我们想感谢雨果韶远试剂和布卢明顿库存中心。我们还要感谢公园Venken韶远，雨果和所有成员的Buszczak Hiesinger实验室的有益讨论。支持这项工作是由美国国家卫生研究院ACR（T32GM083831）的，公屋（RO1EY018884）的赠款和MB（RO1GM086647），授予德州癌症预防研究所以MB和公屋（RP100516），韦尔奇基金会（I-1657）公屋。 MB是在生物医学研究和公屋EE和格里尔加森•弗格森学者尤金·麦克德莫特学者在得克萨斯大学西南医学中心生物医学研究中。

1。选型的BAC和地区的目标<ol><li>要收购的BAC感兴趣的基因（GOI）（这里CG32095作为一个例子），搜索CG32095 <a href="http://www.flybase.org" target="_blank">www.flybase.org的</a> 。在部分股票和试剂，检查一节题为"基因组克隆"BAC的含CG32095。确保BAC包括至少10 KB的上游和下游5 kb的感兴趣的基因。或者，也可以被发现在P [ACMAN]载体7的克隆在<a href="http://www.pacmanfly.org/libraries.html" target="_blank">http://www.pacmanfly.org/libraries.html</a> 。 </li><li> BAC克隆在EPI300细胞DH10B细胞中的P [ACMAN]克隆。基因组克隆需要转化为SW102细胞，这是适合的重组工程。的"脏抽提"（Qiagen公司，而无需使用一列，并用异丙醇沉淀DNA小量制备）开始从5毫升的O / N培养接种细胞含有BAC。新鲜DNA沉淀再悬浮在20微升的蒸馏水2 O后，稀释1微升到100微升的蒸馏水2 O和使用1微升电穿孔电穿孔SW102。为了使电穿孔SW102使用以下协议: </li><li><ol><li> SW102细胞接种到4毫升LB。文化成长在32°CO / N在250转摇。 重要:SW102细胞应该永远不会被暴露在温度高于32°C，除非重组机制的诱导需要。 </li><li>接种1毫升到44毫升LB饱和SW102文化和生长在32℃摇床外径600 0.2-0.3之间（通常约1-1.5小时） 注:其他重组工程协议建议在一个较小的体积不断增长的文化OD 600 0.6-0.7，但是较低的密度生长的细菌在一个更大的体积显着增加变换efficie回馈金12。此外，这一修改减少了等待时间。 </li><li>冷却文化在冰水煤浆5分钟。 重要提示:在下面的步骤中的细胞必须在低温下保存。获得优质的电穿孔细胞，这是至关重要的。 </li><li>在1500×g离心10分钟，在4℃下离心培养。弃上清，洗涤沉淀在25ml冷的（4℃）10％甘油13。首先，在5毫升悬浮细胞攻丝和旋流管冰水煤浆（ 不要吸管向上和向下 ），再加入剩余的10％甘油（20毫升）。 </li><li>离心机在4℃下以1500×g离心10分钟，弃去上清，洗涤沉淀第二次用25毫升冰冷的10％甘油。 </li><li>离心机在4℃下以1500×g离心10分钟，弃上清，洗涤沉淀用25毫升冰冷的10％甘油的第三次。 </li><li>离心在4℃下以1500×g离心10分钟，丢弃线上ernatant。细胞沉淀，可能是非常松散的，在这一点上，和照顾时，应采取弃去上清。将沉淀重悬于1毫升的10％甘油。细胞转移到1.5毫升管中，在4℃下离心30秒，在桌面的离心12000×g离心倒掉大部分离开≈90微升甘油/细胞的上清液。现在已经准备好被电的细胞。 注意:此时，细胞可以储存在-80°C，以备以后使用。然而，这可能会降低能力。 </li><li>电穿孔使用1毫米的比色皿的细胞在1,800 V，25μF，200Ω的DNA导入。电击后，加300μl的SOC，让细胞恢复2小时32°C（晃动）。 LB琼脂板细胞上加25微克/毫升氯霉素（或其它适当的抗生素，根据变换后的BAC或PAC），并在32℃下孵育24-30小时。 </li></ol></li><li>基因打靶同源臂设计的选择ING 10 kb的基因组DNA的上游和下游印尼政府5 KB。选择年底的10 KB和500个基点，5 KB同源臂扩增（见第2节）（ 图1）。这500 bp的片段作为同源臂用于重组的基因组区域，围绕印尼政府为P [ACMAN] KO 1.0。这些片段被称为上游5&#39;同源臂，右臂（RA）的区域相对应的区域，对应于下游的3&#39;同源臂为左手臂（LA）。 重要 :同源臂不应包含BamHI位酶切位点，通过BamHⅠ限制性消化线性化的质粒（在步骤3.2中）来完成。 </li></ol> 2。到的P [ACMAN] KO 1.0同源臂插入<ol><li>设置了两个独立的PCR反应，放大LA和其他扩增的RA。为LA，使用引物经重组I-SCEI-LA-F和BamHI-LA-R使用的引物的BamHI-RA-F和attB2-I-SCEI-RA-R扩增的RA（ 表1）。使用1微升的BAC DNA步骤1.2或0.5微升煮过夜细菌培养，含有BAC利益作为模板。确保使用的DNA聚合酶校对能力。这适用于所有后续的PCR，PCR检查步骤2.8和3.13的例外。 注意:校对DNA聚合酶的延伸时间可能有所不同取决于制造商的常规Taq DNA聚合酶。 PfuUltra II融合热启动，而聚合的速率为15秒/ kb的，可以使用在这些步骤中。 LA / RA PCR的 <table border="1"><tbody><tr><td> 1微升</td><td>薄步骤1.2脏小量制备DNA </td></tr><tr><td> 0.25微升每个</td><td> 20μM引物</td></tr><tr><td> 2.5微升</td><td> 10X PfuUltra II缓冲</td></tr><tr><td> 0.75微升</td><td> 10MM（每个）的dNTP </td></tr><tr><td> 0.5微升</td><td> PfuUltra II融合热启动DNA聚合酶</td></tr><tr><td> 19.75微升</td><td>水</td></tr><tr><td> 25微升</td><td> 总成交量 </td></tr></tbody></table> PCR反应条件 <table border="1"><tbody><tr><td>第一步</td><td> 95℃2分钟以激活酶</td></tr><tr><td>第二步</td><td> 95℃20秒变性</td></tr><tr><td>第三步</td><td> 60°C 20秒退火</td></tr><tr><td>第四步</td><td> 72℃20秒延长</td></tr><tr><td>第五步</td><td>第2步，重复29个循环</td></tr><tr><td>第六步: </td><td> 4°C保持</td></tr></tbody></table></li><li>运行样品在1％琼脂糖凝胶提取产品使用Zymoclean的DNA回收试剂盒。从Zymoclean柱用10微升无菌水洗脱DNA。 </li><li>执行重叠延伸拼接（PCR法SOE）与2.2（ 图2）从每个扩增产物用10-30纳克的总DNA的两个片段。这通常是大约1/20 日的每个纯化的PCR产物: LA / RA PCR-梭 <table border="1"><tbody><tr><td> 0.5微升每个</td><td> LA及RA纯化PCR产物约10-30纳克</td></tr><tr><td> 0.25微升每个</td><td> 20μM引物</td></tr><tr><td> 2.5微升</td><td> 10X PfuUltra II缓冲</td></tr><tr><td> 0.75微升</td><td> 10MM（每个）的dNTP </td></tr><tr><td> 0.5微升</td><td> PfuUltra II融合热启动DNA聚合酶</td></tr><tr><td> 19.25微升</td><td>水</td></tr><tr><td> 25微升</td><td>总成交量</td></tr></tbody></table> PCR反应条件 <table border="1"><tbody><tr><td>第一步</td><td> 95℃2分钟以激活酶</td></tr><tr><td>第二步</td><td> 95℃20秒变性</td></tr><tr><td>第三步</td><td> 55°C 20秒退火（温度下允许武器退火） </td></tr><tr><td>第四步</td><td> 72℃20秒延长</td></tr><tr><td>第五步</td><td>第2步，重复1周期</td></tr><tr><td>第六步: </td><td> 95℃20秒变性</td></tr><tr><td>第七步</td><td> 60°C 20秒退火</td></tr><tr><td>第八步</td><td> 72℃20秒延长</td></tr><tr><td>第九步</td><td>第2步，重复27个循环</td></tr><tr><td>步骤10 </td><td> 4°C保持</td></tr></tbody></table></li><li> PCR样品在琼脂糖​​凝胶上运行，并通过凝胶提取，回收1.0kb的带。这是attB1-LA的BamHⅠ-RA-attB2磁带盒。 </li><li>使用网关BP ClonaseII的酶试剂盒，由制造商提供的协议后设立BP反应。盒2.4将作为供体DNA和的P [ACMAN] KO 1.0作为目标向量。 </li><li>稀释50微升的TransforMax EPI300电穿孔细胞加入30微升的10％甘油或过冷的DH 2 O变换成电穿孔的细胞后，由生产商提供的协议的BP反应的1μl的。在加水稀释盐的浓度相当高的血压反应，以防止在电穿孔过程中的放电（电弧放电）。电穿孔使用1毫米的比色皿的细胞在1,800 V，25μF，200Ω的DNA导入。 </li><li>添加300μl的SOC，让细胞恢复1小时37°C（晃动）。板细胞在LB-琼脂+ 50微克/毫升放大器（的P [ACMAN]-KO 1.0包含的放大器抗性基因的）。板在37°C孵育18-24小时。 </li><li> PCR检查单个菌落使用T3（T3序列是由传统的菌落PCR下游网关盒在P [ACMAN]-KO 1.0（ 图图1）和attB1-I-SCEI-LA-F引物。阳性克隆，将显示一个乐队，运行在1.2 kb。 </li><li>用50μg/ ml的氨苄青霉素的LB中生长的过夜培养的阳性克隆。 </li><li>健全的P [ACMAN]-KO 1.0矢量含有LA及RA在10毫升50微克/毫升氨苄青霉素文化的使用拷贝控制解决方案，制造商的协议（震中）的LB。 </li><li>制造商的协议后，执行Qiagen公司的小量制备。 </li></ol> 3。到的P [ACMAN] KO 1.0重组感兴趣的基因组区域 1天<ol><li>接种SW102细胞含有BAC利益LB加4毫升25微克/毫升氯霉素（CHL）或适当的选择抗生素。文化成长在32°CO / N在250转摇。 重要:除了步骤3.5诱导期间，不应该被暴露SW102细胞的温度高于32°C，高温激活的重组马即Chinery这些细胞。 </li></ol>第2天<ol start="2"><li>精华0.4微克的P [ACMAN]-KO-1.0包含在第2≈20个单位在25μl反应体系的BamHI LA及RA武器。在37°C孵育至少3个小时。 重要:好消化是关键，以确保所有的DNA消化。这将减少误报克隆后。 </li><li>接种1.0毫升饱和SW102/BAC文化成44毫升LB-CHL（25微克/毫升），并在32℃摇床增长0.2-0.3（通常约1-1.5小时）外径600之间。非HS控制包括一个附加的相同的样品。样品将被视为就像实验中的每一个方面例外的HS。这种非热休克培养作为阴性对照。外观的许多殖民地非HS控制板（3.13）通常表示改造未消化的P [ACMAN] KO 1.0 LA/ RA质粒。 </li><li>虽然文化的不断增长，运行的BamHI限制的P [ACMAN] KO凝胶。凝胶提取和10微升的水洗脱DNA升温至55°C。 注意:不要在任何缓冲区洗脱DNA。缓冲区中存在的盐可能导致放电过程中电。 </li><li>热休克的文化正是在42℃水浴15分钟。这将激活的重组工程机械SW102细胞。不要加热控制文化震荡。 </li><li>热休克后立即转移热休克和非恒生控制文化冷冻50毫升规范的管让文化在冰水浆5分钟的寒意。 重要信息:在接下来的步骤中细胞必须保持在低温。取得良好的品质的电穿孔细胞，这是至关重要的。 </li><li>离心10分钟，在1500 XG文化。弃上清，洗涤沉淀在25ml冷的（4℃）10％甘油13。首先，重悬细胞在5毫升冰水煤浆（请勿吸管向上和向下），再加入剩余的10％的甘油（20毫升），攻丝和旋流管。 </li><li>离心机在4℃下以1500×g离心10分钟，弃去上清，沉淀用25毫升冰冷的10％甘油第二次洗涤。 </li><li>离心机在4℃下以1500×g离心10分钟，弃去上清，用25毫升冰冷的10％甘油第三次洗涤沉淀。 </li><li>离心机在4℃下以1500×g离心10分钟，弃去上清液。细胞沉淀，可能是非常松散的，在这一点上，和照顾时，应采取弃去上清。将沉淀重悬于1毫升的10％甘油。细胞转移到1.5毫升管中，在4℃下离心30秒，在桌面的离心12000×g离心丢弃的上清液离开≈90微升水/细胞。 注:此时，细胞可以储存在-80℃下以供以后使用。然而，这可能会降低能力。</li><li>加入2μl的BamHI消化P [ACMAN]（目标为50纳克）的细胞，轻轻混匀，用枪头。细胞转移到1毫米的电比色皿和1,800 V，25μF，200Ω电穿孔。 </li><li>添加300μl的SOC，让细胞恢复2小时32°C（晃动）。 LB琼脂板细胞加50微克/毫升氨苄青霉素，在32°C孵育24-30小时。 </li></ol>第3天<ol start="13"><li>屏幕适当的间隙修复菌落PCR技术。使用T3和RA检查引物RA和T7和LA检查底漆为LA（ 表1）。设计的PCR入住RA和引物PCR入住LA对目标区域100-200个基点（ 表1）。 PCR反应的阳性克隆，采用PCR-T3或PCR入住LA与T7入住RA将显示一个乐队，运行在800-1000 bp的（ 图3）。注:自注册成立以来的一个关键是要测试这两种武器胳膊而不是其他有时观察。 </li><li> PCR验证的殖民地成长在32°C在4毫升LB 50微克/毫升氨苄青霉素过夜。使冻结的库存，这些细胞供以后使用。 </li></ol> 4。更换的基因组区域与目标盒式第4天<ol><li>放大的RFP /简淘汰赛盒PCR使用pENTR-RFP/KAN（原汁原味的ASCI和NheI线性）作为模板和的RFP 5&#39;HA /菅直-F-RFP 3&#39;HA /菅直-R引物（ 表1）。 RFP /简盒可以要求从参考文献11。 </li><li>凝胶纯化的PCR产物在20μl无菌水洗脱。 RFP根纸盒加上同源臂应奔波3 KB。 </li><li>饱和SW102 / P [ACMAN]-KO文化（3.13）接种1毫升到44毫升LB-AMP（50微克/毫升），成长于32℃摇床0.2-0.3之间外径600。非HS控制包括一个附加的相同的样品。此样品将被处理像实验中的每一个方面例外的HS。 </li><li> SW102 /的P [ACMAN] KO文化达到所需的密度后，按照步骤从3.5到3.10。 </li><li> 90微升的电穿孔SW102 /的P [ACMAN] KO细胞靶向卡带插入电穿孔约100纳克。细胞转移到1毫米的电比色皿和1,800 V，25μF，200Ω电穿孔。添加300μl的SOC，让细胞恢复2小时32°C（晃动不是必需的）。 注:强烈建议使用新鲜凝胶纯化盒。 </li><li>板细胞在的LB琼脂+放大器（50微克/毫升）+菅直人（50微克/毫升）和孵育24-30小时，在32°C。 </li></ol>第5天<ol start="7"><li>从5个不同的殖民地文化增长5毫升，在32°CO / N </li></ol>第6天<ol start="8"><li>执行一个"脏小量制备"和运行一半所得到的DNA在1％琼脂糖凝胶，以寻找公关esence的低分子量的质粒。不应该有任何质粒波纹管运行12 KB。 （ 图4） </li><li>制作1:500阳性克隆的DNA，并使用1微升电穿孔成50微升的TransforMax EPI300。 重要提示 :请一定要淡化成一个细胞的DNA，以防止多个质粒电。板细胞在的LB琼脂+放大器（50微克/毫升）+菅直人（50微克/毫升），并孵育18-24小时，在37°C </li></ol> 7天<ol start="10"><li>单个菌落接种于10毫升的LB +成长O / N。 </li></ol>第8天<ol start="11"><li>从步骤4.10饱和文化种子100毫升LB培养基。添加适当的抗生素加100微升1000 X复制控制解决方案和在37°C孵育5-6小时。 </li><li>执行maxiprep，并确认经测序正确的站点中的磁带插入。 </li><li>除了测序，执行限制植物学_AT_植物学ê消化从克隆的DNA不包含任何低分子量质粒的构建及其父母的DNA测试。选定的限制性内切酶将每个基因的不同，但我们的目标是找到一组酶，使研究者表征的recombineered矢量。通过举例的方式，CG32095靶向载体串联用PacI，AscI位，BamHI和的AatII消化。所有预测的波段，没有任何不正确的频带的外观确认，打靶载体已被正确地recombineered（ 图5）。 </li></ol> 5。 体内注入苍蝇和动员盒式<ol><li>注入盒使用fC31整合成一个预先定义的降落地点给出。外部供应商，如虹的转基因品种（ <a href="http://www.rainbowgene.com" target="_blank">http://www.rainbowgene.com</a> ），可用于注射服务。 重要:必须是新鲜的DNA p注射前repared。更高的转化效率在现场观察时，制备DNA，注入同一天。 </li><li>要调动磁带从登陆网站，使用布卢明顿库存号25680或25679。这些股票包含Flippase和I-SCEI的热休克启动子下游，染色体上的两个和三个，分别。此外，它们含有一种hs-隐藏的的转基因上的平衡器染色体和Y染色体上的。收集男性所选择的股票，，处女女性携带的KO盒在登陆网站和交叉。集〜30的十字架和允许女性产卵2-3天。 注:建议选择股票，就是在不同的染色体从目标轨迹。 </li><li>翻转到一组新的30小瓶的父母，热休克幼虫在37℃下1小时，每天两次，连续三天。热冲击激活的酶的生产; flippase和I-SCEI和细胞死亡的基因藏 </em&gt;，杀害平衡器染色体的男性和苍蝇携带。允许父母在新翻转小瓶产卵2-3天，然后重复这些小瓶的热冲击后翻转家长再次到新的小瓶。重复这几个翻转，直到父母不下蛋了。 </li><li>收集处女雌从的后代的热休克苍蝇，穿越到 y - W -男性。瞄准150-200十字架;三个处女和每跨三男。 </li><li>屏幕下的F2代的荧光示波器，使眼内的RFP检测。屏幕RFP积极的眼睛是白色和黄色（Y - W - ）基因突变体。这个筛选过程中积极选择为RFP盒的存在和选择的情况下P [ACMAN]载体（微型白色）和登陆网站，这是标记黄色基因野生型。 注:这是更短的时间内-CON梁漱溟到屏幕RFP的眼睛，比YW苍蝇的第一个屏幕。动员活动是非常有效的，在热冲击，导致少许多苍蝇的眼睛，包含RFP相比Y - W -苍蝇。 </li><li>设置每个 y - W - RFP +飞单对交配，因为每个调动事件可能会有所不同。 RFP的映射到正确的染色体。正确的染色体定位通常&gt; 95％的病例中观察到。 </li><li>建立股票和检查正确的定位事件Southern blot分析使用的标准技术。设计探针上游2 kb的（左手臂）和下游（右臂）正在有针对性的基因，和2 KB（或短如有必要）横跨删除ORF。的前两个探头，将产生一个频带，也就是不存在于野生型，而不会屈服的ORF的频带中的纯合基因敲除的纯合和杂合的敲除。一个快速PCR f的ORF也是可能的，但是这依赖于没有的频带的情况下。 </li></ol>

<ol>
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U.S.A. 100, 2556-2561 (1073).</li><li>Warming, S., Costantino, N., Court, D. L., Jenkins, N. A., Copeland, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+highly+efficient+BAC+recombineering+using+galK+selection.">Simple and highly efficient BAC recombineering using galK selection.</a> Nucleic Acids Res. 33, e36 (2005).</li><li>Copeland, N. G., Jenkins, N. A., Court, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombineering:+a+powerful+new+tool+for+mouse+functional+genomics.">Recombineering: a powerful new tool for mouse functional genomics.</a> Nat. Rev. Genet. 2, 769-779 (2001).</li><li>Venken, K. J., He, Y., Hoskins, R. A., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P[acman]:+a+BAC+transgenic+platform+for+targeted+insertion+of+large+DNA+fragments+in+D.+melanogaster.">P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster.</a> Science. 314, 1747-1751 (2006).</li><li>Venken, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+P[acman]+BAC+libraries+for+transgenesis+studies+in+Drosophila+melanogaster.">Versatile P[acman] BAC libraries for transgenesis studies in Drosophila melanogaster.</a> Nat. Methods. 6, 431-434 (2009).</li><li>Groth, A. C., Fish, M., Nusse, R., Calos, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+transgenic+Drosophila+by+using+the+site-specific+integrase+from+phage+phiC31.">Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31.</a> Genetics. 166, 1775-1782 (2004).</li><li>Bischof, J., Maeda, R. K., Hediger, M., Karch, F., Basler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+transgenesis+system+for+Drosophila+using+germ-line-specific+phiC31+integrases.">An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 3312-3317 (2007).</li><li>Chan, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+discovery+of+Rab+GTPases+with+synaptic+functions+in+Drosophila.">Systematic discovery of Rab GTPases with synaptic functions in Drosophila.</a> Current Biology: CB. 21, 1704-1715 (2011).</li><li>Chan, C. C., Scoggin, S., Hiesinger, P. R., Buszczak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+recombineering+and+ends-out+homologous+recombination+to+systematically+characterize+Drosophila+gene+families:+Rab+GTPases+as+a+case+study.">Combining recombineering and ends-out homologous recombination to systematically characterize Drosophila gene families: Rab GTPases as a case study.</a> Commun. Integr. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenesis+upgrades+for+Drosophila+melanogaster.">Transgenesis upgrades for Drosophila melanogaster.</a> Development. 134, 3571-3584 (2007).</li><li>Dietzl, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+transgenic+RNAi+library+for+conditional+gene+inactivation+in+Drosophila.">A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.</a> Nature. 448, 151-156 (2007).</li></ol>

作者在这里概述的技术方面没有任何竞争权益。

在很大程度上是基于遗传操作的工具，可用于在生物医学研究中的遗传模式生物的力量。小模型C.线虫和果蝇的特别允许在多细胞发育或功能完整的途径和基因家族牵连的廉价和快速的分子遗传分析。近年来出现了显着进步，工具开发，操纵基因在果蝇 14,15。例如，重组工程，它被广泛应用于在小鼠遗传学操纵Bac的DNA构建体，最近为果蝇适应，通过使用的P [ACMAN]...

清洁分子操纵单个基因在他们的内源性基因位点提供了一个非常宝贵的工具来研究真核细胞生物学相关的问题无数。 果蝇反向遗传技术产生损失的功能基因已被证明是具有挑战性的，直到和他的同事介绍Golic 体内基因靶向使用果蝇 1-3同源重组。他们证明，采用线性的DNA片段从一个集成的转基因构造，可以针对特定的基因位点。这种线性的"捐助"的DNA 在体内产生通过FRT-介导的重组（消费作为环状分子从染色体DNA）大范围核酸酶I-SCEI线性。虽然这种方法已成功地用于生成各种定义的病变，该技术并没有轻松扩展平行的许多基因的操纵，因为每个INDIVI双淘汰赛结构需要不同的定制设计。例如，无缝地操纵使用经典的限制性内切酶/结扎克隆或PCR，以及大小的限制，传统的在体内转化载体的大片段的DNA（&gt; 5 kb的） 在体外的困难往往会干扰同源重组靶向快速创建向量。为了克服这些限制，我们结合重组工程/转基因P [ACMAN系统，它允许子克隆和高达100 kb的DNA的转基因的端部从基因打靶方法建立一个有效的和相对快速的平台，使有利于果蝇基因打靶。 重组介导的基因工程（重组工程）是一个强大的同源重组克隆技术4,5。对比常规的限制性内切酶/连接酶克隆，重组工程并不限于序列或SIZE的操纵DNA。重组工程采用了特殊的大肠杆菌coli菌株，港口重组机制提供了一个有缺陷的λ噬菌体4。最近这种技术已被采用，用于在果蝇 6,7。重组工程在果蝇依赖于修改有条件扩增细菌人工染色体（BAC）载体称为P [ACMAN] 6,7。这个载体进行两个的复制起源:oriV的化学诱导后，其产生的高拷贝数的净化需要大量的DNA测序和胚胎注入和ORIS基础条件下，维持低拷贝数。此外，P [ACMAN]载体配有细菌附着（attB位点）的网站。 attB位点作为基板fC31整合酶介导的转基因在果蝇基因组8,9成预定着陆点，允许注册成立的大型DNA片段。 我们已经产生一个P [ACMAN]载体（以下简称为的P [ACMAN]-KO 1.0），可以用作靶向载体的端部出同源重组10,11。要纳入两端的基因打靶技术的系统中，我们加入了两个FRT和两个I-SCEI网站。我们还包括网关盒，在这个修改后的载体，以简化程序纳入到的P [ACMAN] KO 1.0同源臂。这提供了一种快速和简单的方法到目标载体引入任意感兴趣的基因组区域。在这个协议中，我们将描述如何设计的靶向载体，使用P [ACMAN]-KO 1.0，如何调动此载体在体内的内源基因座定位。对于此协议的目的，我们将使用的RFP /简卡带更换感兴趣的基因，但这个协议，可以使用各种含有抗生素选择标记盒。我们已经设计并成功应用于一组磁带基因置换和标记10,11。

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>SW102 Recombination competent bacteria</td>
			<td>NCI-Frederick</td>
			<td>Recombination Bacteria (SW102, SW105 and SW106)</td>
			<td><a href="http://ncifrederick.cancer.gov/research/brb/logon.aspx" target="_blank">http://ncifrederick.cancer.gov/research/brb/logon.aspx</a></td>
		</tr>
		<tr>
			<td>TransforMax EPI300 electrocopmpetent E. coli</td>
			<td>Epicentre</td>
			<td>EC300110</td>
			<td>Includes CopyControl induction solution</td>
		</tr>
		<tr>
			<td>PfuUltra II Fusion HS DNA Polymerase</td>
			<td>Aligent Technology Inc.</td>
			<td>600670</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BamHI-HF</td>
			<td>New England Biolabs</td>
			<td>R3136S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Zymoclean Gel DNA Recovery Kit</td>
			<td>Zymo Research</td>
			<td>D4001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Use Gateway BP ClonaseII Enzyme kit</td>
			<td>Invitrogen</td>
			<td>11789-020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>P[acman]KO1.010</td>
			<td>Buszczak and Hiesinger Labs</td>
			<td>Upon request</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>pENTR RFP-Kan11</td>
			<td>Buszczak and Hiesinger Labs</td>
			<td>Upon request</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Flystocks</td>
			<td>Bloomington stock center</td>
			<td>Stock numbers: 25680, 25679</td>
			<td>y1 w*/Dp(2;Y)G, P{hs-hid}Y; P{70FLP}11 P{70I-SceI}2B snaSco/CyO, P{hs-hid}4 
			y1 w*/Dp(2;Y)G, P{hs-hid}Y; P{70FLP}23 P{70I-SceI}4A/TM3, P{hs-hid}14, Sb1</td>
		</tr>
		<tr>
			<td>Electroporation machine</td>
			<td>Biorad GenePulser Xcell with PC module</td>
			<td>165-2662</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cuvettes</td>
			<td>Fisher Brand</td>
			<td>#FB101</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

recombineering homologous recombination constructs in drosophila

快速，大规模的内源性基因操纵技术 ​​的持续发展，将扩大利用果蝇作为一种遗传模式生物为人类疾病相关的研究。近年来，人们像同源重组和重组工程技术进步。然而，产生明确的的无效突变或标记内源性蛋白仍然是一个重大的努力，大多数基因。这里，我们描述和展示的技术使用基于重组工程的克隆的方法生成，可用于目标和操作在体内的内源基因位点的载体，具体而言，我们已经建立了三种技术的组合:（1）血液中酒精浓度的转基因/重组工程，（ 2）两端的同源重组（3）网关技术，提供了一个可靠，高效和灵活的方法来处理内源性基因位点的。在这个协议中，我们提供了一步一步的详细信息:（1）设计individua升载体，（2）如何克隆大片段的基因组DNA同源重组到载体，使用间隙维修，及（3）如何更换或标记感兴趣的基因，在这些载体中，使用了第二轮的重组工程。最后，我们也将提供如何调动这些录音带在体内产生的淘汰赛，或通过一个标记基因敲的协议。这些方法可以很容易地采用平行的多个目标，并提供一种装置，用于及时和有效的方式操纵果蝇基因组。

LA及RA同源臂的扩增产生500 bp的产品和国有企业的PCR反应应该产生一个1.0 kb的产品（第2.1-2.4; 图2）。 2.5节进行BP反应通常是非常有效率和细菌转化产品产量平均5-100菌落。几乎所有的殖民地PCR测试与检查显示出预期的PCR产物。 在第一轮的重组工程（第3节）期望得到消化的P改造后的20-40殖民地ACMAN]-KO-1.0 SW102细胞含有LA及RA武器。这些克隆中的40％至60％，将携带所需...

Watch this Scientific Journal Video about Recombineering Homologous Recombination Constructs in Drosophila at JoVE.com

Recombineering Homologous Recombination Constructs in Drosophila

同源重组技术大大推进<em&gt;果蝇</em遗传学通过分子精确的突变使创建。最近通过的重组工程允许一个操作大块的DNA，并将其转化为<em&gt;果蝇</em<sup&gt; 6</sup&gt;。这里介绍的方法，这些技术结合起来，以迅速产生大量的同源重组载体。

重组工程同源重组构建<em&gt;果蝇</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

recombineering-homologous-recombination-constructs-in-drosophila

Research

JoVE Journal

Biology

Recombineering Homologous Recombination Constructs in Drosophila

19.1K 次观看.  University of Texas Southwestern Medical Center at Dallas. 同源重组技术大大推进果蝇

视频: Recombineering Homologous Recombination Constructs in Drosophila

Wolbachia Bacterial Infection in Drosophila

My name is Voia Friedman. I am associate researcher at Princeton University. And today I'm going to tell you about a few protocols that I developed to trans infect, to I initially to isolate, to go back from the heli of infected flies, and later on help to inject the heli feed to unaffected flights.So basically, this is a very simple protocol to isolate him oly for basics. And you will use this device from, from mepo that contains a filter that goes on top of an E.So you basically needs to put you, we start from around a hundred flies. You anize the flies, you put them into the filter device, you can just use a funnel and that will add our other fit very easily on this device and wash it with 70%ethanol.You just take the, take the future away, you can throw away the ethanol, and then after three times your last wash you do with molecule water because you don't want have the bacteria from the, with traces of ethanol. Well, and then you just go to a new weapon door and, and then you spin down on a micro, real micro for two minutes And later on how you can inject that. He only feed do unaffected flies.So basically take a double stick tape and usually these double stick tapes have two side, one side is a sticker than the other. So leave the sticker side up because that's the side that you're gonna glue that flies on top of it. And basically you'll, the gas apparatus, if you have this light on top of it, you'll be no gas flow and the flies you wake up if they're on top of the slide.So you can just direct the flow, making a little hood, a little chamber using tape. Then you direct the gas over the flies. So then you put your, this light here with the tape and you, you have your flies.Stick the flies on the top of the tape, all of them the same orientation, and put their head towards the direction that you'll be bringing the needle from. Because then you can just over the, the wing, you make sure since they fly, you don't want to fly. If they woke up during the procedure, don't want them to go away.So make sure that you take a little tip and pressure against the, the, the wings of the fly on the tail. And it's okay if they wings get damaged. And when you get rid, when you take the flies up the injection, most likely they'll lose their wings anyway, so they'll survive.That can, that can be useful because you're not gonna lose the flies that, so he puts a mineral oil on the micrometer for the back of the needle, then the needle injection polar, and then the, you can just adjust the pressure to bring the material up to the tip of the needle. I, I did to inject Somewhere between 10 to 15 nanoliters, but as, as long, as long as I'm consistents. Okay, so one important detail there is a cushion of this experiment is that the, the fly, once the apply is being injected when you saw the needle needs to come as far as you can through the upper part of the abdomen.Because here, basically when the needle penetrates, you have a, a, a, a, a bigger space to go up and down. And basically if it's close, closer to the cuticle, it's less likely they're gonna penetrate vital organs in the flight. You, it is very likely unlikely that you're gonna punch a hole in your gut, for instance, if that is gonna kill the flight that you are performing the surgery.My name is Voia Friedman. I am associate researcher at Princeton University in the lab of Eric Hels. And today I would like to tell you about this protocol that you just saw, the demonstration.So this protocol was done because we wanted to prove that UBA is capable of crossing tissues. So UBA is one of the most successful intracellular paras on the face of the earth. Infects from 20 to 70%of insects and many particles.And also is, is very important in terms of human diseases because also infect nematodes that transmit diseases like river blindness or s and ssis. And so, and CCA is our obligatory endo in, in these worms. And there's a great, a very big interest in using CCA also as means to control parasitic diseases transmitter by mosquito and Danny and this and by mosquito such as malaria and dengue.So this, this, this protocol basically was developed to show that once CCA inside the abdominal cavity of a fly, they can cross tissues and reach the germ. So basically what you can see here is the oph of the drosophila ovary and go back once present in the he leaf it, it can cross a pink peronial. She that is around the ovary.And then they cross a muscle layer. And actually one of the first places that they, they go inside the ovary is the somatic stem cell image. And then they can reach the germline.And this bacteria is transmitted the same way that mitochondria is transmitted is through, is through the mother transmitted, through the mother cyop plants present in the egg. So of course they needs to be present into the germline. So that's why this protocol is developed.And this protocol is, can be very useful to basically traditionally the way that people would try to trans infect wbaa basically take wbaa and introduce a new insect species. It would be through doing micro injections into the, into the embryo. But those are much more complicated.And basically here we are just taking advantage of the o biology since they are capable to cross tissues and moving into the stem cell tissues from, they can pass to the, through to the germline. So that's make easier taking UBA from certain, from, from certain species of insects that are infected and they can be transmitted to uninfected species. Prob I think it's the injection itself.Basically you need to be careful to mobilize the fly as well as you can. We are using double sided stick tape and you need to penetrate the needle basically as close to the, as parallel to the fly as possible so you don't go to deep. And it's less likely that we're gonna kill the fly after they're performing the surgery.

在果蝇的Wolbachia细菌感染

科研

生物学

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

制作生肌工程的组织结构

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

生产 C。线虫</em通过重组工程与转基因 galK选择标记

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

使用内源性野生型BRCA1基因的细胞表达，确定同源重组的BRCA1基因突变的影响

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

影像生活细胞的形状变化果蝇胚胎

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

基因改造和重组唾液的腺体器官培养

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the Mgm101 protein is involved in the recombinational repair of mitochondrial DNA. Recent investigations have indicated that Mgm101 is related to the Rad52-type recombination protein family. These proteins form large oligomeric rings and promote the annealing of homologous single stranded DNA molecules. However, the characterization of Mgm101 has been hindered by the difficulty in producing the recombinant protein. Here, a reliable procedure for the preparation of recombinant Mgm101 is described. Maltose Binding Protein (MBP)-tagged Mgm101 is first expressed in Escherichia coli. The fusion protein is initially purified by amylose affinity chromatography. After being released by proteolytic cleavage, Mgm101 is separated from MBP by cationic exchange chromatography. Monodispersed Mgm101 is then obtained by size exclusion chromatography. A yield of ~0.87 mg of Mgm101 per liter of bacterial culture can be routinely obtained. The recombinant Mgm101 has minimal contamination of DNA. The prepared samples are successfully used for biochemical, structural and single particle image analyses of Mgm101. This protocol may also be used for the preparation of other large oligomeric DNA-binding proteins that may be misfolded and toxic to bacterial cells.

The yeast mitochondrial nucleoid protein, Mgm101, is a Rad52-type recombination protein that forms large oligomeric rings. A protocol is described to prepare soluble recombinant Mgm101 using the Maltose Binding Protein (MBP)-tagging strategy coupled with cation exchange and size exclusion chromatography.

MBP基于标记策略的Mgm101重组蛋白的制备

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

亚克隆加插入（SPI） - 一种新型重组工程方法基因打靶载体的快速构建

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.