完全支持这项工作是由美国国立卫生研究院，国家老龄问题研究所的院内研究计划。我们感谢SGS1表达质粒的酵母菌株和布拉德约翰逊博士（美国宾夕法尼亚大学医学院，费城，宾夕法尼亚）的博士罗德尼罗斯坦（哥伦比亚大学）。

1。酵母菌株菌株与野生型SGS1 TOP3（WT; W303 - 1A，基因型， 共同商定一个 ADE2 1 CANL - 100 HIS3 - 11，15 LEU2 - 3，112 trpl，升URA3 - 1）[2]，一个sgs1突变（W1292 - 3C ;基因型共同商定 一个 SUP4 - Ø::URA3 sgs1 - 25 ADE2 1 CAN1 - 100 HIS3 - 11，15 LEU2 - 3，112 TRP1 - 1 URA3 - 1 RAD5 - 535）和一个sgs1 TOP3突变（W1058 - 11C，基因型，MAT 一个 SUP4:URA3 sgs1 - 25 TOP3 - 2::HIS3 ADE2 - 1 CAN1 - 100 HIS3 - 11，15 LEU2 - 3，112 TRP1 - 1 URA3 - 1 RAD5 - 535）已被特点[3]，并分别请提供罗德尼罗斯坦博士（哥伦比亚大学）。 2。质粒DNA的建设以下所讨论的计划是WRN基因的克隆在YEp112SpGAL 。 <ol><li>定点突变WRN（E84A核酸外切酶的死，K577M解旋死了，R834C，和K1016A）在质粒YEp195SpGAL WRN [4]使用快速转换二XL定点突变试剂盒（Stratagene公司）的诱变引物和一个标准的协议Lofstrand实验室（马里兰州盖瑟斯堡）。 </li><li>从各自的YEp195SpGAL WRN结构双酶切后用 Sal I和MLU一凝胶纯化DNA片段编码WRN及其相关变种</li><li>凝胶纯化的片段，然后克隆到萨尔我MLU我的网站，2微米的多拷贝质粒含有一个TRP1选择标记，构建一个半乳糖苷酶诱导启动子的控制下 YEp112SpGAL WRN或WRN的变种，如在图1所示的矢量YEp112SpGAL 。 </li><li>取得的构造，分别命名为YEp112SpGAL - WRN，YEp112SpGAL WRN E84A，YEp112SpGAL WRN K577M，YEp112SpGAL WRN K1016A和YEp112SpGAL WRN R834C 。 </li></ol> 3。转型酵母培养物生长，使用标准的协议和被执行的转换，使用醋酸锂为基础的协议Gietz等[5]。简单地说，下面的步骤进行。 <ol><li>上述菌株培养于5 ml YPD（酵母提取物，蛋白胨，葡萄糖）培养基过夜。 reinoculated于50 ml YPD培养基培养，并获准直到OD 600增长1.0 - 1.2达成。 </li><li>收获培养物，用20毫升水冲洗，并在1 100毫米醋酸锂（LiAc）毫升悬浮。细胞培养在30℃10分钟。 </li><li>细胞沉淀悬浮于100毫米LiAc到终体积为500μL，这是足以做十转换。分装50μL的细胞悬液，离心收集细胞沉淀。 </li><li>细胞沉淀中添加了以下顺序:250μL50％聚乙二醇（PEG）36μL，25μL2 mg / ml的单链DNA（鲑鱼精子DNA），5毫升的质粒DNA（100纳克1米LiAc 1000纳克），和50μl水。细胞沉淀，振荡1分钟。 </li><li>细胞，然后培养于30 ° C为30分钟，热休克在42 ° C为20分钟。 </li><li>收获细胞，并在1毫升水中悬浮。由于质粒YEp112SpGAL包含TRP作为一个可选的标记，所有窝藏突变株的构建将TRP精通。因此，选择SC（合成完整）缺乏葡萄糖媒体色氨酸和孵化30板电镀暂停转化° C为3-4天，直到出现的殖民地。 </li></ol> 4。 WRN增长缓慢的表型恢复的遗传分析转化sgs1 TOP3应变。 1。数分析: <ol><li>为了研究上生长的野生型的父母（W303 - 1A），sgs1，或sgs1 TOP3株，与YEp112SpGAL或YEp112SpGAL WRN到SC减去色氨酸板，内含2％葡萄糖挑染相应株 WRN表达的影响（ GLU）或2％半乳糖（GAL）。作为对照，sgs1 TOP3 / YEp112SpGAL - SGS1被列入。板在30 ° C孵育2天。 </li><li>要确定的影响WRN蛋白的表达水平较低的增长速度sgs1 TOP3应变能力WRN，sgs1 TOP3应变转化与YEp112SpGAL，YEp112SpGAL WRN，或YEp112SpGAL - SGS1挑染到SC减去色氨酸板含有不同浓度的GAL低至0.005％，高达2％。板，然后在30 ° C孵育2天。 </li><li>为了评估WRN的功能要求， 恢复sgs1 TOP3背景增长缓慢的表型，sgs1 TOP3 YEp112SpGAL WRN（E84A，K577M，R834C，K1016A）转化是streaked到SC减去色氨酸板包含在指定的浓度GLU或GAL。对于控件，sgs1 TOP3应变YEp112SpGAL，YEp112SpGAL WRN或YEp112Sp的GAL - SGS1转化。板，然后在30 ° C孵育2天。 </li></ol> 2。液体培养分析: <ol><li>要评估转化sgs1 TOP3液体培养菌株的生长，酵母细胞生长在SC RAF减去色氨酸在30 ° C过夜。文化reinoculated在SC RAF减去色氨酸和生长在30 ° C年初数期（ 外径 600〜0.5）。文化，然后在SC媒体含有2％的GAL和增长 0.05外径reinoculated其次，在指定的时间间隔测量吸光度。 </li></ol> 5。 WRN羟基脲或methylmethane磺酸敏感性的遗传分析转化酵母菌株。 <ol><li>为了确定sgs1或sgs1 TOP3株，株转化与YEp112SpGAL或YEp112SpGAL WRN MMS和胡灵敏度WRN表达的影响，生长在SC RAF减去色氨酸在30 ° C </li><li>文化reinoculated在SC RAF减去色氨酸和生长在30 ° C至年初数期（外径600〜0.6至0.8）。 </li><li>这些菌株十倍连续稀释准备使用缺乏色氨酸的SC培养基。从文化的大幅增长，5 × 10 6细胞和连续稀释10,000倍。四，每个稀释度微升到SC GAL减去色氨酸板含有胡或MMS表示浓度被发现。在30 ° C孵育板</li><li>作为对照，野生型菌株（W303 - 1A）转化YEp112SpGAL或YEp112SpGAL WRN，或sgs1 TOP3 / YEp112SpGAL SGS1治疗如上所述。 </li></ol> 6。 WRN细胞周期分布改变sgs1 TOP3应变。 <ol><li>为了研究细胞周期分布的WRN转化sgs1 TOP3应变，转化YEp112SpGAL，YEp112SpGAL WRN，或YEp112SpGAL - SGS1 sgs1 TOP3的文化，以及矢量转化野生型菌株（W303 - 1A）的资深大律师皇家空军的成长零下30 ° TRP在彗星为通宵。 </li><li>文化reinoculated在外径0.05和增长TIL年初数期（外径0.5 -0.6 600）。WRN诱导表达增加2％终浓度的GAL 。文化，然后让其生长在30 ° 6小时彗星</li><li>文化当时的DAPI（4&#39;，6 diamidino - 2 -苯基吲哚）染色处理。 <ol><li>文化收获离心10分钟在室温（RT）在5800 XG。 </li><li>洗涤细胞一次1X磷酸盐缓冲液（PBS），在室温为20分钟，然后被固定在70％的乙醇。 </li><li>固定细胞1 × PBS洗两次，最后悬浮在1X PBS。 </li></ol></li><li>细胞分别置于幻灯片，并在Vectashield安装，安装用DAPI（1微克/毫升）（矢量，美国）的媒介。一个AXIOVERT 200米显微镜（蔡司镜头100X）和复合微分干涉对比（DIC）的细胞研究。荧光图像分析使用AxioVision，3.0版程序（蔡司）。 </li></ol> 7。免疫印迹分析。 1。要确定的WRN蛋白的表达，或转化 sgs1 或sgs1 TOP3株是 WRN变种，准备下面的方法，通过immunblotting分析蛋白质裂解液。 <ol><li> SC RAF减去色氨酸转化sgs1或sgs1 TOP3株生长在30℃过夜。 </li><li>文化reinoculated和孵育30 ° C直到年初数期（外径600 0.5 - 0.8），然后加入2％终浓度的GAL诱导。文化，然后让其生长在30 ° 6小时彗星</li><li>离心收集细胞（3毫升），用PBS冲洗。在碱性裂解液悬浮细胞沉淀[50毫米的NaOH，pH为10.5，2毫米EDTA，1毫米苯甲基磺酰氟（PMSF），2％SDS，10％甘油，5％2 - 巯基乙醇和蛋白酶抑制剂（罗氏分子生化）]煮沸5分钟，离心澄清，并与1 M盐酸中和。 </li><li>从细胞裂解液相当于量的8-16％SDS聚丙烯酰胺凝胶蛋白解决。 </li></ol> 2。要确定不同的半乳糖浓度的影响下，WRN蛋白的表达水平，文化如下处理。 <ol><li> TOP3 sgs1转化株生长在30 ° C过夜SC RAF减去色氨酸。 </li><li>文化是reinoculated，并在30 ° C直到年初数期（ 外径 600 0.5 - 0.8），进而诱发加入GA孵育l至2％终浓度。文化，然后让其生长在30 ° 6小时彗星</li><li>离心收集细胞，重悬于缓冲液中（50毫米的Tris - CL，pH值7.5，1％去氧胆酸钠，1％TRITON X - 100，0.1％十二烷基硫酸钠[SDS]）含有蛋白酶抑制剂（PMSF 0.05％，0.05微克/ ml的亮肽素）。 </li><li>玻璃珠（425至600微米）的混合物，涡旋破碎细胞。 </li><li>在15000 XG和由此产生的匀浆，离心5 min，收集上清。 </li><li> Bradford法蛋白在裂解馏分估计。 </li><li>从不同的裂解液组分的蛋白质浓度相同的是8-16％SDS聚丙烯酰胺凝胶上解决。 </li></ol> 3。由西方印迹使用一个WRN鼠单克隆抗体针对一个抗原决定簇的指示，在纯化的C -末端片段WRN WRN或WRN突变蛋白的表达[6]（1:1000，春谷实验室）。 4。其次是基层抗体孵育孵化与二级（辣根过氧化物酶标记）抗体（1:5000）。杂交的ECL Plus西方的检测系统，每个制造商的协议进行了处理。 5。对于定量免疫印迹分析，提高纯化的重组凝胶后的印迹的量化标准线性曲线可以产生包括标签全长WRN蛋白的浓度。此外，细胞从细胞或酵母裂解液样品20微克的量相当于提取物被装上凝胶估计 WRN浓度。 6。 ImageQuant然后进行分析，生成凝胶和标准的线性曲线。 7。 WRN蛋白的浓度在不同的半乳糖苷酶浓度，使用标准的线性曲线产生上述估计。 8。代表性的成果在这项研究中使用的所有菌株色氨酸营养缺陷型。因此，sgs1，sgs1 TOP3和野生型W303 - 1A与YEp112SpGAL或YEp112SpGAL WRN转化株，其成长能力存在SC减去色氨酸媒体的基础上选定的。要研究WRN表达转化sgs1 TOP3突变细胞生长的影响，文化挑染到含有2％的半乳糖苷酶SC减去色氨酸媒体诱发 WRN表达的板块。 TOP3 decatenates交织Sgs1解旋酶生成的复制过程中[1，2] DNA分子，因此，在TOP3的情况下，扭转应力是不能解除导致增长缓慢和超重组。 sgs1遗传功能是抑制增长缓慢的一个TOP3突变表型。如果WRN可替代SGS1与遗传相互作用TOP3，TOP3 sgs1 TOP3增长缓慢的表型恢复将预期。 如图2a所示，是WRN转化sgs1 TOP3应变向量相比增长明显缓慢转化sgs1 TOP3应变。sgs1 TOP3转化的YEp112SpGAL SGS1质粒作为阳性对照（图2A），这表明，野生型表示Sgs1 sgs1 TOP3突变基因互补的生长表型。同样在半乳糖苷酶缺乏增长的转变sgs1 TOP3株为2天（图2A）所示。 WRN表达对父母的野生型（W3031A） 或 sgs1株（图2B）增长没有影响，表明细胞生长的影响，WRN具体sgs1 TOP3突变背景观察。进行的遗传研究表明，WRN解旋酶，但没有外切酶活性恢复 TOP3增长的表型（ 图 3B） WRN变种。中WRN自然发生错义多态性与解旋酶的活性的干扰，取消其恢复 TOP3增长缓慢，表型的能力。 TOP3突变株在细胞周期的后期S/G2阶段延迟[1]，可能占到其增长缓慢的一个特点。 TOP3背景SGS1基因突变，抑制在细胞周期的S/G2阶段的延迟。如果WRN表达可以恢复在S/G2阶段sgs1 TOP3的细胞周期的延迟，不可分割的核出芽细胞升高的人口将预计sgs1表达WRN TOP3突变的细胞。正如图4所示，大芽生细胞的百分比明显高于sgs1 TOP3 比sgs1 TOP3 /矢量/ WRN，建议恢复S/G2 DE<html>打下TOP3特点。 sgs1 TOP3双突变是不太敏感的MMS或胡比TOP3单一突变[2]。自sgs1 TOP3 WRN受影响的细胞的生长，我们未来methylmethane的磺酸（MMS，烷化剂）和羟基脲（HU，复制抑制剂）。sgs1 TOP3 / WRN显示这两个药物的敏感性与sgs1 TOP3的敏感性研究其效果/ SGS1（图5）。 WRN变种的遗传分析表明，WRN解旋酶/ ATP酶，但不 WRN外切酶活性，其灵敏度MMS和胡锦涛的影响。 <img src="/files/ftp_upload/1639/1639fig1tmb.jpg" alt="图1" border="0" /> 图1。 YEp112SpGAL载体克隆WRN / WRN变种。原理演示 ，请<a href="https://www.jove.com/files/ftp_upload/1639/1639fig1.jpg">点击这里</a>为图1的放大版本。 <img src="/files/ftp_upload/1639/1639fig2tmb.jpg" alt="图2" border="0" /> 图2。WRN在sgs1 TOP3表达恢复增长缓慢，表型的TOP3。 小组，sgs1 TOP3应变YEp112SpGAL或YEp112SpGAL WRN转化SC -色氨酸板，内含2％或2％GLU GAL条纹。作为对照sgs1 TOP3应变改造YEp112SpGAL SGS1的是挑染两个板块。板在30 ° C孵育2天，然后拍照。 小组B，野生型菌株W303 - 1A或sgs1应变YEp112SpGAL或YEp112SpGAL WRN转化SC -色氨酸板块挑染或者在含2％葡萄糖或2％ GAL 。板在30℃孵育4天，然后拍照。板块组成，分别在A和B组小组。请<a href="https://www.jove.com/files/ftp_upload/1639/1639fig2.jpg">点击这里</a>看大图图2。 <img src="/files/ftp_upload/1639/1639fig3tmb.jpg" alt="图3" border="0" /> 图3。WRN ATP酶/解旋酶，但没有外切酶活性，需要恢复增长缓慢，表型的TOP3 sgs1 TOP3背景 。sgs1 TOP3应变转化与ATP酶/解旋死（YEp195SpGAL WRN K577M），外切酶的死（YEp195SpGAL WRN E84A），RQC突变（YEp195SpGAL WRN K1016A），多态突变（YEp195SpGAL - WRN R834C）挑染SC -色氨酸要么是2％的葡萄糖（C组 ）或2％ 加仑（B组 ）的板块。板在30 ° C孵育2天，然后拍照。板块组成，在小组答:请<a href="https://www.jove.com/files/ftp_upload/1639/1639fig3.jpg">点击这里</a>图3看到一个更大的版本。 <img src="/files/ftp_upload/1639/1639fig4tmb.jpg" alt="图4" border="0" /> 图4。WRN表达诱导S/G2逮捕sgs1 TOP3细胞。sgs1 TOP3 YEp112SpGAL，YEp112SpGAL WRN，或YEp112SpGAL SGS1转化株对数增长的文化，和矢量转化野生型菌株在半乳糖苷酶浓度为2 ％诱导6小时文化收获，加工材料和方法中所述的DAPI染色和使用AXIOVERT 200米显微镜（蔡司镜头100X）观察。显示的是YEp112SpGAL（左上角）转化sgs1 TOP3与YEp112SpGAL WRN（右上）的DAPI染色。箭头显示一心一意核细胞。 G1（单细胞）和S/G2（芽生细胞）的细胞分布在下部面板上显示。图4看到一个更大的版本， <a href="https://www.jove.com/files/ftp_upload/1639/1639fig4.jpg">请点击</a>这里。 <img src="/files/ftp_upload/1639/1639fig5tmb.jpg" alt="图5" border="0" /> 图5。对数增长sgs1 TOP3 YEp112SpGAL，YEp112SpGAL WRN，死亡外切酶（YEp112SpGAL WRN E84A），ATP酶/解旋死（YEp112SpGAL WRN K577M），转化的应变文化 WRN表达对MMS和胡锦涛sgs1 TOP3应变灵敏度 。 RQC突变（YEp112SpGAL WRN K1016A），多态突变（YEp112SpGAL - WRN R834C），YEp112SpGAL SGS1和载体转化野生型株父母发现一个到SC -色氨酸含葡萄糖或半乳糖苷酶和MMS或胡板10倍的系列稀释在指定的浓度。培养板在30 ° C为2天（控制盘），第4天（MMS和胡板），然后拍照。请9/1639fig5.jpg"&gt;点击这里看大图图5。

<ol>
	<li>Aggarwal, M., Brosh, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WRN+helicase+defective+in+the+premature+aging+disorder+Werner+Syndrome+genetically+interacts+with+Topoisomerase+3+and+restores+the+top3+slow+growth+phenotype+of+sgs1+top3.">WRN helicase defective in the premature aging disorder Werner Syndrome genetically interacts with Topoisomerase 3 and restores the top3 slow growth phenotype of sgs1 top3.</a> Aging. 1, 219-233 (2009).</li><li>Gangloff, S., McDonald, J. P., Bendixen, C., Arthur, L., Rothstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yeast+type+I+topoisomerase+Top3+interacts+with+Sgs1,+a+DNA+helicase+homolog:+a+potential+eukaryotic+reverse+gyrase.">The yeast type I topoisomerase Top3 interacts with Sgs1, a DNA helicase homolog: a potential eukaryotic reverse gyrase.</a> Mol Cell Biol. 14, 8391-8398 (1994).</li><li>Shor, E., Gangloff, S., Wagner, M., Weinstein, J., Price, G., Rothstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+homologous+recombination+genes+rescue+top3+slow+growth+in+Saccharomyces+cerevisiae.">Mutations in homologous recombination genes rescue top3 slow growth in Saccharomyces cerevisiae.</a> Genetics. 162, 647-662 (2002).</li><li>Sharma, S., Sommers, J. A., Brosh, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+function+of+the+conserved+non-catalytic+domain+of+Werner+syndrome+helicase+in+DNA+replication.">In vivo function of the conserved non-catalytic domain of Werner syndrome helicase in DNA replication.</a> Hum Mol Genet. 13, 2247-2261 (2004).</li><li>Gietz, R. D., Schiestl, R. H., Willems, A. R., Woods, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+transformation+of+intact+yeast+cells+by+the+LiAc/SS-DNA/PEG+procedure.">Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure.</a> Yeast. 11, 355-360 (1995).</li><li>von Kobbe, C., Thoma, N. H., Czyzewski, B. K., Pavletich, N. P., Bohr, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Werner+syndrome+protein+contains+three+structure+specific+DNA+binding+domains.">Werner syndrome protein contains three structure specific DNA binding domains.</a> J Biol Chem. 278, 52997-53006 (2003).</li></ol>

利用酵母作为模型系统的优势之一是在定义的DNA复制和修复的途径，酵母和人类之间的保守突变体的可用性。进一步选择窝藏的特定基因的转化是很容易的和可靠的实验室菌株营养缺陷型突变体，并与营养缺陷型标记载体，都是现成的。使用这些载体的基因产物的表达可诱导启动子的控制（例如，半乳糖苷酶诱导启动子，等）置于受。考虑到这些优势，我们开发的酵母为基础的模型系统研究沃纳?...

genetic studies of human dna repair proteins using yeast as a model system 

了解人类DNA修复蛋白的遗传途径的角色，是一个严峻的挑战，许多研究者。在哺乳动物系统中的遗传研究已经有限的，由于缺乏现成的工具，包括定义的突变基因的细胞株，表达系统的监管，并适当的可选标记。为了规避这些困难，在低等真核生物的遗传模型系统功能保守的DNA修复蛋白和途径的研究已成为有吸引力的选择。我们已经开发了一个模型酵母系统来研究，沃纳综合征的解旋酶，核酸定义不清的基因功能（<em&gt; WRN</em&gt;）在核酸代谢。与定义的遗传突变背景相关的细胞表型，可以进行调查，以澄清的细胞和分子功能<em&gt; WRN</em通过其催化活性和蛋白质的相互作用。人类<em&gt; WRN</em&gt;基因和相关的变体，克隆DNA质粒在酵母中的表达，可以放在监管质粒元素的控制下。表达式构造，然后可以转换成相应的酵母突变体的背景下，通过多种方法检测的遗传功能。使用这种方法，我们确定<em&gt; WRN</em&gt;，其相关的RecQ家庭成员BLM和Sgs1一样，工作在TOP3依赖途径可能为基因组稳定性的重要。这是在最近出版的[1]在描述的<a href="http://www.impactaging.com"&gt; www.impactaging.com</a&gt;。本文提供详细的方法，具体分析在酵母遗传互补研究。

Watch this Scientific Journal Video about Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System at JoVE.com

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

可在酵母的遗传研究调查的人类基因在细胞DNA的代谢的分子和细胞的功能。方法描述为人类的遗传特征<em&gt; WRN</em&gt;早衰障碍沃纳综合征在使用酵母作为一个听话的模型系统的功能保守的途径有缺陷的基因产物。

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

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Research

JoVE Journal

Biology

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

17.9K 次观看.  National Institute on Aging, NIH. 可在酵母的遗传研究调查的人类基因在细胞DNA的代谢的分子和细胞的功能。方法描述为人类的遗传特征 WRN早衰障碍沃纳综合征在使用酵母作为一个听话的模型系统的功能保守的途径有缺陷的基因产物。

视频: Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

作为一个型号的遗传生物鳉胚胎显微注射

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

科研

生物学

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development.
Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope.

DT40, a model vertebrate genetic system, provides a powerful tool to analyze protein function. Here we describe a simple method that allows qualitative analysis of parameters that influence DNA synthesis during the S-phase in DT40 cells at the single molecule level.

使用可视化的DNA复制DNA纤维的技术，在脊椎动物的模型系统DT40

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

改良酵母双杂交系统，以确定蛋白与生长因子Progranulin的相互作用

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

成像生物膜损伤和参与亚细胞修复过程

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded &#180;indefinitely&#180;. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

遗传条形码与荧光蛋白的复用的应用程序

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

在联合电子能谱成像miniSOG标记的DNA修复蛋白可视化（ESI）

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

DNA电穿孔，肌纤维的分离和成像

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

多酶筛选使用高通量基因酶的筛选系统

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...