这项工作是部分资金由国家儿童健康和人类发展研究所（NICHD的 - R21 HD059015）的生殖科和查尔斯H.胡德基金会青年研究者儿童健康研究奖（马萨诸塞州波士顿）。

1。定点突变的基因序列高度富含GC KISS1R <ol><li>模板：Myc基因标记与融合其N -末端的人类KISS1R完整的cDNA序列。这个序列克隆到的pCS2 +的表达载体，这是其后用于转染哺乳动物细胞线兼容。这表达载体被称为本pCS2 + Myc基因KISS1R。 </li><li>引物设计：引物设计进行所需的突变，根据Quikchange定点突变试剂盒（Stratagene公司）的说明。总结： <ul><li>两者（正向和反向引物）必须包含所需的突变和退火对面链的质粒以相同的顺序（如正向和反向引物的互补性） <img src="/files/ftp_upload/2897/step1.jpg" alt="第1步" /></li><li>底漆应25至45个碱基长，最终在一个或多个C或G基地 <img src="/files/ftp_upload/2897/step2.jpg" alt="第2步" /> <img src="files/ftp_upload/2897/step3.jpg" alt="第3步" /></li><li>引入的突变（S）应在底漆中，正确的顺序〜10-15基地两侧两侧</li><li>引物的熔点温度（TM）应等于或大于78 ° C。使用下列公式来估算的Tm： <ul><li>当引入突变：TM = 81.5 + 0.41（％GC） - 675 / N - ％的失配（N是在基地的引物长度） </li><li>当引入插入或缺失：TM = 81.5 + 0.41（％GC） - 675 / N（n不包括基地正在插入或删除） </li><li>使用脱盐引物（没有必要的进一步纯化）。 <img src="/files/ftp_upload/2897/step4.jpg" alt="第4步" /></li></ul></li></ul></li><li>最好的扩增条件包括除PCRx增强解决方案（Invitrogen公司）和DMSO。至少有2浓度的DMSO，如4％和8％，应进行测试。我和图1和一个有代表性的PCR扩增的pCS2 + MycKISS1R的结果，如图2所示，PCR条件列于表。 </li><li>消除父母的DNA甲基化的DNA与DpnI消化扩增产物为1小时30分钟在37 ° C：2μL10X NEBuffer 4 DpnI0.5μL（10U; New England Biolabs公司）的PCR产物的离心管17.5μl混合。 </li><li>变换DpnI处理的PCR产物：XL10金Ultracompetent大肠杆菌45μlDpnI处理的DNA混合2μL 大肠杆菌中预冷15 ml细胞培养管（Stratagene公司）。 β-巯基乙醇添加2μL，并按照Stratagene公司的指示。板100 -400μl转化菌在LB琼脂板含100μg/ml氨苄青霉素和孵化，在37 ° C。 </li><li>小量2至4个单个菌落分离质粒DNA。确认成功引进孤立的DNA测序和分析所需的突变。 </li></ol> 2。 MycKISS1R到HEK - 293细胞的瞬时转染<ol><li>以下实验进行人类胚胎肾细胞（HEK - 293）中的CO 2培养箱（5％CO 2）培养37 ° C的10％胎牛血清（FBS）和1％青霉素/链霉素的DMEM补充。 </li><li>种子的HEK - 293细胞在6孔板在2.5 × 10 5细胞/ ml，让他们成长在37℃过夜°转染前彗星。注：（i）使用一式三份每个实验条件下的井;（二）在转染时的理想的细胞汇合30％-50％。 </li><li>转染的HEK - 293细胞使用的GenePorter转染试剂（Genlantis），根据制造商的指示：将一半的无血清DMEM0.5μgpCS2 + MycKISS1R加0.5μg控制（空）向量总和的DNA /1μg。每转微克的DNA加入10μl转染试剂混合的另一半。注意：理想的质粒DNA的浓度可能会有所不同。 </li></ol> 3。细胞治疗和裂解<ol><li>转染24小时后，细胞培养基取代1毫升DMEM培养液含2.5％胎牛血清（降低细胞代谢）。注意：这可能有助于减少血清中和/或放大的检测结果。 </li><li>直接添加到每一个完整的6孔板以及溶酶体抑制剂（100μg/孔的亮肽素）。孵育37℃，6或16 h（或其他所需的时间） </li><li>直接添加到所有井的两个整个6孔板新鲜准备的蛋白酶抑制剂（10μM/的MG132）。孵育于37℃，2，4，6或16小时（或所需的时间）。新增车辆的第四次6孔板（0时间点）的所有水井和37 ° C为16H </li><li>当孵化结束后，移动板冰，冰，以防止蛋白质的降解和执行整个裂解过程： <ul><li>为了增加蛋白质的产量，结合在一个单一的CENTR 6孔板一式三份ifuge管</li><li>吸冰冷的磷酸1毫升中期和洗细胞一次缓冲液（PBS） </li><li>添加含有蛋白酶抑制剂（1X鸡尾酒含有100MM AEBSF盐酸，80μm的抑肽酶，5MM bestatin冰冷的裂解液100μL（20MM HEPES，pH值7.4，1％NP - 40，150mm的氯化钠，为1mM EDTA，0.25％脱氧胆酸钠）， 1.5MM E - 64，0.5M EDTA，2MM亮肽素和胃酶抑素一个1MM，加上2mm的PMSF），每孔</li><li>与取出一个细胞刮刀和转移细胞，细胞裂解液离心管</li><li>细胞通过20号针头传递〜10倍。注意：不要超声用于膜蛋白免疫印迹检测的样品。超声导致膜蛋白的聚集，不会在电泳迁移</li><li>细胞裂解液在4 ° C孵育一个摇摆的平台上为1h </li><li>离心机细胞裂解液4 ° C为10分钟10,000 × RPM和上清转移到新管。注意：请勿打扰颗粒在此步骤</li><li>确定使用的BCA法的上清中加入10μl的蛋白浓度（皮尔斯） </li><li>稀释裂解液中含有蛋白酶抑制剂的裂解缓冲1mg/ml的。 </li></ul></li></ol> 4。免疫沉淀和Western blot检测的MycKISS1R <ol><li>执行以下冰免疫步骤（或在4 ° C）： <ul><li>冰冷PBS洗两次琼脂糖标记的抗myc抗体（2.5μg/样品）适量添加裂解液蛋白400μg。 </li><li>免疫沉淀裂解液的MycKISS1R一夜之间在4 ° C在一个轻轻摇动摇椅平台。 </li><li>离心脉冲离心琼脂糖珠在4 ° C（高达10,000 × RPM） </li><li>吸并丢弃上清液（不扰民的颗粒） </li><li>一次用冰冷的裂解液和两次冰冷PBS清洗珠。纺前，轻轻地反转管</li><li>在2X上样缓冲液含10％β-巯基乙醇含有抗体的约束MycKISS1R Ressuspend珠。 </li></ul></li><li> MycKISS1R免疫复合物的免疫印迹： <ul><li>热样品，30分钟在37 ° C。 注意 ：不要煮沸用于膜蛋白免疫印迹检测的样品。如超声，煎煮，也导致这些蛋白质的聚集</li><li> 5分钟立即移动到冰管</li><li>经SDS - PAGE分离蛋白在4-15％梯度凝胶。 </li><li>在25V的Immobilon - FL PVDF膜（红外线检测）30分钟传输缓冲区使用Bio - Rad公司的半干转移（Tris碱48MM，39mm的甘氨酸，1.2mm的SDS，20％甲醇，pH值9.2）仪器</li><li>洗膜在室温下5分钟的TRIS缓冲液（TBS）和1H块在室温阻断上的摇摆平台（或者可用于阻止非特异性结合，5％的牛奶的TBS）与Licor奥德赛。 </li><li>在4℃过夜孵育膜° C的兔抗Myc抗体阻断含有0.1％Tween - 20的解决方案（1:500） </li><li>删除的主要抗体，洗膜5分钟3次用TBS含有0.1％Tween - 20的（TBST） </li><li>在室温与山羊红外线RedDye ® 800CW标记的抗兔IgG（1:10,000）在封闭液含0.1％Tween - 20的0.01％SDS孵育1h后膜</li><li>删除第二抗体，洗膜5分钟3次的TBS（删除剩余的吐温20）用TBST和最后一次</li></ul></li><li>使用LI - COR的奥德赛红外线成像仪成像和定量MycKISS1R： <ul><li>膜MycKISS1R将使用LI - COR的奥德赛红外线成像仪成像。首先，放置在底部左上角奥德赛扫描器膜，使其与电网。封面用的橡胶垫，理顺与滚子的气泡，并盖上盖子</li><li>在计算机上创建一个新的项目文件。命名该文件，点击“完成”，然后进入扫描仪“扫描”登录。尺寸扫描仪控制台框，以适应你的膜，然后选择169μm分辨率和图像质量中等</li><li>选择强度设置为700（红色）和800（绿色）通道，根据每个信号的强度预计。这是信号可视化的目的，并不会影响量化。点击“开始扫描”。 </li><li>名称和保存的扫描，然后点击“确定”，打开一个新窗口定量。 MycKISS1R单体应该是可见的的，在约43kD </li><li>使用的“盒子”的工具（左侧侧边栏上），一个盒子周围绘制的第一个乐队。拖动框周围，以确保所有频段适合，然后“复制”和“粘贴”框中的所有频段</li><li>选择所有盒使用“Ctrl + A键”，然后选择选项减去中位数的背景。点击顶部菜单上的“报告”，并与量化值的电子表格将出现。这里显示的代表结果是represented倍以上未经处理的细胞增加（零时间） </li></ul></li></ol> 5。代表性的成果： <ol><li>定点突变的基因序列高度富含GC KISS1R： 表1显示的试剂组合，以提高KISS1R放大效率。这种组合被成功地用于引进几个突变针对KISS1R cDNA序列不同的区域，以及为这个高度富含GC的基因扩增。图1显示了KISS1R诱变骑自行车和扩增条件。这些条件已被修改，从QuikChange定点突变试剂盒（Stratagene公司）。 图2显示了一个代表性的结果，使用这种优化的协议。另外4％或8％DMSO结合PCRx增强，显着提高富含GC KISS1R的cDNA含有质粒的扩增产量。在此代表的实验，提供4％DMSO扩增条件稍微好一点，比8％DMSO。 DpnI处理的扩增产物转化大肠杆菌ultracompetent 大肠杆菌通常产量100 1000多个殖民地，并成功引进所需的突变率是80-90％，通过DNA测序确定。 </li><li> MycKISS1R结构，来研究受体生理： 在此代表的实验中，野生型pCS2 + MycKISS1R扩增根据优化的协议，这里描述的是用来研究在体内KISS1受体的退化有关的细胞株中瞬时表达MycKISS1R（HEK - 293）。治疗溶酶体（亮肽素）或蛋白酶（MG132）抑制剂的方法中所描述的协议，转染HEK - 293细胞后，细胞裂解和免疫印迹处理。图3顶部面板显示扫描MycKISS1R单体，而底部面板中显示的顶部面板上显示的频段的量化。定量分析表明，既不6H也不亮肽素治疗16H影响MycKISS1R蛋白水平的乐队。相反，治疗与MG132在MycKISS1R蛋白在这些细胞中的时间依赖性增加，这与孵化后16H受体的45倍，与MG132积累高潮。这些观测结果表明，不同于大多数G蛋白偶联受体，KISS1R退化的蛋白酶体（而非溶酶体）。 </li></ol><table border="1"><tbody><tr><td></td><td></td><td> 二甲基亚砜 </td><td></td></tr><tr><td> 试剂 </td><td> 0 </td><td> 4％ </td><td> 8％ </td></tr><tr><td>水</td><td> 35 </td><td> 33 </td><td> 31 </td></tr><tr><td> 10X PFU超Taq酶缓冲</td><td> 5 </td><td> 5 </td><td> 5 </td></tr><tr><td> dNTP混合液（10MM） </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td>引物感（25pmol/μl） </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td>引物反义（25pmol/μl） </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td> 10X PCRx增强解决方案</td><td> 5 </td><td> 5 </td><td> 5 </td></tr><tr><td>二甲基亚砜</td><td> 0 </td><td> 2 </td><td> 4 </td></tr><tr><td> PFU超Taq DNA聚合酶（2.5U/μl） </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td>质粒DNA（20ng/μl） </td><td> 1 </td><td> 1 </td><td> 1 </td></tr></tbody></table> 表1。试剂组合，成功地用于变异和放大GC 丰富 KISS1R <img src="/files/ftp_upload/2897/2897fig1.jpg" alt="图1" /> 图1。骑自行车成功的突变和扩增GC丰富KISS1R条件：一个2分钟的热启动18秒30的融化周期在95 ° C，1分钟退火，在55 ° C和6时68分延长° C。额外的10的68分钟延长° C被添加在最后一个周期的结束。这些设置进行了调整，从原诱变QuikChange II XL -定点突变试剂盒（Stratagene公司）的协议。 <img src="/files/ftp_upload/2897/2897fig2.jpg" alt="图2" /> 图2。富含GC pCS2 + Myc基因KISS1R DMSO的存在或缺乏扩增的可视化：五pCS2μL等份+ Myc基因扩增在0存在KISS1R，4个或8％DMSO被装在此代表的1％琼脂糖凝胶与乙锭染色溴化物和可视化使用紫外线灯。 6KB质粒带装载在4％存在PCR产物和8％DMSO两个车道上可见，但不是在第一线，这是用PCR加载二甲基亚砜的情况下扩增的产品。 <img src="/files/ftp_upload/2897/2897fig3.jpg" alt="图3" /> 图3。亮肽素或MG132对HEK - 293细胞中的MYC KISS1R蛋白水平：HEK - 293细胞表达Myc基因KISS1R分别用100μg/ml亮肽素或10μMMG132在37 ° C时指定的时间。 Myc基因细胞裂解液400μgKISS1R是2.5μg琼脂糖标记的抗myc抗体免疫沉淀和免疫印迹分析。 （一）LI - COR的奥德赛Myc基因KISS1R用兔抗- Myc基因标记抗体的检测与IRDye 800CW标记的抗兔孵化后免疫印迹孵化;（二）Myc基因KISS1R频带的量化所示： （一）使用LI - COR的奥德赛量化软件。结果表示为倍，增加了未经处理的细胞（时间0）。

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Betaine+improves+the+PCR+amplification+of+GC-rich+DNA+sequences.">Betaine improves the PCR amplification of GC-rich DNA sequences.</a> Nucleic Acids Res. 25, 3957-3958 (1997).</li><li>Sahdev, S., Saini, S., Tiwari, P., Saxena, S., Singh Saini, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplification+of+GC-rich+genes+by+following+a+combination+strategy+of+primer+design,+enhancers+and+modified+PCR+cycle+conditions.">Amplification of GC-rich genes by following a combination strategy of primer design, enhancers and modified PCR cycle conditions.</a> Mol Cell Probes. 21, 303-307 (2007).</li><li>Kong, K. C., Poyner, D. R., Wheatly, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+10.">Chapter 10.</a> MSTA. in G Protein-Coupled Receptors: Essential Methods. , 197-204 (2010).</li><li>Hall, R. A., George, S. R., O'Dowd, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+9.">Chapter 9.</a> G protein-Coupled Receptor-Protein Interactions. , 170-171 (2005).</li><li>Chaturvedi, K., Bandari, P., Chinen, N., Howells, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteasome+Involvement+in+Agonist-Induced+Down-Regulation+of+Mu+and+Delta+Opioid+Receptors.">Proteasome Involvement in Agonist-Induced Down-Regulation of Mu and Delta Opioid Receptors.</a> J Biol Chem. 276, 12345-12355 (2001).</li><li>Shenoy, S. K., McDonald, P. H., Kohout, T. A., Lefkowitz, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Receptor+Fate+by+Ubiquitination+of+Activated+Beta+2-Adrenergic+Receptor+and+Beta-Arrestin.">Regulation of Receptor Fate by Ubiquitination of Activated Beta 2-Adrenergic Receptor and Beta-Arrestin.</a> Science. 294, 1307-1313 (2001).</li></ol>

定点突变已被用于研究蛋白质的功能，通过引进超过三十年的靶基因的编码序列的核苷酸变化。原来的技术是于1978年由英国加拿大化学家和诺贝尔经济学奖得主迈克尔史密斯10。迈克尔史密斯分享1993年诺贝尔化学奖与雨霏，美国生物化学家穆利斯是谁发明的聚合酶链式反应（PCR）11。迈克尔史密斯所描述的原始方法多年来已改善。此过程中的高精确度和成功率，以及在一个相对较...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> 10X PCRx增强解决方案</td><td> Invitrogen公司</td><td> 52391 </td></tr><tr><td> PfuUltra高保真DNA聚合酶替代洗涤剂</td><td> Stratagene公司</td><td> 600385 </td></tr><tr><td> DPN -我</td><td> New England Biolabs公司</td><td> R0176 </td></tr><tr><td> XL10金Ultracompetent 大肠杆菌大肠杆菌细胞</td><td> Stratagene公司</td><td> 200314 </td></tr><tr><td> DMEM培养液</td><td> Cellgro </td><td> 10 - 013 - CV </td></tr><tr><td>胎牛血清</td><td>亚特兰大生物</td><td> S11550 </td></tr><tr><td> Geneporter转染试剂</td><td> Genlantis </td><td> T201007 </td></tr><tr><td>亮肽素</td><td> Calbiochem </td><td> 108975 </td></tr><tr><td> MG132 </td><td> Calbiochem </td><td> 47491 </td></tr><tr><td> 10xPBS </td><td> Ambion公司</td><td> AM9625 </td></tr><tr><td>蛋白酶抑制剂的鸡尾酒和PMSF </td><td>圣克鲁斯生物技术</td><td> SC - 24948 </td></tr><tr><td>皮尔斯BCA蛋白检测试剂盒</td><td> Thermo Scientific的</td><td> 23225 </td></tr><tr><td>抗- Myc的标签（克隆4A6）琼脂糖共轭</td><td> Millipore公司</td><td> 16-219 </td></tr><tr><td> 2X加载缓冲区</td><td> BioRad公司</td><td> 161-0737 </td></tr><tr><td>标准的Tris - HCl，4-15％的坡度预制凝胶</td><td> BioRad公司</td><td> 345-0028 </td></tr><tr><td>的Immobilon - FL PVDF膜</td><td> Millipore公司</td><td> IPFL00010 </td></tr><tr><td>奥德赛封闭液</td><td> COR Biosciences公司</td><td> 927-40000 </td></tr><tr><td> 10xTBS </td><td> BioRad公司</td><td> 170-6435 </td></tr><tr><td>兔抗Myc抗体</td><td>细胞信号转导</td><td> 2272 </td></tr><tr><td>羊抗兔IRDye 800CW </td><td> COR Biosciences公司</td><td> 926-32211 </td></tr><tr><td>奥德赛成像红外系统</td><td> COR Biosciences公司</td><td></td></tr><tr><td>暂停蛋白酶抑制剂（100X） </td><td> Thermo Scientific的</td><td> 78430 </td></tr></tbody></table>

mutagenesis and analysis of genetic mutations in the gc-rich kiss1 receptor sequence identified in humans with reproductive disorders

kisspeptin受体（KISS1R）确认为青春期的触发和生殖能力的成年 1,2,3调节G蛋白偶联受体。已与iodiopathic hypogonadotropic性腺功能低下症（IHH）1,2和性早熟4的患者确定KISS1R失活的突变。这些突变体的功能研究为我们理解再生产的这种受体的调节，以及背后的机制是至关重要的那些塑造的疾病结果，导致异常KISS1R信令和功能。然而，的高度富含GC序列的KISS1R基因，使得它相当困难的引入突变或扩增PCR这种受体的基因编码。 在这里，我们描述了一种方法，引入利益的突变，这种高度富含GC的序列，已成功地用于产生超过十几KISS1R突变体在我们的实验室。我们已经优化了PCR反应条件，以方便KISS1R包括在KISS1R序列的替换，删除或插入的突变体，放大。的另外一个PCR增强剂解决方案，以及二甲基亚砜的一个很小的比例，尤其是有利于提高放大。这种优化过程可能是有用的，以及其他富含GC的模板。 表达载体编码KISS1R是被用来描述这种受体的信号和功能，以了解如何突变可能会改变KISS1R功能，导致生殖相关的表型。因此，定点突变产生KISS1R的突变体的潜在应用1,4,5,6,7,8许多研究可以说明。作为一个例子，在KISS1R（Arg386Pro），这是与性早熟有关，获得性功能突变已被证明延长受体的反应配体刺激4以及改变的降解率KISS1R 9 。有趣的是，我们的研究表明，KISS1R被蛋白酶体降解，而不是大多数G蛋白偶联受体 9所描述的经典的溶酶体降解。在这里介绍的例子，KISS1R退化是在人胚胎肾细胞（HEK - 293）瞬时表达Myc基因标记KISS1R（MycKISS1R）调查和与蛋白酶或溶酶体抑制剂治疗。使用琼脂糖标记的抗myc抗体免疫印迹分析免疫沉淀的细胞裂解液。使用LI - COR的奥德赛红外线系统进行检测和定量上的污点MycKISS1R。这种方法可能是有用的，以及其他感兴趣的蛋白质降解的研究。

Watch this Scientific Journal Video about Mutagenesis and Analysis of Genetic Mutations in the GC-rich KISS1 Receptor Sequence Identified in Humans with Reproductive Disorders at JoVE.com

Mutagenesis and Analysis of Genetic Mutations in the GC-rich KISS1 Receptor Sequence Identified in Humans with Reproductive Disorders

kisspeptin受体（KISS1R）的突变与患者的生殖系统紊乱。在这里，我们描述如何引入利益的突变的KISS1R富含GC的序列以及使用KISS1R结构特点免疫沉淀和免疫印迹的受体的降解途径

在富含GC KISS1受体序列与人类生殖系统紊乱中确定的诱变和基因突变分析

The kisspeptin receptor (KISS1R) is a G protein-coupled receptor recognized as the trigger of puberty and a regulator of reproductive competence in ...

mutagenesis-analysis-genetic-mutations-gc-rich-kiss1-receptor

Research

JoVE Journal

Biology

13.9K 次观看.  University of Miami Miller School of Medicine. kisspeptin受体（KISS1R）的突变与患者的生殖系统紊乱。在这里，我们描述如何引入利益的突变的KISS1R富含GC的序列以及使用KISS1R结构特点免疫沉淀和免疫印迹的受体的降解途径

视频: Mutagenesis and Analysis of Genetic Mutations in the GC-rich KISS1 Receptor Sequence Identified in Humans with Reproductive Disorders

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

在鸡胚神经诱导的含量为

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

科研

生物学

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

原代培养及质粒电穿孔尔蒂小鼠器官。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

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

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

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

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

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

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

另类文化的人多能干细胞的生产，维护和遗传分析

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

遗传和生物化学方法<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;蛋白齐聚评估：兰尼碱受体案例研究

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

与VirusMapper超分辨率显微镜开源单粒子分析

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust technologies that measure RNA biomarkers in situ in clinical specimens. While grind-and-bind assays like RNAseq and quantitative RT-PCR enable highly sensitive gene expression measurements, they also require RNA extraction and thus prevent valuable expression analysis within the morphological tissue context. The in situ hybridization (ISH) assay described here can detect RNA target sequences as short as 50 nucleotides at single-nucleotide resolution and at the single-cell level. This assay is complementary to the previously developed commercial assay and enables sensitive and specific in situ detection of splice variants, short targets, and point mutations within the tissue. In this protocol, probes were designed to target unique exon junctions for two clinically important splice variants, EGFRvIII and MET&#916;14. The detection of short target sequences was demonstrated by the specific detection of CDR3 sequences of T-cell receptors &#945; and &#946; in the Jurkat T-cell line. Also shown is the utility of this ISH assay for the distinction of RNA target sequences at single-nucleotide resolution (point mutations) through the visualization of EGFR L858R and KRAS G12A single-nucleotide variations in cell lines using automated staining platforms. In summary, the protocol shows a specialized RNA ISH assay that enables the detection of splice variants, short sequences, and mutations in situ for manual performance and on automated stainers.

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Here, we describe an in situ hybridization assay which enables sensitive and specific detection of sequences as short as 50 nucleotides with single-nucleotide resolution at the single-cell level. The assay, which can be performed manually or automatically, can enable visualization of splice variants, short sequences, and mutations within the tissue context.