我们感谢环境服务协会教育信托基金，在剑桥SynBio基金合成生物学和社会正义和赋权，印度政府部，给予资金支持。

1.培养基的制备<ol><li>根据Castenholz，1988年17准备BG11培养基。 <ol><li>准备100X BG11的储备液，微量元素铁的股票（ 表1）。 </li><li>制备磷酸盐股票的单独的溶液， 的 Na 2 CO 3的库存，N - [三（羟甲基）甲基] -2-氨基乙磺酸（TES）缓冲液和碳酸氢钠 （ 表1）。 </li><li>高压灭菌的磷酸盐和Na 2 CO 3的股票。过滤消毒TES缓冲液和碳酸氢钠 0.2微米的过滤器。 </li><li>通过组合976毫升的水，将10毫升100倍BG11的1毫升痕量元素和1ml的铁库存准备BG11和高压灭菌的溶液中。后该溶液冷却到室温，加入1毫升磷酸盐库存，将1ml 的 Na 2 CO 3的库存和10毫升碳酸氢钠的。 </li><li>为BG11固体培养基，加入琼脂15克和700毫升水于一个FLASK。向第二个烧瓶中，加入的 Na 2 S 3克2- O 3 226毫升水，将10毫升100倍BG11的1毫升痕量元素和1ml的铁库存。高压灭菌两种解决方案。之后这些解决方案已经冷却至室温，将它们合并并加入1毫升磷酸盐库存，将1ml 的 Na 2 CO 3的库存，将10毫升TES缓冲液，和10毫升碳酸氢钠的。 注意:解决方案是单独制备，以避免某些盐的沉淀。 </li></ol></li><li>对蔗糖选择，制备50％（重量/体积）的蔗糖溶液。过滤消毒用0.2微米的过滤该溶液，并加入到BG11（100毫升50％的蔗糖到900毫升BG11的），以产生BG11 / 10％蔗糖平板上。 注意:不要碳酸氢钠添加到BG11 / 5％蔗糖琼脂平板上。加的 Na 2 CO 3为正常。 </li><li>对于聚球藻的培养加入10毫升的1摩尔的4-（2-羟乙基）哌嗪-1-乙磺酸，N-的- （2-羟乙基）哌嗪NR42 - （2-乙磺酸）（HEPES）和1ml维生素B 12（ 表1）的1升BG11培养基中。 注意:在市售的BG11培养基中培养菌株的转化是显著少效率比在这里描述的BG11培养基配方，因此不建议。 </li></ol> 2.蓝藻菌生长<ol><li>在100毫升锥形瓶中培养菌株为50毫升最大体积并在120rpm摇动。密封用Parafilm和穿刺三个小的孔BG11板在板的一侧，以允许气体交换。孵育在30℃下，在光生物反应器的荧光灯所有菌株在光强度20-40微摩尔光子微米之间-2秒-1。 </li><li>最好使用无菌技术。处理层流罩所有蓝藻菌。 注意:这是特别重要的，当菌株是用含有蔗糖的培养基中培养，可以很容易地沾污ated。 </li></ol> 3.质粒构建的生成<ol><li>设计组引物，包括所需的限制酶位点，使用引物设计软件，例如引物3（http://frodo.wi.mit.edu/primer3/），以扩增的两〜1 kb的区域的5&#39;和3&#39;感兴趣的基因。通过Cyanobase咨询蓝藻物种的基因组序列（http://genome.kazusa.or.jp/cyanobase）。 见表2此处使用的所有引物。设计引物时考虑以下因素: <ol><li>确保扩增区域包括将要突变的基因， 例如 ， 图1的5&#39;和3&#39;区域。 </li><li>不发生变异间隔区，以避免反义和非编码RNA的无意的突变。用于产生在集胞藻突变体，是指在Mitschke 等记载的转录起始位点的列表。，2011 18，以避免反义的突变或非编码RNA。 </li><li>当选择侧翼区不包括相邻基因作为这些基因在大肠杆菌中的表达的整个开放阅读框可以与克隆干扰。 </li></ol></li><li>扩增按照制造商的说明使用高保真DNA聚合酶产品通过PCR。 注:在我们的经验，这种酶会产生一些错误。 <ol><li>建立含有HF缓冲液和DMSO的任0，1.5或3微升50微升PCR反应。使用100纳克每反应的基因组DNA。使用由98℃初始变性步骤的程序，30秒，35轮的98℃10秒，67℃，30秒，72℃30秒，随后的72℃最后延伸步骤下进行5分钟。这通常给予一致的产品。 </li></ol></li><li>验证与核酸内切酶消化通过凝胶电泳正确大小的PCR产品和样品。运行1％（重量/体积）含0.02％琼脂糖凝胶上（体积/体积）溴化乙锭45分钟在100伏 注意:溴化乙锭是一种潜在的诱变剂，应使用适当的保护处理。 </li><li>纯化使用根据制造商的说明的DNA纯化试剂盒的PCR产物。还可以使用此试剂盒质粒片段，包括从琼脂糖凝胶裁片的净化。在水14微升洗脱纯化的DNA。 </li><li>用于克隆步骤，根据制造商的说明孵育限制性内切酶反应的混合物在37℃下为&gt; 1小时在30微升的总体积。 </li><li>用于连接的步骤，在室温下在20μl的总体积&gt; 1小时后，含有5微升纯化消化的质粒，12微升纯化消化的插入物，缓冲液2微升和1微升连接酶连接DNA片段。 </li><li>根据以下方法制备大肠杆菌 DH5α转化体细胞。 <ol><li>成长隔夜E.大肠杆菌 </em&gt;文化在10毫升卢里亚BERTANI（LB）的媒体。 </li><li>接种400毫升的LB中含有6毫升的1摩尔的MgCl 2（ 表1）用1毫升过夜培养物的1升锥形瓶中。 </li><li>生长的培养在37℃下以220rpm进行约4小时或直至OD 600nm处达到0.4-0.6。 </li><li>细胞置于冰上1小时。 </li><li>离心机在2,800×g离心10分钟以沉淀细胞在4℃。 </li><li> 160 ml溶液A（ 表1）取出上清，重悬并在冰上孵育20分钟。 </li><li>离心机在2,800×g离心10分钟以沉淀细胞在4℃。 </li><li>在4毫升溶液A +甘油（ 表1）取出上清，重悬。 </li><li>准备50微升等分，冻结液态氮，储存在-80℃。 </li><li>混合5微升连接混合物用50μl感受态细胞，并孵育在冰上1小时。 </li><li>热休克细胞在42℃下90秒，弗洛通过在冰上温育结婚2分钟。 </li><li>添加950微升LB培养基（ 表1）中，并在37℃孵育1小时。 </li><li>等分试样50和200微升在平板上用适当的抗生素，或者氨苄青霉素（100微克/ ml）和/或卡那霉素（30微克/毫升）。 注意:卡那霉素和氨苄青霉素是有毒的，应适当的保护处理。 </li><li>挑并与合适的抗生素接种2毫升LB培养基孵育单个菌落。 </li></ol></li><li>纯化使用根据制造商的说明一小量制备质粒纯化试剂盒的所有质粒。 </li><li>产生的质粒，在用于敲除cpcC1C2基因这个具体的例子，根据下面的步骤。 <ol><li>扩增使用引物cpcC1C2leftfor和cpcC1C2leftrev（参见步骤3.2， 表2）的1012 bp的5&#39;侧翼区（左片段）。除去PCR反应的少量，并确认是否正确尺寸产品已通过凝胶电泳（步骤3.3）被放大。消化该片段和pUC19用Xba I和Bam HI（步骤3.5）。 </li><li>既净化制剂（步3.4），结扎（步骤3.6），转换（步骤3.7），并通过小量（步骤3.8）设置了4个2毫升LB与质粒净化独立的殖民地氨苄青霉素（100微克/毫升）的液体培养。 </li><li>检查通过的XbaⅠ/ 巴姆 HI消化和凝胶电泳（步骤3.3）片段到pUC19的插入。 2,660 bp和1012 bp的条带指示正确引入插入到质粒。 </li><li>扩增使用引物cpcC1C2rightfor和cpcC1C2rightrev（参见步骤3.2， 表2）的1016 bp的3&#39;侧翼区（右片段）。除去PCR反应的少量，并确认是否正确尺寸产品已通过凝胶电泳（步骤3.3）被放大。消化该片段和pUC19用Sac I和EcoRⅠ（STEP 3.5）。 </li><li>既净化制剂（步3.4），结扎（步骤3.6），转换（步骤3.7），并通过小量（步骤3.8）设置了4个2毫升LB与质粒净化独立的殖民地氨苄青霉素（100微克/毫升）的液体培养。 </li><li>检查通过萨克 I / RI 生态消化（步骤3.5）和凝胶电泳（步骤3.3）片段到pUC19的插入。 2,660基点人和1 016 bp的条带指示正确引入插入到质粒。 注:XbaⅠ位 / 巴姆 HI位点&#39;为3的克隆区域和萨克 I / RI 生态 &#39;区域5克隆到pUC19尽可能使用。如果可行的话，始终包含在对于5&#39;区域或3正向引物&#39;区的反向引物一个BamHⅠ位点，以确保以后的克隆步骤是更容易执行。 </li><li>序列的两个插入件，以确定该序列使用引物跨越插入部位， 例如 ，M1为正确3前进和M13反 ​​向（ 见表2）。顺序必须是正确的，以确保没有错误被引入侧翼区。 </li><li>消费的来自pUC19通过的XbaⅠ/ 巴姆 HI消化左侧的片段。消化的pUC19 +正确的片段用XbaI / 巴姆 HI（步骤3.5）。 </li><li>从净化通过使用手术刀刀片DNA切除的琼脂糖凝胶（步骤3.3）的1012 bp的左片段和3,676基点的pUC19 +右片段。 </li><li>既净化制剂（步3.4），结扎（步骤3.6），转换（步骤3.7），并通过小量（步骤3.8）设置了4个2毫升LB与质粒净化独立的殖民地氨苄青霉素（100微克/毫升）的液体培养。 </li><li>检查片段插入通过的XbaⅠ/ 巴姆 HI消化（步骤3.5）和凝胶电泳（步骤3.3）的pUC19 +右片段的插入。 3,676 bp和1012 bp的条带指示插入正确插入质粒（请参阅本质粒B）。 </li><li&gt;从香港海关19 pUM24cm通过巴姆 HI消化NPT1 /卡带的sacB。摘要质粒B带巴姆 HI（步骤3.5）。 注意:NPT1 /的sacB盒不必从琼脂糖凝胶中纯化，因为pUM24cm编码赋予氯霉素抗性的蛋白质。因此，如果菌落在LB生长/氨苄青霉素/卡那霉素的琼脂平板上的唯一可能的组合，这将导致抗性菌落是掺入NPT1 /的sacB盒的入质粒B. </li><li>净化两种制剂（步3.4），结扎（步骤3.6），转换（步骤3.7），并设置4个2毫升LB与质粒净化独立的殖民地氨苄青霉素（100微克/毫升）和卡那霉素（30微克/毫升）的液体培养通过小量（步骤3.8）。 </li><li>检查通过巴姆 HI消化（步骤3.5）和凝胶电泳（步骤3.3）的NPT1 /卡带的sacB插入到质粒B点。 4,688 bp和3894 bp的条带表明日的正确插入E将到质粒（请参阅本质粒A）。 </li><li>可替代地，钝端NPT1 /的sacB盒和克隆到质粒B的左侧和右侧片段之间的不同限制性内切酶位点的NPT1 /的sacB盒必须在左边和右边的片段之间进行克隆。 注:如果需要的外盒的表达那么这应该质粒B的左，右片段此质粒然后在未标记的基因敲除步骤中使用之间插入。 </li></ol></li></ol> 4.生成标记的蓝藻和聚球藻的突变体<ol><li>通过接种环满格为30-50毫升BG11培养基中建立了一个新的文化。生长2-3天的文化OD 750nm的 = 0.2〜0.6。 注意:通常单个菌落太小用于接种和单个细胞暴露于甚至低光水平将导致光抑制和选择光耐药突变。 </li><li>离心机1-2毫升培养2,300×g离心5分钟并弃上清。在&gt; 2300 XG不要离心任何蓝藻文化，因为这可能会损坏电池。以BG11培养基洗涤沉淀一次。 注意:不要涡旋，因为这可能会导致菌毛这对DNA的摄取必需的损失悬浮细胞。轻柔吹打悬浮细胞。 </li><li> BG11培养基添加到100微升的最终体积。转移细胞到14毫升的圆底管中。 </li><li>添加1微克质粒A对细胞和通过轻轻敲击混合。添加&lt;10微升质粒。 注:优选质粒应是&gt; 100毫微克/微升的浓度，但浓度低于此是足够的成功转型。 </li><li>在孵化器水平放置管子下来。孵育培养4-6小时。 注:细胞可以通过点击每1-2小时进行简单混合，但是这不是必不可少的。样品可以被放置在一个振荡培养箱虽然这并不显著提高效率。 </li><li>散布在BG11琼脂平板上的细胞培养/质粒DNA混合物的等分无抗生素。通常20微升和80微升等分被分散在不同的板块。 </li><li> 〜24小时后，加水含卡那霉素2.5-3毫升0.6％琼脂溶液（每次20毫升0.12克琼脂，100微升的100毫克/毫升卡那霉素）琼脂板上。冷却此溶液至〜42℃，并加入到琼脂平板的边缘。倾斜板，使该溶液形成表面上的偶数&#39;顶层琼脂&#39;层。 </li><li>孵育琼脂平板上的时间再延长一段时间。经过大约7天后殖民地应该是可见的。 注:琼脂平板可以堆叠3高的孵化器。通常情况下200个菌落都是每变换得到。 </li><li>上BG11 +卡那霉素（30微克/毫升）的琼脂平板上划线单个菌落。划分琼脂平板成6个扇区，用钝端牙签出来连胜殖民地放在每个单独的部门。获得单菌落不是转化的重要，只是增长。 </li><li>确认根据制造商的说明使用Taq DNA聚合酶通过PCR标记基因敲除。加入2微升氯化镁 （25毫米），每反应。 <ol><li>除去细胞的一小部分，并转移到含有50微升水和〜20 425-600微米的玻璃珠的管中。撼动振动器在〜2000转5分钟。离心机以15,700xg xg离心5分钟，并用5微升上清液每50μlPCR反应。 注意:不要悬浮液。细胞碎片需要留在管的底部。 </li></ol></li><li>验证突变<ol><li>利用引物设计软件（如:引物），它涵盖了淘汰赛区域设计引物。设计引物起始于淘汰赛地区〜200 bp的两侧。 注意:引物用于验证cpcC1C2突变体在表2中概述和被称为cpcC1C2for和cpcC1C2rev。 </li><li>扩增使用程序由95℃的初始变性步骤中2分钟，35轮95℃1分钟，60℃1分钟，72℃每序列kb的1分钟，随后一个产品5分钟的72℃最后延伸步骤。包括野生型对照。这通常给予一致的产品。 </li><li>验证通过凝胶电泳基因型。标记基因敲除的转化体将呈现〜4kb的（从左边两个和右侧片段加上NPT1 /的sacB盒0.2 kb的）和不存在的野生型带（ 图2）的频带。 注意:在某些情况下，一个〜4kb的频带中没有明显的突变体观察到的由于大尺寸该PCR产物。但是，如果没有则观察到对应于野生型的预期大小的带通常该菌株是一个显着的敲除。 </li></ol></li><li>如果野生型带仍然存在，然后重新条纹上的应变新鲜BG11 +卡那霉素（30微克/毫升）琼脂制版及重复PCR。重复重新划线过程，直到使得没有野生型带在PCR反应中观察到的突变体是分开。 注意:卡那霉素的量增加至50微克/ ml的浓度，然后100微克/毫升，有时为了充分偏析显着的突变是必不可少的。 </li><li>如果应变显示了通过PCR有明显的突变配置文件，然​​后重新上连胜新鲜BG11 +卡那霉素（30微克/毫升）琼脂平板上。使用该菌株生成无人盯防的淘汰赛。 注意:该协议可以被用于产生与只抗生素抗性盒标记的突变体，即通过从pUC18K 20只与NPT1盒更换的左，右片段之间的NPT1 /的sacB盒。 </li></ol> 5.代无标记蓝藻突变体<ol><li>通过接种环全C的设置标记基因敲除的新鲜培养厄尔成30-50毫升BG11培养基。生长2-3天的文化OD 750nm的 = 0.2〜0.6。 </li><li>离心机将10ml培养物2300×g离心5分钟并弃上清。以BG11培养基洗一次。 注意:不要涡旋，因为这可能会导致菌毛这对DNA的摄取必需的损失悬浮细胞。轻柔吹打悬浮细胞。 </li><li>添加BG11到200微升的最终体积。转移细胞到14毫升的圆底管中。 </li><li>添加1微克质粒B核酸向细胞通过轻轻敲击混合。 </li><li>孵育样品4-6小时。水平放置管子下来。 注:细胞可以通过点击每1-2小时进行简单混合，但是这不是必不可少的。样品可以被放置在振荡培养箱尽管这并不提高效率。 </li><li>添加1.8毫升BG11培养基中孵育样品，共4天振荡培养。这是足够的时间，以允许在多个染色体副本发生重组。 </li><li>上BG11 / 5％蔗糖琼脂平板转化混合物的板等分试样。板50微升，10微升和每琼脂板1微升。如果一个殖民地的草坪出现在所有这些琼脂平板上稀释新鲜平板解决方案进一步和分装。经过大约7天后殖民地应该是可见的。 </li><li>补丁琼脂平板上首次+ BG11卡那霉素（30微克/毫升）30-50单个菌落和BG11 / 5％蔗糖琼脂平板上第二，使用钝端牙签。生长在BG11 / 5％蔗糖板但不BG11 +卡那霉素板块都是潜在的无标记击倒任何细菌。细菌在两个平板上生长很可能是蔗糖抗性由于sacB基因的突变。 </li><li>验证使用相同的引物和方法，被用于检查标记击倒未标记击倒。 例如 cpcC1C2for和cpcC1C2rev（ 表2）用于检验cpcC1C2无标记基因敲除。一个没有标记的淘汰赛将显示在琼脂糖凝胶相应吨乐队Ø野生型大小减去删除区（ 图2）。 </li><li>如果应变示出通过PCR未标记的突变体轮廓（步骤4.11.2）和凝胶电泳（ 图2），然后重新条纹上的新鲜BG11琼脂平板上无抗生素。 </li></ol> 6株长期储存<ol><li>通过接种环满格为30-50毫升BG11培养基一起成立了应变的一个新的文化。生长3-4天的文化OD 750nm的 = 0.4〜0.7。 </li><li>在约2ml BG11的BG11和悬浮细胞洗涤一次。 </li><li>添加0.8毫升浓缩细胞的一个管。然后加入0.2毫升80％过滤消毒的甘油。 </li><li>可选0.93毫升浓细胞添加到另一个管。添加0.07毫升DMSO中至该管。 注意:DMSO是有毒的，应使用适当的保护处理。 </li><li>储存在-80℃的两个管。为了重振株取出管刮去部分细胞用钝齿挑到琼脂平板无抗生素。条纹出正常使用无菌循环。 </li></ol><img alt="图1" src="/files/ftp_upload/54001/54001fig1highres.jpg" width="600" /> 图1:质粒构建新一代标记和未标记击倒， 如 cpcC1和cpcC2在集胞藻 （A） 集胞基因组，其中（B）cpcC1和cpcC2和邻近的基因位于的区域。以黑色突出显示是在突变要被删除的基因组的区域。这是通过PCR扩增的基因组中的（C）的网站。 5&#39;侧翼区（蓝色表示）和3&#39;侧翼区（以红色表示）被放大用限制性内切酶位点克隆到pUC19上。 5&#39;（或3&#39;）的侧翼区域被切除了的pUC19的并插入pUC19中+ 3&#39;（或59）侧翼区质粒产生质粒B.（D）从pUM24的NPT1 /卡带的sacB通过巴姆 HI消化切和插入的5&#39;和3&#39;的侧翼区产生质粒A. <a href="https://www.jove.com/files/ftp_upload/54001/54001fig1highres.jpg" target="_blank">请点击这里查看大图版本这个数字。</a>

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The authors declare that they have no competing financial interests.

在无人盯防的产生突变体的最重要的步骤是:1）精心设计载体，以确保只有目标区域被改变; 2）确保样品保持无菌，尤其是当上培养的蔗糖; 3）电镀转化最初在缺乏抗生素，随后加入琼脂加抗生素24小时后的BG11琼脂平板上标注的突变体产生的细胞; 4）培养标志着前4整天突变体上镀BG11加蔗糖琼脂平板:5）确保标记突变体是完全隔离和6）确认彻底突变株的基因型。对于这最后的步骤中，设计用于扩增...

蓝藻是地球上几乎每一个自然环境中的细菌的进化古老而多样化的门。在海洋生态系统中，他们都特别丰富，许多营养循环中发挥关键作用，约占一半的固碳1，在海洋中的大多数固氮2和数百万吨的油气产量3每年。叶绿体，细胞器负责在真核藻类和植物的光合作用，很可能已经从由宿主生物体4吞噬蓝藻演变。蓝藻已经证明是有用的模式生物进行光合作用，电子传递5和生化途径，其中许多植物中是保守的研究。此外蓝藻越来越多地被用于生产食品，生物燃料6，电力7和工业化合物8，由于它们的喜的水和CO 2 ghly有效转化生物质使用太阳能9。许多物种可以在非耕地用最少的营养物和海水中培养，这表明蓝藻可能在大规模生长，而不会影响农业生产。某些物种也是天然产品，包括抗真菌剂，抗菌剂和抗癌化合物10,11的源。 产生突变的能力是关键了解蓝藻光合作用，生物化学和生理学，以及必不可少的菌株用于工业用途的发展。大多数已发表的研究中产生遗传上通过抗生素抗性盒的插入修饰的菌株到感兴趣的部位。这限制了可被引入到菌株，因为只有少数的抗生素抗性盒可用于在蓝藻使用突变的数量。含有基因的菌株抗生素赋予重sistance不能用于工业生产中的开放的池塘，这很可能是唯一的成本效益的方法，以产生生物燃料和其它低价值产品12。未标记突变体的产生克服了这些限制。未标记的突变体不包含外源DNA的，除非有意包括在内，并且可以操作多次。因此，可以根据需要在一个菌株，以产生尽可能多的改变。此外，在变形例的网站的下游基因极性影响可以最小化，从而使机体13的更精确的修饰。 为了产生突变体菌株，自杀质粒在蓝藻染色体含有两个DNA片段相同的区域侧翼的基因被删除（称为5&#39;和3&#39;侧翼区）的第一构造。然后两个基因这些侧翼区域之间插入。其中的一个编码抗生素抗性蛋白;第二编码将SacB，这督促集体企业果聚糖蔗糖酶的化合物，赋予蔗糖敏感性。在此过程中的第一阶段中，标记的突变体，含有一些外源DNA 即菌株产生。该质粒构建体与蓝藻细胞混合和该DNA是由生物体吸收自然。转化体是由生长在含有适当的抗生素和通过PCR验证了突变体基因型琼脂平板上选择。自杀质粒不能感兴趣的菌株中复制。因此，任何抗生素抗性菌落，将导致从由此感兴趣的基因中插入染色体中的重组事件。以产生未标记突变体中，明显的突变，然后用仅包含5&#39;和3&#39;侧翼区的第二自杀质粒混合。然而，如果需要的外源DNA的插入，由5&#39;和3&#39;侧翼用含有这些DNA片段之间插入目的基因的盒的区域的质粒，可以使用。塞莱ction是，通过含有蔗糖琼脂平板上生长。作为当sacB基因产物被表达蔗糖是致死的细胞，该存在的唯一的细胞是其中已经发生了第二次重组事件，由此，蔗糖敏感性基因，除了在抗生素抗性基因，已被重组出的染色体上的质粒。作为重组交换的结果，侧翼区和它们之间的任何DNA被插入到染色体。 我们已经成功地使用这些方法来产生在集胞藻的同一菌株的多个染色体突变。 6803（以下简称为集胞藻属 ）13,14，引入单点突变到感兴趣13的基因和用于基因盒的表达。而代无标记击倒之前已经提供了工作在集胞藻 15,16，详细的方法证实，由辅助的关键步骤的视觉呈现，是不公开的。我们还以另一种模式蓝藻， 聚球藻申请代明显击倒同样的方法。 PCC7002（以下简称为聚球藻 ）。该协议提供了产生突变体和用于验证和存储这些菌株快速协议的清晰，简单的方法。

<table border="0" cellpadding="0" cellspacing="0" width="645">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:237px;">NaNO3</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">S5506</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgSO4.7H2O</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">230391</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CaCl2</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">C1016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">citric acid</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">C0759</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2EDTA</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">EDT002</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">H3BO3</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">339067</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MnCl2.4H2O</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">M3634</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">ZnSO4.7H2O</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">Z4750</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2MoO4.2H2O</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">331058</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CuSO4.5H2O</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">209198</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Co(NO3)2.6H2O</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">239267</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ferric ammonium citrate</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">F5879</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">K2HPO4</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">P3786</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2CO3</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">SODC001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TES</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">T1375</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaHCO3</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">SODH001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cyanocobalamin</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">47869</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2S2O3</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">72049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Bacto agar</td>
			<td style="width:123px;">BD</td>
			<td style="width:119px;">214010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Sucrose</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">SUC001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Petri dish 90 mm triple vented</td>
			<td style="width:123px;">Greiner</td>
			<td style="width:119px;">633185</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2 &#181;m filters</td>
			<td style="width:123px;">Sartorius</td>
			<td style="width:119px;">16534</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">100 mL conical flasks</td>
			<td style="width:123px;">Pyrex</td>
			<td style="width:119px;">CON004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Parafilm M 100 mm x38 m</td>
			<td style="width:123px;">Bemis</td>
			<td style="width:119px;">FIL003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phusion high fidelity DNA polymerase&#160;</td>
			<td style="width:123px;">Phusion</td>
			<td style="width:119px;">F-530</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td style="width:123px;">Melford</td>
			<td style="width:119px;">MB1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA purification kit&#160;</td>
			<td style="width:123px;">MoBio</td>
			<td style="width:119px;">12100-300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Restriction endonucleases</td>
			<td style="width:123px;">NEB</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">T4 ligase</td>
			<td style="width:123px;">Thermo Scientific</td>
			<td style="width:119px;">EL0011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Luria Bertani broth</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:119px;">12795-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">MES</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">M8250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Kanamycin sulfate</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">60615</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Ampicillin</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">A9518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">GeneJET plasmid miniprep kit</td>
			<td style="width:123px;">Thermo Scientific</td>
			<td style="width:119px;">K0503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">14 mL round-bottom tube</td>
			<td style="width:123px;">BD falcon</td>
			<td style="width:119px;">352059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">GoTaq G2 Flexi DNA polymerase</td>
			<td style="width:123px;">Promega</td>
			<td style="width:119px;">M7805</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">425-600 &#181;m glass beads</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">G8772</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glycerol</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">DMSO</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">D8418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Fluorescent bulbs</td>
			<td style="width:123px;">Gro-Lux</td>
			<td style="width:119px;">69</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">HT multitron photobioreactor</td>
			<td style="width:123px;">Infors</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of marked and markerless mutants in model cyanobacterial species

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation of cyanobacteria, especially model organisms such as Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002, is a key tool for both basic and applied research. Generation of unmarked mutants, whereby chromosomal alterations are introduced into a strain via insertion of an antibiotic resistance cassette (a manipulatable fragment of DNA containing one or more genes), followed by subsequent removal of this cassette using a negative selectable marker, is a particularly powerful technique. Unmarked mutants can be repeatedly genetically manipulated, allowing as many alterations to be introduced into a strain as desired. In addition, the absence of genes encoding antibiotic resistance proteins in the mutated strain is desirable, as it avoids the possibility of 'escape' of antibiotic resistant organisms into the environment. However, detailed methods for repeated rounds of genetic manipulation of cyanobacteria are not well described in the scientific literature. Here we provide a comprehensive description of this technique, which we have successfully used to generate mutants with multiple deletions, single point mutations within a gene of interest and insertion of novel gene cassettes.

质粒设计是成功产生两个标记和未标记的突变体是至关重要的。 图1给出了质粒A的一个例子和B用于产生在集胞藻属基因cpcC1和cpcC2 13的缺失突变体。在每种情况下，5&#39;和3&#39;侧翼区是约900-1,000碱基对。可用于降低侧翼区虽然我们已经试验成功最小的已经约500基点。质粒B也可以可以包含5&#39;和3&#39;〜1kb的侧翼区或天然基因序列的修...

Watch this Scientific Journal Video about Generation of Marked and Markerless Mutants in Model Cyanobacterial Species at JoVE.com

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Introducing multiple genomic alterations into cyanobacteria is an essential tool in the development of strains for industrial and basic research purposes. We describe a system for generating unmarked mutants in the model cyanobacterial species Synechocystis sp. PCC6803 and marked mutants in Synechococcus sp. PCC7002.

在型号蓝藻种类标记和无标记突变体的产生

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation ...

generation-marked-markerless-mutants-model-cyanobacterial

Research

JoVE Journal

Biology

11.9K 次观看.  University of Cambridge. Introducing multiple genomic alterations into cyanobacteria is an essential tool in the development of strains for industrial and basic research purposes. We describe a system for generating unmarked mutants in the model cyanobacterial species Synechocystis sp. PCC6803 and marked mutants in Synechococcus sp. PCC7002.

视频: Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the anatomy and connectivity of its nervous system has been determined from electron micrographs and refined by pharmacological assays. In this video, we describe how two complementary neural stimulants, an acetylcholinesterase inhibitor, called aldicarb, and a gamma-aminobutyric acid (GABA) receptor antagonist, called pentylenetetrazole (PTZ), may be employed to specifically characterize signaling at C. elegans neuromuscular junctions (NMJs) and facilitate our understanding of antagonistic neural circuits.
Of 302 C. elegans neurons, nineteen GABAergic D-type motor neurons innervate body wall muscles (BWMs), while four GABAergic neurons, called RMEs, innervate head muscles. Conversely, thirty-nine motor neurons express the excitatory neurotransmitter, acetylcholine (ACh), and antagonize GABA transmission at BWMs to coordinate locomotion. The antagonistic nature of GABAergic and cholinergic motor neurons at body wall NMJs was initially determined by laser ablation and later buttressed by aldicarb exposure. Acute aldicarb exposure results in a time-course or dose-responsive paralysis in wild-type worms. Yet, loss of excitatory ACh transmission confers resistance to aldicarb, as less ACh accumulates at worm NMJs, leading to less stimulation of BWMs. Resistance to aldicarb may be observed with ACh-specific or general synaptic function mutants. Consistent with antagonistic GABA and ACh transmission, loss of GABA transmission, or a failure to negatively regulate ACh release, confers hypersensitivity to aldicarb. Although aldicarb exposure has led to the isolation of numerous worm homologs of neurotransmission genes, aldicarb exposure alone cannot efficiently determine prevailing roles for genes and pathways in specific C. elegans motor neurons. For this purpose, we have introduced a complementary experimental approach, which uses PTZ.
Neurotransmission mutants display clear phenotypes, distinct from aldicarb-induced paralysis, in response to PTZ. Wild-type worms, as well as mutants with specific inabilities to release or receive ACh, do not show apparent sensitivity to PTZ. However, GABA mutants, as well as general synaptic function mutants, display anterior convulsions in a time-course or dose-responsive manner. Mutants that cannot negatively regulate general neurotransmitter release and, thus, secrete excessive amounts of ACh onto BWMs, become paralyzed on PTZ. The PTZ-induced phenotypes of discrete mutant classes indicate that a complementary approach with aldicarb and PTZ exposure paradigms in C. elegans may accelerate our understanding of neurotransmission. Moreover, videos demonstrating how we perform pharmacological assays should establish consistent methods for C. elegans research.

This video demonstrates how to employ two neural stimulants, aldicarb and pentylenetetrazole (PTZ), in complementary ways to study synaptic function in the nematode, C. elegans. This complementary approach may also be used to shed light on evolutionarily conserved mechanisms for modulating neuronal synchrony and has implications for epilepsy and seizures.

为线虫突触传递的突变体的药理特性的范式

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the ...

科研

生物学

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Neurocircuit检测癫痫发作癫痫突变果蝇</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

在斑马鱼和镜头移植在眼突变分析中的应用

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

一种无创性的发采样技术，以获得难以捉摸的小型兽类高质量的DNA

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

侵入细胞DISC1蛋白种类的产生，纯化及表征

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

检测，可视化和定量活性氧的生成在变形虫模型系统

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

快速，高效的斑马鱼基因分型采用PCR与高分辨率熔体分析

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

从人类体细胞与仙台病毒高效的新一代人类诱导多能干细胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

使用两步PCR和下一代16S rRNA基因测序进行微生物群分析

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...