作者想感谢她的帮助下，劳伦斯的描述电室的建设卡伦。

1。电室的建设<ol><li>订购一个microslide，BTX型号453与3.2毫米差距（哈佛器械＃45-0105）（ 图2A）。金属导轨，应完全密封，以幻灯片的底部。 </li><li>使用DREMEL工具切断一个处理一个塑料离心管架。切成小5，每个与以下尺寸的矩形件的处理:长度0.8厘米，高度0.6厘米，宽度0.3厘米（ 图 2B ）。这些塑料件将用于模具microslide室单井的可重复使用的垫片。 </li><li> microslide金属导轨之间的间隔均匀插入塑胶垫片。垫片应紧贴（ 图2C）。 </li><li>切掀起了P200的枪头的尖端，适合尖端到3毫升注射器，和100％的硅橡胶水族馆密封胶填充注射器。填补塑胶垫片与密封胶之间的差距。务必填写之间的密封插头和microslide的基础形式，所以，无气泡从底部的空白。 </li><li>上盖的塑胶垫片放置一根金属棒，并用长尾夹，使垫片地方举行，而密封剂干燥（图2D - E ）。让密封胶干通宵。 </li><li>删除长尾夹，金属棒，塑料垫片（ 图2F）。使用手术刀刀片清理关闭的金属导轨的顶部密封胶。使用解剖显微镜检查的microslide，确保无气泡在硅水坝底部。删除任何密封膜，在裸机露出里面的水井井可能已形成。填写水，确保水不会泄漏到邻井（S）。重复所有油井。 </li><li>完成microslide适合在塑料盘，与毗邻的菜端的窗口（图2G）的金属杆，最终将电极连接到金属杆。 </li></ol> 2。 DNA的制备<ol><li>质粒DNA加入到1.5ml离心管放在冰上，并把音量与蒸馏水150μl。可合并多个质粒物种合作电（ 例如 ，实验DsRed的构造和控制GFP的构建）。当计算量的DNA添加到管，记住，最终的DNA等分量将60μl。通常情况下，每个构造是在最后的0.5μg/μl集中使用。 </li><li>加入15μl3M醋酸钠（pH值5.2）和450μl100％的乙醇沉淀的DNA。反转或自来水管多次混合。 </li><li>自旋的DNA，在4℃，13200 RPM，30分钟。用70％乙醇洗涤沉淀，然后旋转，再在4℃，13200 RPM，15分钟。空气干燥颗粒，直到半透明，约7分钟，然后悬浮在54μl无菌水。加入6μl无菌10X PBS液（pH 7.4）和混合。 </li></ol> 3。眼的集合<ol><li>消毒电室，并用70％乙醇的所有文书。虽然无须执行在组织培养罩眼收集和解剖，消毒手套和台式整个过程中保持无菌条件下与乙醇和尝试。 </li><li>夹层中（比例为1:1的DMEM:F12，100U/ml青霉素，100μg/ml链霉素，0.29mg/ml L -谷氨酰胺，和5μg/ml胰岛素）:准备两个培养皿35毫米菜3毫升中期和60毫米菜6毫升介质。这一步应该是在一个组织文化罩。 </li><li>消毒新生儿的头部和颈部（产后一天0）70％的乙醇小狗鼠标。快速斩首用剪刀和头部转移到无菌的100毫米菜。 </li><li>切去头皮，露出眼睛的小剪刀。用弯钳轻轻舀眼轨道，并将其放置在一个35mm菜含有夹层介质的眼睛。它可能有助于在低功耗的解剖显微镜下取出眼睛。 </li><li>重复步骤，直到所有的眼睛已收集3.3和3.4。而夹层，夹层介质的眼睛保持在室温。您将需要3-4％的DNA等分眼睛。 </li></ol> 4。视网膜夹层<ol><li>使用70％乙醇消毒刀片和无菌塑料移液管的包装。剪下叶尖关闭吸管，以便它可以吸了全眼球。存放在不使用时，塑料包装的吸管。 </li><li>将一只眼睛从35mm的菜60毫米菜。在高功率的解剖显微镜下，用细镊子从眼睛表面，以消除任何组织，如眼外肌和脂肪。然后，取出捏基地的视神经。 </li><li>为了分离视网膜，捅了一个小洞，我n在角膜缘的巩膜。从钳都对插脚插入成孔（视网膜表面相切），轻轻撕开巩膜/ RPE。白化小鼠，巩膜和视网膜色素上皮出现视网膜组织，它是一种均匀的哑光灰色的光泽相对。在色素的小鼠中，RPE是黑色的。离开的镜头到位。 </li><li>使用移液管移动到其他介质35毫米菜解剖视网膜。 </li><li>重复步骤4.2至4.4，直到所有的目光都被解剖。 </li><li>商店的视网膜组织，37℃培养箱内培养，直到你准备electroporate。 </li></ol> 5。准备为电<ol><li>中等准备35毫米菜。对于每一个DNA等分被电，你将需要一盘夹层介质和培养基的培养皿（夹层中加10％胎牛血清）。适当标签的菜肴。 </li><li>使用P200的移液器和无菌1X PBS洗出电盘分庭。每个分庭拥有量为60 -100μL。洗出每个分庭三次。 </li><li>填写与DNA等份分庭。任何未使用的商会应填写与60μl1X PBS。连接电极的电盘。 </li><li>上electroporator使用以下设置:模式，LV电压，30V; 50毫秒脉冲长度，脉冲，5数;时间间隔为950毫秒;极性，单极。 </li></ol> 6。电穿孔<ol><li>用细镊子把握镜头的视网膜，并转移到电商会。每室最多可容纳3-4鼠标视网膜（ 图3）。 </li><li>使用镊子排队，这样的镜头对连接到正极的金属条倾斜的视网膜。 Kimwipe与每次传输后，以避免从一室的DNA结转到下一清洁镊子。 </li><li>一旦所有的视网膜是一致的，按"开始"上electroporator。微小气泡的形式连接到负电极的金属条。 </li><li>断开电极和关闭electroporator。 </li><li>使用镊子轻轻移动视网膜离室的墙壁。 </li><li>使用无菌移液管从商会转移到视网膜含有夹层介质的35mm菜。 </li><li>洗出每个分庭无菌1X PBS 3次，然后用无菌水冲洗。喷雾用70％乙醇的菜。 </li></ol> 7。配售视网膜上的文化过滤器<ol><li>使用移液管转移到含有培养基的35mm菜视网膜。 </li><li>标签无菌6孔培养板的孔，并填写3毫升培养基每口井。 </li><li>使用无菌钳Nuclepore一轮的Whatman过滤器，有光泽的一面之上中期，在每口井的地方。 </li><li>在解剖显微镜下，用无菌移液管，过滤器，镜头面朝下将其转移到视网膜。如果视网膜土地镜头朝上，把它捡起来用移液管，并试图再次将它放在。不要超过4视网膜上放置一个过滤器，并确保每个视网膜周围介质的水滴保持独立于其他液滴。请注意，我们的文化视网膜镜头完好，虽然其他协议调用到这一步9之前拆除的镜头。 </li><li>将培养板在37 °彗星组织文化培养箱（5％的CO 2）和成长所需的时间量，通常为八天。根据我们的经验，改变介质在为期8天的文化时期是不必要的，虽然中期的变化可能需要更长的文化时期。 </li></ol> 8。收获和flatmounting荧光视网膜植<ol><li>替换为4％多聚甲醛/ 1X PBS在每口井的培养基。如果视网膜仍然坚持到过滤器，使用产钳翻转过滤器，并轻轻地揭下过滤网的视网膜。多聚甲醛孵育30分钟。在室温下。视网膜免受光线，以避免漂白的荧光。 </li><li> 10分钟在1X PBS冲洗两次视网膜。 </li><li>使用一次性吸管转移到视网膜的PBS的小水滴在载玻片。一种荧光解剖显微镜下，使用镊子翻动，使他们的视网膜电穿孔端（ 即镜头端）。 </li><li>放置玻璃"脚"，从粉碎盖玻片，在幻灯片的角落（直径约1-2毫米的玻璃碎片），这些脚防止视网膜展平。幻灯片，以便它涵盖的视网膜和依靠脚放在一个完整的玻璃盖玻片。如果有必要，使用吸管添加更多的幻灯片和盖玻片之间的PBS。 </li></ol> 9。 flatmount成像和荧光定量<ol><li>使用单色相机的图像在红色和绿色通道的低功耗（4X目标）flatmounted视网膜荧光化合物显微镜配备。所有的视网膜成像与一个给定的荧光通道相同的曝光时间，使荧光强度的比较。确保不饱和像素中的任何图像，否则将无法准确定量。导出图像在灰度TIFF格式。 </li><li>打开一个视网膜ImageJ软件（（ 即 ..，红色通道和绿色通道）设置图像<a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a> ）。为了本教程，绿色荧光通道（绿色荧光蛋白），是控制的构造，它是整个在实验中所有视网膜不断。红色荧光通道（DsRed的蛋白）是实验性的构造，每个组的视网膜变化。应以灰度图像。 </li><li> ImageJ，选择控制的绿色形象，并指定圆直径为100个单位（分析/工具/投资回报率经理/ /指定）的利益。重复共创建八界这个圈子（经理/新增的投资回报率）。移动各界1-5选择"五个统一电穿孔地区"，避免视网膜和地区覆镜头（ 图 4A）外侧边缘。此外，选择三个地区以外的视网膜/镜头来测量背景荧光（圆圈6-8）。选择红色的形象，联合国检查"显示所有"中的投资回报率经理框，并重新检查框。所有八个圈，应该会出现在红色的形象。取消选择所有的圆坐标的投资回报率经理。 </li><li>选择与红色的图像，记录感兴趣的各界人士的平均像素值（投资回报率经理/测量），测量1-8应该出现，其中1-5是红色的视网膜测量和6-8是红色背景测量。选择绿色形象，并记录平均像素值;测量9-16应该会出现，其中9-13是绿色的视网膜测量和14-16绿色背景测量。复制到Excel中的测量数据进行分析（ 图5）。 </li><li>平均在两个红色的三个背景测量和绿色通道。在红色通道中的五个视网膜测量中减去红色背景的平均水平;重复的绿色通道。对于每个视网膜区域的利益，分化的背景减去绿色的背景测量中减去红色测量以控制绿化水平（ 图5）实验的红色水平正常化。 </li><li>确定全部归为一个给定的DsRed的构造（例如，5％测量视网膜时间3个独立的视网膜）的测量的平均值和标准偏差。为了定量比较在不同的日子进行electroporations的结果，总是包括一个"标准"在每个电设置DsRed的/绿色荧光蛋白沉淀。整个实验的相对表达值可以比较正常化的表达水平，"标准"。 </li></ol> 10。代表性的成果: 一个良好的电结果在视网膜表面的DNA结构（S）（ 图4A）跨越1 / 4到1 / 3的表达。由于在特别棒的光感受器有效地转，这种技术是理想的量化感光特定的启动子活性（ 图 4B ）。我们以前使用这种方法，量化杆特定的Rho和Gnat1 位点10启动子变异的范围。我们发现，这是可以量化的启动子活性，超过了近300倍的范围。 图5是从一个单一的电穿孔视网膜的样本数据集。在这个特殊的例子，在红色通道中的实验构建pNrl（1.1kb）- DsRed的测量和控制结构pNrl（3.2kb）- GFP的绿色通道。 6-9以这种方式测量的视网膜将包括一个完整的数据集的pNrl（1.1kb）- DsRed的构建，基于"DsRed的标准化绿色荧光蛋白"的价值观和标准差计算。例如，如果我们比较pNrl表达水平（1.1kb）- DsRed的，pNrl（0.8kb）- DsRed的，然后两个结构需要具有相同的GFP控制（ 例如 ，pNrl（3.2kb）电穿孔- GFP）和成像在相同的曝光时间。这是可能池中的数据收集在不同的日子，如果一个标准DsRed的/ GFP电是每天进行（例如，pNrl（3.2kb）- DsRed的+ pNrl（3.2kb）- GFP）的。对于每个实验构造，随后将被归归DsRed的水平，以"标准"（pNrl（3.2kb）- DsRed的）DsRed的标准化水平。 我们在这里描述的是该技术主要用于量化活动感光CRES 10,13 。细胞类型特定的顺式调控的活动也可以被量化罕见的视网膜细胞类型，如双极细胞14，但是这通常需要在垂直的截面选择，而不是在flatmount筹备工作，是可以量化的地区利益。同样是真正的驱动器在多种细胞类型，如感光细胞和双极细胞表达的CRES。实验过程相似。 <img src="/files/ftp_upload/2821/2821fig1.jpg" alt="图1"/> 图1。视网膜植体电穿孔程序的概述。首先，整个眼睛是孤立的，从产后一天0鼠标幼仔和视网膜剥离（P1）。二，视网膜被放置在与DNA电穿孔（P2），充满商会。三，视网膜放置在过滤器和八天（P3），培养。第四，是固定的视网膜植，装在幻灯片上，和成像。 ImageJ软件（P4）的荧光强度测量。第五，ImageJ数据处理，电子表格程序来量化各发起人（P5）的活动的区别。 <img src="/files/ftp_upload/2821/2821fig2.jpg" alt="图2"/> 图2电盘建设。一）从哈佛器械，BTX模型453（产品编号45-0105）未修改的microslide室。 B）DREMEL工具是用来切断塑料管架处理。处理是切成长方形垫片的尺寸如下:长度0.8厘米，高度0.6厘米，宽度0.3厘米。三）塑胶垫片，装成在相同的时间间隔microslide室。水族馆密封剂注入垫片（未显示）之间的差距。 D）金属栏是放置在间隔。五）酒吧和垫片是长尾夹夹紧密封胶烘干举行一切都在一夜之间发生到幻灯片。 F）的垫片被删除，井进行测试，以确保它们是水密。 G）的成品幻灯片适合与毗邻的菜端的窗口的金属条的塑料盘。 <img src="/files/ftp_upload/2821/2821fig3.jpg" alt="图3"/> 图3图与视网膜电盘。该商会都充满了DNA的解决方案（一次最多五个不同的解决方案）。视网膜被放置在商会和导向，使镜片是靠在连接到正极的金属条;，将三个或四个视网膜适合在五个商会。电流会导致带负电荷的DNA分子转移到视网膜细胞。 <img src="/files/ftp_upload/2821/2821fig4.jpg" alt="图4"/> 图4。）ImageJ测量flatmount视网膜荧光水平。在DsRed的（实验）和绿色荧光蛋白（对照组）渠道ImageJ软件打开flatmount图像灰度;注意，这些图像已有色仅用于说明目的。 5个测量界（1到5）放置在均匀电穿孔的地区，避免边缘和镜头（虚线）。三个测量界（6至8）以外的视网膜，以确定背景荧光水平放置。二）高功率电穿孔在视网膜外植体的横截面图像。外植体在出生后8 cryoprotected在30％sucrose/1X PBS在4℃过夜，° C，在OCT包埋固定，并在12μm冷冻切片。外核层（ONL）感光细胞的荧光结构pNrl（1.1kb）- DsRed和pNrl（3.2kb）- GFP的表达。 INL，内核层;保利协鑫，神经节细胞层。 <img src="/files/ftp_upload/2821/2821fig5.jpg" alt="图5"/> 图5。使用Excel的荧光数据的处理。在第1步，平均每个测量圆的像素值复制到电子表格（单元格B3，维生素B12，F3 - F5 H3 - H5）。测量＃1-5 DsRed的视网膜值和＃6-8 DsRed的背景值，测量GFP视网膜值＃9-13＃14-16 GFP背景值。注意测量＃1和＃9，对应于相同的测量圈，因为这样做测量＃2和＃10，等等。在第2步，DsRed和GFP通道的平均背景值计算（细胞F6，H6）。在第3步，从每视网膜测量中减去平均背景（单元格C3 - C12）。在第4步，每一个背景减去DsRed的测量是归其相应的GFP测量（单元格D3 - D7）。

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+and+RNA+interference+in+the+rodent+retina+in+vivo+and+in+vitro.">Electroporation and RNA interference in the rodent retina in vivo and in vitro.</a> Proc Natl Acad Sci U S A. 101, 16-22 (2004).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+expression+of+transgenes+introduced+by+in+vivo+electroporation.">Controlled expression of transgenes introduced by in vivo electroporation.</a> Proc Natl Acad Sci U S A. 104, 1027-1032 (2007).</li><li>LoTurco, J., Manent, J. B., Sidiqi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+and+improved+tools+for+in+utero+electroporation+studies+of+developing+cerebral+cortex.">New and improved tools for in utero electroporation studies of developing cerebral cortex.</a> Cereb Cortex. 19, 120-125 (2009).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+gene+function+in+the+retina.">Analysis of gene function in the retina.</a> Methods Mol Biol. 423, 259-278 (2008).</li><li>Lee, J., Myers, C. A., Williams, N., Abdelaziz, M., Corbo, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+fine-tuning+of+photoreceptor+cis-regulatory+elements+through+affinity+modulation+of+transcription+factor+binding+sites.">Quantitative fine-tuning of photoreceptor cis-regulatory elements through affinity modulation of transcription factor binding sites.</a> Gene Ther. 17, 1390-1399 (2010).</li><li>Carter-Dawson, L. D., LaVail, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rods+and+cones+in+the+mouse+retina.+II.+Autoradiographic+analysis+of+cell+generation+using+tritiated+thymidine.">Rods and cones in the mouse retina. II. Autoradiographic analysis of cell generation using tritiated thymidine.</a> J Comp Neurol. 188, 263-272 (1979).</li><li>Young, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+differentiation+in+the+retina+of+the+mouse.">Cell differentiation in the retina of the mouse.</a> Anat Rec. 212, 199-205 (1985).</li><li>Corbo, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRX+ChIP-seq+reveals+the+cis-regulatory+architecture+of+mouse+photoreceptors.">CRX ChIP-seq reveals the cis-regulatory architecture of mouse photoreceptors.</a> Genome Res. 20, 1512-1525 (2010).</li><li>Kim, D. S., Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+core+paired-type+and+POU+homeodomain-containing+transcription+factor+program+drives+retinal+bipolar+cell+gene+expression.">A core paired-type and POU homeodomain-containing transcription factor program drives retinal bipolar cell gene expression.</a> J Neurosci. 28, 7748-7764 (2008).</li><li>Karra, D., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+techniques+for+neuronal+cells.">Transfection techniques for neuronal cells.</a> J Neurosci. 30, 6171-6177 (2010).</li><li>Onishi, A., Peng, G. H., Chen, S., Blackshaw, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pias3-dependent+SUMOylation+controls+mammalian+cone+photoreceptor+differentiation.">Pias3-dependent SUMOylation controls mammalian cone photoreceptor differentiation.</a> Nat Neurosci. 13, 1059-1065 (2010).</li></ol>

外植体电穿孔是一种简单的方法量化顺式调控活动在发展中国家的小鼠视网膜。通过鼠标转基因顺式调控分析相比，电便宜得多，需要唯一的新生小鼠的幼崽，DNA，解剖仪器，电/组织培养设备。这也是耗时更短的时间内:一个实验需要的准备时间只有几个小时，一个约八天的文化时期，和几个小时，成像和数据分析实验结束。植电也优于细胞培养为基础的顺式调控的分析，因为实际利用视网膜组织。视网膜的发展相当正常植文化，形成了三个不同的细胞层（外核层，内核层和神经节细胞层），虽然光感受器失败阐述外节。 另一个优点是，植体的电高度重复性。同样构造成不同的视网膜电穿孔，甚至在​​不同的日子，通常在相同的相对表达水平的结果。此外，因为电穿孔质粒被认为是保持在核episomally并没有纳入到染色体，他们不似乎是受同一个集成的现场效果，困扰在转基因小鼠进行的顺式调控分析。 植电确实有一些局限性。首先，仍然只在细胞周期中的细胞可以有效地转电 15 。在P0，棒和其他以后出生的视网膜细胞类型（双极细胞，无长突细胞，男LLER神经胶质细胞）的主要细胞，这种方法的目标人群。 P0电锥光感受器电穿孔已报告16，但效率低。第二个限制是，外植体文化，超过两个星期，结果在视网膜逐步畸形，因此不推荐。如果发起人量化晚的时间点， 然而，在体内电穿孔9可能被执行，视网膜剥离所需的时间点，平面安装的解剖视网膜，和定量分析，如第9条所述。第三个限制是，只有适度的高通量检测。与细胞培养为基础的分析，可以测试数以百计的结构在一次实验中，在本议定书中所描述的技术要求最低的每一个构造整个小鼠视网膜。因此，只有一对夫妇十几结构可以合理地在一天内被电。 使用本办法的启动子活性的定量方面的额外需要注意的是，有一个潜在的"出血"到GFP通道DsRed的荧光，尤其是当化验非常强的促销员。这样做的原因是发射光谱部分重叠DsRed的是绿色荧光蛋白。为了规避这个问题，优化排放过滤器应使用，最大限度地减少DsRed和GFP的光谱重叠。当这种优化的过滤器设置不到位，另一个潜在的解决方案是使用代替GFP的一个蓝移荧光蛋白（例如，BFP或CFP） 。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>电穿孔菜，Microslide 453 </td><td> BTX的哈佛装置</td><td> 45-0105 </td><td>请参阅修改协议第1条</td></tr><tr><td> 100％的硅橡胶水族馆水泥</td><td> Perfecto制造</td><td></td><td></td></tr><tr><td>塑料微管架</td><td> Fisher Scientific则</td><td> 05-541 </td><td>只有将用于机架处理</td></tr><tr><td> DREMEL工具</td><td></td><td></td><td>切断塑料管架处理</td></tr><tr><td> DMEM培养液</td><td> GIBCO / Invitrogen公司</td><td> 11965 </td><td></td></tr><tr><td> F12 </td><td> GIBCO / Invitrogen公司</td><td> 11765 </td><td></td></tr><tr><td> L-Glu/pen/strep </td><td> GIBCO / Invitrogen公司</td><td> 10378-016 </td><td> 100X浓度</td></tr><tr><td>胰岛素</td><td> Sigma - Aldrich公司</td><td>我的6634 </td><td> 1000X股票，悬浮在5mg/ml 5mm的HCl和过滤消毒</td></tr><tr><td>胎牛血清</td><td> GIBCO / Invitrogen公司</td><td> 26140-079 </td><td></td></tr><tr><td>流脑830平方波electroporator </td><td> BTX的哈佛装置</td><td></td><td></td></tr><tr><td> Nuclepore过滤器</td><td>瓦特曼</td><td> 110606 </td><td> 25MM，0.2微米</td></tr><tr><td>组织文化的孵化器</td><td></td><td></td><td> 37℃，5％的CO 2 </td></tr><tr><td>盖玻片＃1.5 </td><td> Fisher Scientific则</td><td> 12 - 544E </td><td> 0.16毫米厚</td></tr><tr><td>荧光化合物显微镜与摄像头</td><td></td><td></td><td>相机应该是单色（ 例如 ，ORCA - ER相机滨松） </td></tr><tr><td> EGFP / DsRed的过滤器设置复式显微镜</td><td>色度科技公司</td><td> 86007 </td><td>此过滤器设置红色和绿色chanenels之间的最小化bleedthrough </td></tr><tr><td> ImageJ软件</td><td>美国国立卫生研究院</td><td></td><td> <a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a> </td></tr></tbody></table>

quantifying the activity of cis-regulatory elements in the mouse retina by explant electroporation

在细胞基因网络转录因子控制的时空格局和它们的靶基因的表达水平 ，结合顺式调控元件（CRES），短（300-600 bp）的基因组DNA，可以躺在上游，下游延伸，或在他们所控制的基因的内含子。 CRES（ 即 ，增强/促销员）通常由多个群集的结合位点的转录激活和阻遏 1-3 。他们作为转录输入的逻辑集成在spatiotemporally精确，定量准确的启动子活性的形式单一输出。大多数哺乳动物顺式调控的研究，依靠转基因小鼠化验 CRES 4-5增强剂功能的一种手段。这种技术是费时，昂贵，插入现场效果的帐户上，基本上不定量。另一方面，对哺乳动物的综合招聘考试功能的定量分析已开发的组织培养系统 （例如，双荧光素酶检测），但在体内的这些结果的相关性往往是不确定的的。 电穿孔技术提供了一个很好的替代传统的鼠标，因为它允许的顺式调控哺乳动物组织生活活动的时空和定量评估的转基因。此技术已在独联体调节中枢神经系统的分析，特别有用，尤其是在大脑皮层和视网膜 6-8 。虽然在体内和体外小鼠视网膜电，已开发和广泛松田和Cepko 6-7,9描述，我们最近开发出一种简单的方法来量化电穿孔小鼠视网膜10的感光特定CRES活动。由于进入视网膜，电引入的DNA的量可能有所不同，从实验到实验，这是必要的，包括共同电穿孔"内参"在所有的实验。在这方面，该技术是非常相似的双荧光素酶检测，用量化培养的细胞中启动子活性。 化验感光独联体监管活动时，电通常是在新生小鼠（出生后0天，P0），这是高峰棒生产11-12。一旦视网膜细胞类型，成为有丝分裂后，电要少得多有效。鉴于杆出生在新生小鼠和事实棒构成超过70％在成年鼠的视网膜细胞的高利率，多数是在P0电穿孔细胞棒。出于这个原因，棒感光器是最简单的视网膜细胞类型，研究通过电。我们在这里描述的技术主要是有用的量化感光CRES活动。

Watch this Scientific Journal Video about Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation at JoVE.com

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

本协议描述了一个简单而廉价的方式来量化的顺式调控元件的活动（即，增强/促销员）通过外植体电小鼠视网膜生活。 DNA的制备，视网膜剥离，电，视网膜外植体文化，和固定后的分析和定量描述。

量化的活动顺</em在小鼠视网膜>调控元件外植体电穿孔

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

quantifying-activity-cis-regulatory-elements-mouse-retina-explant

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

14.4K 次观看.  Washington University School of Medicine. 本协议描述了一个简单而廉价的方式来量化的顺式调控元件的活动（即，增强/促销员）通过外植体电小鼠视网膜生活。 DNA的制备，视网膜剥离，电，视网膜外植体文化，和固定后的分析和定量描述。

视频: Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

科研

神经科学

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

转基因小鼠内耳的发展<em&gt;在体内</em&gt;电穿孔

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

遗传操纵鼠标的发展下丘脑通过<em&gt;在子宫内</em&gt;电穿孔

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

血管再生在中枢神经系统中使用的小鼠视网膜评估

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay.
In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain.
Compared to the traditional TRAP assay that utilize 32P labeled radioactive dCTP&#39;s for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

在小鼠大脑的各个区域端粒酶活性:非放射性端粒酶重复序列扩增法（TRAP）分析

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

成像钙<sup&gt; 2+</sup&gt;在小鼠视网膜锥光感受器轴突终末动态

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

体内基因转移到雪旺细胞在啮齿动物坐骨神经电穿孔

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

哺乳动物轴突再生: 成年小鼠背根神经节体内电穿孔的研究

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a protein, it is vital to also determine its distribution. Here, we describe a protocol employing immunofluorescence, confocal microscopy, and computer-based analysis to determine the distribution of the synaptic protein Mover (also called TPRGL or SVAP30). We compare the distribution of Mover to that of the synaptic vesicle protein synaptophysin, thereby determining the distribution of Mover in a quantitative manner relative to the abundance of synaptic vesicles. Notably, this method could potentially be implemented to allow for comparison of the distribution of proteins using different antibodies or microscopes or across different studies. Our method circumvents the inherent variability of immunofluorescent stainings by yielding a ratio rather than absolute fluorescence levels. Additionally, the method we describe enables the researcher to analyze the distribution of a protein on different levels: from whole brain slices to brain regions to different subregions in one brain area, such as the different layers of the hippocampus or sensory cortices. Mover is a vertebrate-specific protein that is associated with synaptic vesicles. With this method, we show that Mover is heterogeneously distributed across brain areas, with high levels in the ventral pallidum, the septal nuclei, and the amygdala, and also within single brain areas, such as the different layers of the hippocampus.

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

免疫荧光定量小鼠脑突触蛋白的异种分布

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...

量化的活动顺调控元件外植体电穿孔

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