR-pDNOR-CMV）和2K7慢病毒结合在酶反应以产生含有CMV启动一个2K7矢量，感兴趣的基因，一个内部核糖体进入位点和GFP（ 图1）。该Gateway克隆2K7构建体结合PMD2.G病毒包膜和pBR8.91病毒包可以被共转染到HEK293T细胞，以产生慢病毒随后可以用于转导的OPCs。一旦感染了慢病毒的OPCs的表达感兴趣的蛋白质的高水平。这些OPCs的就可以接种于DRG神经元的文化和效果的表达的高水平的所需蛋白质的施加在髓鞘可以询问。共同评价培养物用免疫印迹分析髓鞘蛋白表达和可视化的免疫细胞化学的形成髓鞘的轴突段。

<table><tbody><tr><td>2K7 lentivector&amp;nbsp;</td><td></td><td></td><td>Kind gift from Dr Suter&lt;sup&gt;9&lt;/sup&gt;</td></tr><tr><td>5-Fluoro-2&amp;prime;-deoxyuridine</td><td>Sigma-Aldrich</td><td>F0503-100mg</td><td></td></tr><tr><td>Alexa Fluor 488 Goat anti-mouse IgG</td><td>Jackson Immunoresearch</td><td>115545205</td><td></td></tr><tr><td>Alexa Fluor 488 goat anti-rabbit IgG (H+L)</td><td>Life Technologies</td><td>A11008</td><td></td></tr><tr><td>Alexa Fluor 594 goat anti-mouse IgG (H+L)</td><td>Life Technologies</td><td>A11005</td><td></td></tr><tr><td>Alexa Fluor 594 goat anti-rabbit IgG (H+L)</td><td>Life Technologies</td><td>A11012</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma-Aldrich</td><td>A9518-5G</td><td></td></tr><tr><td>B27 - NeuroCul SM1 Neuronal Supplement</td><td>Stem Cell Technologies&amp;nbsp;</td><td>5711</td><td></td></tr><tr><td>BDNF (Human)</td><td>Peprotech</td><td>PT450021000</td><td></td></tr><tr><td>Biotin (d-Biotin)</td><td>Sigma Aldrich</td><td>B4639</td><td></td></tr><tr><td>Bradford Reagent</td><td>Sigma Aldrich</td><td>B6916-500ML&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A4161</td><td></td></tr><tr><td>Chloramphenicol</td><td>Sigma-Aldrich</td><td>C0378-100G</td><td></td></tr><tr><td>CNTF</td><td>Peprotech</td><td>450-13020</td><td></td></tr><tr><td>DAKO fluoresence mounting media</td><td>DAKO</td><td>S302380-2</td><td></td></tr><tr><td>DMEM, high glucose, pyruvate, no glutamine</td><td>Life Technologies</td><td>10313039</td><td></td></tr><tr><td>DNase</td><td>Sigma-Aldrich</td><td>D5025-375KU</td><td></td></tr><tr><td>DPBS</td><td>Life Technologies</td><td>14190250</td><td></td></tr><tr><td>DPBS, calcium, magnesium</td><td>Life Technologies</td><td>14040182</td><td></td></tr><tr><td>EBSS</td><td>Life Technologies</td><td>14155063</td><td></td></tr><tr><td>EcoRI-HF</td><td>NEB</td><td>R3101</td><td></td></tr><tr><td>Entry vectors for promoter and gene of interest</td><td></td><td></td><td>Generate as per protocols 1-2</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>12003C</td><td></td></tr><tr><td>Forskolin&amp;nbsp;</td><td>Sigma Aldrich</td><td>F6886-50MG</td><td></td></tr><tr><td>Glucose (D-glucose)</td><td>Sigma-Aldrich</td><td>G7528</td><td></td></tr><tr><td>Glycerol</td><td>Chem Supply</td><td>GL010-500M</td><td>See stock solutions</td></tr><tr><td>Goat Anti-Mouse IgG</td><td>Jackson ImmunoResearch</td><td>115005003</td><td></td></tr><tr><td>Goat Anti-Mouse IgM&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>115005020</td><td></td></tr><tr><td>Goat Anti-Rat IgG</td><td>Jackson ImmunoResearch</td><td>112005167</td><td></td></tr><tr><td>Hoechst 33342</td><td>Life Technologies</td><td>H3570</td><td></td></tr><tr><td>Igepal</td><td>Sigma Aldrich</td><td>I3021-100ML&amp;nbsp;</td><td></td></tr><tr><td>Insulin&amp;nbsp;</td><td>Sigma Aldrich</td><td>I6634&amp;nbsp;</td><td></td></tr><tr><td>Kanamycin</td><td>Sigma-Aldrich</td><td>60615</td><td></td></tr><tr><td>Laminin&amp;nbsp;</td><td>Life Technologies</td><td>23017015</td><td></td></tr><tr><td>LB Medium</td><td></td><td></td><td>See stock solutions</td></tr><tr><td>LB-Agar</td><td></td><td></td><td>See stock solutions</td></tr><tr><td>L-Cysteine</td><td>Sigma-Aldrich</td><td>C-7477</td><td></td></tr><tr><td>Leibovitz&#039;s L-15 Medium</td><td>Life Technologies</td><td>11415064</td><td></td></tr><tr><td>L-Glutamate</td><td>Sigma-Aldrich</td><td>G1626</td><td></td></tr><tr><td>L-Glutamine- 200 mM (100x) liquid</td><td>Life Technologies</td><td>25030081</td><td></td></tr><tr><td>LR Clonase II Plus enzyme</td><td>Life Technologies</td><td>12538-120</td><td></td></tr><tr><td>MEM, NEAA, no Glutamine</td><td>Life Technologies</td><td>10370088</td><td></td></tr><tr><td>Mouse &amp;alpha; &amp;beta;III Tubulin&amp;nbsp;</td><td>Promega</td><td>G7121</td><td></td></tr><tr><td>Mouse &amp;alpha;MBP (monoclonal)</td><td>Millipore</td><td>MAB381</td><td></td></tr><tr><td>Na pyruvate&amp;nbsp;</td><td>Life Technologies</td><td>11360-070</td><td></td></tr><tr><td>NAC</td><td>Sigma Aldrich</td><td>A8199</td><td></td></tr><tr><td>NcoI-HF</td><td>NEB</td><td>R3193S</td><td></td></tr><tr><td>NEBuffer 4</td><td>NEB</td><td>B7004S</td><td></td></tr><tr><td>Neurobasal medium</td><td>Life Technologies</td><td>21103049</td><td></td></tr><tr><td>NGF (mouse)</td><td>Alomone Labs</td><td>N-100</td><td></td></tr><tr><td>NT-3</td><td>Peprotech</td><td>&amp;nbsp;450-03</td><td></td></tr><tr><td>O1 antibody - Mouse anti-O1</td><td>Millipore</td><td>MAB344</td><td>Alternative if O1 hybridoma cells are unavailable</td></tr><tr><td>O1 hybridoma cells</td><td></td><td></td><td>Conditioned medium containing anti-O1 antibody to be used for immunopanning</td></tr><tr><td>O4 antibody - Mouse anti-O4</td><td>Millipore</td><td>MAB345</td><td>Alternative if O4 hybridoma cells are unavailable</td></tr><tr><td>O4 hybridoma cells</td><td></td><td></td><td>Conditioned medium containing anti-O4 antibody to be used for immunopanning</td></tr><tr><td>Competent cells</td><td>Life Technologies</td><td>A10460</td><td></td></tr><tr><td>One Shot Stbl competent cells</td><td>Life Technologies</td><td>C7373-03</td><td></td></tr><tr><td>Papain Suspension</td><td>Worthington/Cooper</td><td>LS003126</td><td></td></tr><tr><td>pBR8.91</td><td></td><td></td><td>Kind gift from Dr Denham&lt;sup&gt;10&lt;/sup&gt;</td></tr><tr><td>PDGF-AA (Human)</td><td>Peprotech</td><td>PT10013A500&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Penicillin-streptomycin 100x solution</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>pENTRY4IRES2GFP</td><td>Invitrogen</td><td>11818-010&amp;nbsp;</td><td></td></tr><tr><td>pMD2.G</td><td>Addgene</td><td>12259</td><td></td></tr><tr><td>Poly-D-lysine</td><td>Sigma</td><td>P6407-5MG</td><td></td></tr><tr><td>Polyethylenimine (PEI)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>408727-100ML</td><td></td></tr><tr><td>Poly-L-ornithine&amp;nbsp;</td><td>Sigma Aldrich&amp;nbsp;</td><td>P3655</td><td></td></tr><tr><td>Progesterone&amp;nbsp;</td><td>Sigma Aldrich</td><td>P8783</td><td></td></tr><tr><td>Protease inhibitor tablet (Complete mini)</td><td>Roche</td><td>11836153001</td><td></td></tr><tr><td>Proteinase K</td><td></td><td></td><td>Supplied with Clonase enzyme</td></tr><tr><td>Putrescine</td><td>Sigma Aldrich</td><td>P-5780</td><td></td></tr><tr><td>Rabbit &amp;alpha; neurofilament</td><td>Millipore</td><td>AB1987&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Rabbit &amp;alpha;MBP (polyclonal)</td><td>Millipore</td><td>AB980</td><td></td></tr><tr><td>Ran2 hybridoma cells</td><td>ATCC</td><td>TIB-119</td><td>Conditioned medium containing anti-Ran2 antibody to be used for immunopanning</td></tr><tr><td>Rat anti CD140A/PDGFRa antibody</td><td>BD Pharmingen</td><td>558774</td><td></td></tr><tr><td>SacII</td><td>NEB</td><td>R0157</td><td></td></tr><tr><td>SOC medium</td><td></td><td></td><td>Supplied with competent bacteria</td></tr><tr><td>Sodium selenite&amp;nbsp;</td><td>Sigma Aldrich</td><td>S5261</td><td></td></tr><tr><td>Spe I</td><td>NEB</td><td>R0133S</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>NEB</td><td>M0202S</td><td></td></tr><tr><td>T4 DNA Ligase Buffer</td><td>NEB</td><td>B0202S</td><td></td></tr><tr><td>TE buffer pH8</td><td></td><td></td><td>See stock solutions</td></tr><tr><td>TNE lysis buffer</td><td></td><td></td><td></td></tr><tr><td>Trace Elements B</td><td>Cellgro&amp;nbsp;</td><td>99-175-CI</td><td></td></tr><tr><td>Transferrin (apo-Transferrin human)</td><td>Sigma-Aldrich</td><td>T1147</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284&amp;nbsp;</td><td></td></tr><tr><td>Trypsin</td><td>Sigma-Aldrich</td><td>T9201-1G</td><td></td></tr><tr><td>Trypsin Inhibitor From Chicken Egg White</td><td>Roche</td><td>10109878001</td><td></td></tr><tr><td>Trypsin-EDTA (1x), phenol red (0.05%)</td><td>Life Technologies</td><td>25300-054</td><td></td></tr><tr><td>Unconjugated Griffonia Simplicifolia Lectin BSL-1</td><td>Vector laboratories&amp;nbsp;</td><td>L-1100</td><td></td></tr><tr><td>Uridine</td><td>Sigma-Aldrich</td><td>U3003-5G</td><td></td></tr></tbody></table>

production and use of lentivirus to selectively transduce primary oligodendrocyte precursor cells for in vitro myelination assays

髓鞘是一个复杂的过程，涉及神经元和髓鞘形成神经胶质细胞，在中枢神经系统（CNS）的少突胶质细胞和雪旺氏细胞在周围神经系统（PNS）。我们使用一个体外髓鞘形成测定中，已建立的模型用于研究的CNS髓鞘体外 。要做到这一点，少突胶质细胞前体细胞（OPCs的）被加入到该纯化的初级啮齿类背根神经节（DRG）神经元，以形成髓鞘形成共培养物。为了专门询问角色由少突胶质细胞表达的特定蛋白质发挥对髓鞘形成，我们已经开发协议被接种到DRG神经元在此之前，有选择地使用转导的慢病毒过表达的OPC野生型，组成积极或显性负蛋白质。这让我们专门询问在调节髓鞘少突胶质细胞，这些蛋白质的作用。该协议也可以在加时赛的应用研究她的细胞类型，从而提供了一种方法，允许以期望的细胞类型表达的蛋白质，如少突胶质细胞用于信令和补偿机制的目标研究的选择性操纵。总之，结合慢病毒感染的OPC 体外髓鞘检测提供了参与髓鞘形成的分子机制进行分析的战略工具。

旗标记细胞外信号相关 ​​激酶1（标志-ERK1）构建体用于慢病毒的生产用限制性验证酶消化所用的构建体，包括2K7构建体和所需的病毒生产的包装和辅助构建体两者的（ 图3） 。 <img alt="图3" src="/files/ftp_upload/52179/52179fig3highres.jpg" /> 图3:DNA构建验证 。所需的慢病毒生产所有的DNA构建体从Stbl3细菌中纯化，并通过限制性?...

Watch this Scientific Journal Video about Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays at JoVE.com

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

生产和使用慢病毒的有选择性地转导原少突胶质前体细胞<em&gt;在体外</em&gt;髓鞘测定

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

production-use-lentivirus-to-selectively-transduce-primary

Research

JoVE Journal

Developmental Biology

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

14.4K 次观看.  The University of Melbourne. Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

视频: Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating diseases such as multiple sclerosis and transverse myelitis. Myelination is required to not only facilitate rapid impulse propagation within the central nervous system, but also to provide trophic support to underlying axons. Oligodendrocyte progenitor cells (OPCs) can be studied in vitro to help identify factors that may promote or inhibit oligodendrocyte differentiation. To date, many of the methods available to evaluate this process have either required large numbers of cells, thus limiting the number of conditions that can be investigated at any one time, or labor-intensive methods of quantification. Herein, we describe a protocol for the isolation of large numbers of highly pure OPCs together with a fast and reliable method to determine oligodendrogenesis from multiple conditions simultaneously. OPCs are isolated from P5-P7 neonatal rat cortices and grown in vitro for three days prior to differentiation. Four days after differentiation, oligodendrogenesis is evaluated using a dual-infrared fluorescence-scanning assay to determine expression of the myelin protein.

Oligodendrocytes are the myelinating cells of the central nervous system. This protocol describes a method for the isolation and culture of their precursors, oligodendrocyte progenitor cells, from rat cortices, as well as a fast and reliable quantitative method to evaluate oligodendrogenesis in vitro in response to experimental factors.

大鼠少突胶质祖文化的制备和使用双红外荧光扫描Oligodendrogenesis的量化

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating ...

科研

发育生物学

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

慢病毒载体平台, 用于将表观基因编辑工具高效地传递到人类诱导的多能干细胞衍生疾病模型中

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

遗传学

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

工程神经前体宫内移植,用于Vivo神经结构研究

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

从中枢神经系统转导细胞的慢病毒载体生产

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

神经科学

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

来自祖，人脑少突胶质细胞培养系统

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

胚胎干细胞分化成少突胶质前体

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

生物学

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture.

小鼠初级少突胶质的快速特异磁分离

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

从多能干细胞生成人类神经元和少突胶质细胞，用于模拟神经元-少突胶质细胞相互作用

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of primary human bronchial epithelial cells.

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

优化慢病毒生产和细胞培养必要条件，成功转导初级人支气管上皮细胞

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of ...

生产和使用慢病毒的有选择性地转导原少突胶质前体细胞

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