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

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

该<em&gt;果蝇</em&gt;成虫盘瘤模型:基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

科研

癌症研究

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

在Vivo功能研究的疾病相关的稀有人类变异使用果蝇

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

遗传学

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

洗涤剂辅助重组重组果蝇阿拉他素到脂质混合测定脂质体

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

生物化学

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of cells or tissues in model organisms. These systems contain two components as separate transgenic lines. A driver line expresses a transcriptional activator under the control of tissue-specific promoters/enhancers, and a reporter/effector line harbors a target gene placed downstream to the binding site of the transcription activator. Animals harboring both components induce tissue-specific transactivation of a target gene expression. Precise spatiotemporal expression of the gene in targeted tissues is critical for unbiased interpretation of cell/gene activity. Therefore, developing a method for generating exclusive cell/tissue-specific driver lines is essential. Here we present a method to generate highly tissue-specific targeted expression system by employing a &#34;Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated&#34; (CRISPR/Cas)-based genome editing technique. In this method, the endonuclease Cas9 is targeted by two chimeric guide RNAs (gRNA) to specific sites in the first coding exon of a gene in the Drosophila genome to create double-strand breaks (DSB). Subsequently, using an exogenous donor plasmid containing the transactivator sequence, the cell-autonomous repair machinery enables homology-directed repair (HDR) of the DSB, resulting in precise deletion and replacement of the exon with the transactivator sequence. The knocked-in transactivator is expressed exclusively in cells where the cis-regulatory elements of the replaced gene are functional. The detailed step-by-step protocol presented here for generating a binary transcriptional driver expressed in Drosophila fgf/branchless-producing epithelial/neuronal cells can be adopted for any gene- or tissue-specific expression.

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Here, we present a method for generating tissue-specific binary transcription systems in Drosophila by replacing the first coding exon of genes with transcription drivers. The CRISPR/Cas9-based method places a transactivator sequence under the endogenous regulation of a replaced gene, and consequently facilitates transctivator expression exclusively in gene-specific spatiotemporal patterns.

基于基因组编辑的果蝇组织特异二进制转录系统的高效生成策略

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of ...

发育生物学

用于果 蝇 菌株长期保存的 PGC 冷冻保存

Primordial Germ Cell Cryopreservation and Revival of Drosophila Strains

Drosophila strains must be maintained by the frequent transfer of adult flies to new vials. This carries a danger of mutational deterioration and phenotypic changes. Development of an alternative method for long-term preservation without such changes is therefore imperative. Despite previous successful attempts, cryopreservation of Drosophila embryos is still not of practical use because of low reproducibility. Here, we describe a protocol for primordial germ cell (PGC) cryopreservation and strain revival via transplantation of cryopreserved PGCs into agametic Drosophila melanogaster (D. melanogaster) host embryos. PGCs are highly permeable to cryoprotective agents (CPAs), and developmental and morphological variation among strains is less problematic than in embryo cryopreservation. In this method, PGCs are collected from approximately 30 donor embryos, loaded into a needle after CPA treatment, and then cryopreserved in liquid nitrogen. To produce donor-derived gametes, the cryopreserved PGCs in a needle are thawed and then deposited into approximately 15&#160;agametic host embryos. A frequency of at least 15% fertile flies was achieved with this protocol, and the number of progeny per fertile couple was always more than enough to revive the original strain (the average progeny number being 77.2 &#177; 7.1), indicating the ability of cryopreserved PGCs to become germline stem cells. The average number of fertile flies per needle was 1.1 &#177; 0.2, and 9 out of 26 needles produced two or more fertile progeny. It was found that 11 needles are enough to produce 6 or more progeny, in which at least one female and one male are likely included. The agametic host makes it possible to revive the strain quickly by simply crossing newly emerged female and male flies. In addition, PGCs have the potential to be used in genetic engineering applications, such as genome editing.

作者聚焦：PGC 冷冻保存 – 一种长期保存果蝇菌株的替代方法 

A long-term preservation method for Drosophila strains as an alternative to the frequent transfer of adult flies to fresh food vials is highly desirable. This protocol describes the cryopreservation of Drosophila primordial germ cells and strain revival via their transplantation to agametic host embryos.

果蝇菌株的原始生殖细胞冷冻保存和复兴

Drosophila strains must be maintained by the frequent transfer of adult flies to new vials. This carries a danger of mutational deterioration and ...

果蝇中 phiC31 整合酶介导的胚胎转基因

Embryo Microinjection for Transgenesis in Drosophila

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the construction of transgenic flies. Microinjection requires some types of equipment, including a microinjector, a micromanipulator, an inverted microscope, and a stereo microscope.&#160;Plasmids isolated with a plasmid miniprep kit are qualified for microinjection. Embryos at the pre-blastoderm or syncytial blastoderm stage, where nuclei share a common cytoplasm, are subjected to microinjection. A cell strainer eases the process of dechorionating embryos. The optimal time for dechorionation and desiccation of embryos needs to be determined experimentally. To increase the efficiency of embryo microinjection, needles prepared by a puller need to be beveled by a needle grinder. In the process of grinding needles, we utilize a foot air pump with a pressure gauge to avoid the capillary effect of the needle tip. We routinely inject 120-140 embryos for each plasmid and obtain at least one transgenic line for around 85% of plasmids. This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and&#160;presents a detailed protocol for embryo microinjection for transgenesis in Drosophila.

作者聚焦:优化胚胎显微注射以促进果蝇的转基因

This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and presents an optimized protocol for embryo microinjection,&#160;a crucial step for creating transgenic flies.

胚胎显微注射用于果蝇转基因

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the ...

重组工程同源重组构建

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在Vivo功能研究的疾病相关的稀有人类变异使用果蝇

洗涤剂辅助重组重组果蝇阿拉他素到脂质混合测定脂质体

基于基因组编辑的果蝇组织特异二进制转录系统的高效生成策略

果蝇菌株的原始生殖细胞冷冻保存和复兴

果蝇菌株的原始生殖细胞冷冻保存和复兴

果蝇菌株的原始生殖细胞冷冻保存和复兴

胚胎显微注射用于果蝇转基因

胚胎显微注射用于果蝇转基因

胚胎显微注射用于果蝇转基因