这项工作是由乔治亚癌症联盟授予- R9028支持。

理由利用使用的Thermo Scientific Dharmacon寡核苷酸，50到80个碱基，在这里我们提出一个过程，基于一个基因校正实验，寡核苷酸可以遗传信息转移到酵母细胞的基因组DNA后，退火目标染色体DNA。通过寡核苷酸的成功目标是打进显示预期的表型酵母殖民地的外观。如果所需的基因改造进行在寡核苷酸序列中的一个或多个核苷酸，遗传信息流直接从RNA道染色体的目标DNA，因此，在染色体水平在定位过程中的RNA / DNA杂交的形式。一个的Thermo Scientific Dharmacon的RNA含寡核苷酸/ S可以作为模板，通过DNA修复合成基因打靶或可以嵌入到DNA为模板，在DNA复制服务。在这些实验中，我们使用的酵母/酵母/真核细胞模型系统，然而，类似的方法还可以应用在其他生物体或细胞类型。在这个视频中，我们使用热科学Dharmacon寡核苷酸的同源性设计目标酵母的基因组位点，并包含在中间的一个核苷酸，以正确的靶基因的突变。改造后的RNA含有寡，我们观察到目标站点的修正，在一定的频率，这表明，道寡核苷酸的RNA可以纳入染色体DNA作为DNA合成的模板，在DNA复制。内的RNA道进行稳定的遗传信息传送到下面的单元格几代人。在这里，我们描述了酵母细胞转化的Thermo Scientific Dharmacon RNA含寡核苷酸和方法步骤，以检测RNA信息转移到染色体DNA。 答:在本实验中使用的RNA含寡糖的设计我们设计的一种RNA寡核苷酸，纠正在酵母的遗传缺陷s 酵母染色体DNA trp5轨迹。在协议中使用的酵母菌株包含一个突变 trp5基因两碱基缺失和一个无义突变（图1A） 。这种酵母是一种色氨酸营养缺陷型突变体，并不构成没有色氨酸媒体上的殖民地。在酵母基因组数据库（http://www.yeastgenome.org/）中的TRP5基因的DNA序列是可用的。的Thermo Scientific Dharmacon RNA含寡核苷酸在本议定书中使用一个65 - MER与DNA插入一个2基地，旨在纠正的缺失突变，旨在纠正 trp5等位基因（图1A）无义突变的核苷酸。寡核苷酸序列设计如下: 5&#39; - AAAAGGGTTTTGATGAAGCTGTCG - CG - GATCCCACATTCTG - RG - GAAGACTTCAAATCCTTGTATTCT。这是合成寡核苷酸的Thermo Scientific Dharmacon（拉斐特，CO）在200纳米尺度，是脱盐，deprotected没有页面净化使用。 B.制备酵母转化RNA含有寡糖（Storici 等修改，2007年1） <ol><li>擦拭将用于实验，包括寡管，移液器，旋涡，衣架，实验区，并与核糖核酸酶净化解决方案，以消除潜在的RNase污染的一切开始之前研究员戴手套的材料。使用无RNase水，化学试剂，试管和枪头在所有步骤。在这个实验中的每一个步骤应该是无RNase。 </li><li>重悬的Thermo Scientific Dharmacon RNA含有寡收到该公司以250 pmoles /μL无RNase的水涡大力溶解沉淀原液。储存在-80 ° C。 </li><li>改造前，立即在冰上解冻的RNA寡核苷酸和无RNase在无RNase管的水稀释50 pmoles /μL。每个转换需要1 nmole含有RNA的寡核苷酸。 </li><li>变性的选择金额在100℃2分钟加热块含有RNA的寡核苷酸，以消除对寡核苷酸二级结构。 </li><li>变性后立即放置在冰上管。在冰上保持到转型。 </li><li>在实验中控制了相应的DNA只寡核苷酸用于。这寡核苷酸是从-20 ° C解冻，并转变为寡核苷酸的RNA含准备，如上面所述。 </li></ol> C.酵母细胞的转化，使用RNA含有寡糖<ol><li> trp5突变的酵母细胞接种5毫升丰富的YPD液体培养基中，成长在30 ° C过夜（见材料）。 </li><li>转移到YPD液体培养基中50毫升1.5毫升的隔夜文化。 </li><li>在30℃摇床（225转）4H中孵育细胞。 </li><li>准备解决方案1紧接在无RNase管改造和解决方案2（见材料）。 </li><li>将细胞培养50毫升无RNase管，并在3000转，对应1562年克的2分钟，旋转。细胞沉淀的沉淀约。 0.3厘米3。 </li><li>取出50毫升无RNase水，并在2分钟3000转的旋转上清，洗涤细胞。 </li><li> 5次重复第6步，培养基和核糖核酸酶，可以在中尽可能摆脱。 </li><li>取出5毫升的解决方案1和2分钟的转速在3000旋转上清，悬浮细胞。 </li><li>删除的解决方案1 ​​250μL上清和悬浮细胞。这种细胞的数量是足够7-8转换。 </li><li>分装50μL细胞悬液，在无RNase离心管，加入20μl的RNA含寡工作的解决方案（1 nmole），或20μL的DNA寡核苷酸工作的解决方案（1 nmole），或20μL无菌水没有为阴性对照寡核苷酸。每个转化反应，然后加入300μL的解决方案2。有没有需要添加在转型过程中的鲑鱼精子DNA，寡核苷酸为载体本身的行为。 </li><li>涡大力混合元件均匀。 </li><li> 30 ° C时30分在摇床孵育转化反应。 </li><li>热休克在42 ° C为15分钟。 </li><li>在5000转，相当于2236克4分钟，离心细胞。 </li><li>取出100μL无菌水上清和悬浮细胞。 </li><li>这个细胞悬液等分，并用无菌水稀释十万倍和YPD使用约板的板细胞。 15无菌玻璃珠和孵化在30 ° C时2天。 </li><li>板从每改造一个合成色氨酸（SC -色氨酸）使用约不完整的固体培养基的Petri菜反应中的所有重悬细胞。 15无菌玻璃珠和孵化在30 ° C时4-5天（图2）。 </li></ol> D.分析RNA含寡糖基因校正<ol><li>计数的选择性培养基（图2），以及YPD培养基上生长的菌落计算的RNA寡核苷酸基因校正频率，唯一的DNA寡核苷酸和无寡控制数量。比较后所得的数字。 trp5两个碱基的缺失和无义突变的等位基因的自发回归率小于10-9使用的酵母菌株，因此，我们期望寡核苷酸时没有添加到细胞选择性培养基上无菌落形成。 </li><li>随机挑选了几个数转化到YPD培养基殖民地获得单菌落分离。等待两天菌落生长，然后采取几个单菌落（至少5次），使YPD在选择性培养基上的补丁。 </li><li>设计一对引物扩增区域（250-1,000 BP）的菌落PCR（图1B），含有RNA的寡核苷酸的目标。菌落PCR的步骤如下（Storici和雷斯尼克修改，2006 年 2） 。 <ol><li>从单个修补程序的50μL水悬浮细胞（大约1 mm3的），并添加1个单位的lyticase。在室温下孵育10分钟，随后在一个加热块孵化100 ° C为5分钟，打破了细胞和基因组DNA在溶液中释放。 </li><li> PCR条件:PCR反应包括10μL的细胞再悬浮溶液，正向和反向引物各50 pmoles，1μL10毫米dNTPs，1个单位的Taq DNA聚合酶，5μL10倍的缓冲和调整用无菌水到终体积50μL。 PCR程序是3分，30个循环95章第30 ° C °，55章第30，在72分钟和1℃;的最后72℃延伸7分钟时间° C，然后样品在95℃保存在4 ° C。这种反应的一个延伸时间是1分/ KB假设。 </li><li>继PCR检测，样品的1％或2％（取决于预期大小的PCR产物）观察PCR产物琼脂糖凝胶（图3）上运行。 </li></ol></li><li>如果遗传信息的RNA含有寡核苷酸转移产生的一个新的限制在酵母基因组的目标区域网站（图1B），它是可以用适当的限制性内切酶消化PCR产物，以核实信息的正确传输。如果没有任何限制的网​​站是由RNA含寡核苷酸的生成，请转至步骤6。精华PCR产物使用特定的限制性内切酶。消化反应包括6μLPCR产物，缓冲区，牛血清白蛋白（可能不需要某些酶，所用的酶的指令），限制性内切酶0.5μL，无菌水15μL。样品孵育的温度所用的酶的特定的1小时。 </li><li>运行未消化的样本连同同一行2％琼脂糖凝胶上的消化样品观察遗传修饰的RNA的RNA含寡核苷酸（图3）道转移。 </li><li>使用PCR纯化试剂盒纯化PCR产物和DNA测序作好准备。提交样本用于放大的产品相同的引物序列。 </li><li>分析DNA测序结果，使用软件，它允许多个序列与选择的参考序列（图4）对齐。 </li></ol> E.碱处理RNA含寡核苷酸（图5） <ol><li>对于每一个反应，加入到1.5 ml管nmole 1 RNA含寡核苷酸，或DNA的寡核苷酸（4 250 pmolesμL/μL原液）。 </li><li>加入4μL的1 M氢氧化钠进行水解，或另外添加4μLH 2 O为阴性对照，并在65℃水浴中孵育1小时然后将冰水浴。 </li><li>与2μL1.2 M盐酸，1中号的Tris - HCl，4μLH 2 O 4μL，或H 2 O和另外6μL，4μL1米的Tris - HCl为阴性对照压制。在冰上保持到转型。 </li></ol><img src="/files/ftp_upload/2152/2152fig1.jpg" alt="图1" /> 图1。有缺陷的trp5基因和基因的RNA含有寡纠正TRP5示意图一）trp5突变基因包含2碱基缺失（黑色三角形）和1基义突变（星号）。的单链RNA含插入2基"（蓝色回路）和1 - RNA的碱基替换（红色矩形）生成一个 Van91我限制性内切酶（支架）寡核苷酸酵母细胞转化为正确的遗传缺陷的trp5基因。 b）在TRP5基因是由RNA含寡核苷酸（蓝色矩形表示更正基地）修复，Van91我的网站是到TRP5基因产生的。一个278 bp的片段，包括只有一个Van91我TRP5基因的酶切位点，是PCR扩增，由一对引物（P1和P2）。 P1和P2的Van91 PCR产物消化后产生的177 bp和101 bp的片段，我也所示。 <img src="/files/ftp_upload/2152/2152fig2.jpg" alt="图2" /> 图2。含有的RNA寡核苷酸转化的结果。）无寡核苷酸转化的酵母细胞不形成SC -色氨酸中的任何殖民地，看到板AB 。 B）板CG显示酵母殖民地SC -色氨酸越来越多后，细胞转化与1 RNA含寡nmole。 C）板HL显示酵母菌菌落SC -色氨酸越来越多后，细胞转化与相应的DNA只控制寡核苷酸1 nmole。 <img src="/files/ftp_upload/2152/2152fig3.jpg" alt="图3" /> 图3。检测遗传信息转移到RNA的寡核苷酸经酶切消化的PCR扩增目标基因组区域。P1和P2扩增，并与 Van91我限制性内切酶消化PCR检测样本的2％琼脂糖凝胶电泳产品含有酵母染色体DNA。泳道1，8和15，尺寸为100，200，300，400和500 bp的DNA阶梯上标明左巷，2巷3，PCR扩增PCR 产物 trp5突变株的基因组DNA扩增trp5轨迹产品从激进党+唯一的DNA寡针对性的殖民地的基因组DNA扩增;巷4-7来自色氨酸+含有RNA的寡有针对性的殖民地的基因组DNA; 9日至14巷，Van91我酶切扩增，PCR产物从2至7车道的PCR产物。 Van91我的完整无缺的PCR产品在10至14车道的乐队的存在可以解释TRP5轨迹（CCACATTCTGG）部分消化。考虑到切割现场Van91我（CCANNNN NTGG）可以有多个序列，在TRP5生成的网站可能无法酶的最优化的目标。事实上，所有上述的PCR产物以下的DNA测序，我们没有额外的变化超出寡核苷酸进行（见图4）的检测。 Van91 P1和P2引物和消化产品带扩增278 bp的PCR乐队177 bp和101 bp的我都显示在右边的箭头。 <a href="http://www.jove.com/files/ftp_upload/2152/2152fig4.jpg"><img src="/files/ftp_upload/2152/2152fig4tmb.jpg" border="0" alt="图4Thumb" /></a> 图4。 DNA测序结果显示含有的RNA寡核苷酸基因校正。针对一个基因组区域）的DNA电泳的RNA含寡核苷酸。 RNA含寡核苷酸的RG的DNA序列（蓝色盒装）摹来自。此外盒装的是插入的CG基地。从所有其他的PCR产物的测序结果也为骑士AR为这一个远高于背景的荧光信号，。 B）的的色氨酸+的的DNA，只有寡核苷酸（T1），和的RNA，含寡核苷酸（T2 - T5）的顶部共识序列匹配的针对性和转化 TRP5地区的序列是比较的trp5突变细胞针对寡核苷酸（基因组DNA，前红色阴影部分）。修复RNA含在底部的寡核苷酸DNA插入双基和一个碱基（G）的RNA的替代标记为红色。地区盒装在蓝色与黄色渐变显示的RNA以及含有寡核苷酸的DNA寡核苷酸正是纠正在所有被测样品的缺失突变和无意义突变。虚线标记的RNA寡核苷酸序列的位置。 <img src="/files/ftp_upload/2152/2152fig5.jpg" alt="图5" /> 图5。碱处理可以防止基因的寡核苷酸的RNA含校正，由RNA含寡核苷酸（R）的转化频率是在红色的DNA寡核苷酸（四）所示，在蓝色酒吧的酒吧和所示。误差棒代表3寡核苷酸为每个独立的转换的平均值的标准误差。的DNA寡核苷酸显示没有用NaOH处理类似的转换频率。不同，改造RNA含寡核苷酸的频率下降到0以下用NaOH处理。因此，与RNA含寡核苷酸的准备不是只与DNA寡核苷酸污染，因此，观察到的转换频率是特定的RNA含有寡。

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	<li>Storici, F., Bebenek, K., Kunkel, T. A., Gordenin, D. A., Resnick, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-templated+DNA+repair.">RNA-templated DNA repair.</a> Nature. 447, 338-3341 (2007).</li><li>Storici, F., Resnick, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+delitto+perfetto+approach+to+in+vivo+site-directed+mutagenesis+and+chromosome+rearrangements+with+synthetic+oligonucleotides+in+yeast.">The delitto perfetto approach to in vivo site-directed mutagenesis and chromosome rearrangements with synthetic oligonucleotides in yeast.</a> Methods Enzymol. 409, 329-345 (2006).</li><li>Storici, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-mediated+DNA+modifications+and+RNA-templated+DNA+repair.">RNA-mediated DNA modifications and RNA-templated DNA repair.</a> Curr Opin Mol Ther. 10, 224-230 (2008).</li><li>Cerritelli, S. M., Crouch, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribonuclease+H:+the+enzymes+in+eukaryotes.">Ribonuclease H: the enzymes in eukaryotes.</a> FEBS J. 276, 1494-1505 (2009).</li></ol>

本文的视频制作赞助商赛默飞世尔科技，产生本出版物中使用的试剂和仪器。

事实上，RNA可以直接传输的遗传信息在细胞基因组DNA被发现利用使用合成的RNA含寡核苷酸（Dharmacon合成的寡核苷酸）1。这是原则的证明，细胞可以作为DNA合成的模板中使用含RNA分子或RNA的唯一序列。使用RNA，含有不仅导致示威从RNA遗传信息可直接传送，而不需要一个反转录DNA复制中间的基因组DNA寡核苷酸，但也认为RNA可以作为同源模板使用证明1,3的DNA损伤的修复。寡核苷酸的RNA只或只包含在DNA序列中嵌入一个单核苷酸，在这个例子中，我们这里介绍。 RNA含寡核苷酸具有一个嵌入式设计，以纠正基因缺陷 trp5等位基因在的无义突变的核苷酸。从核苷酸基因组DNA的遗传信息传递的是显示在表型的改变目标细胞（细胞的缺乏色氨酸的培养基上生长）。只有一个控制的DNA寡核苷酸与RNA含有寡基因校正的频率测量和比较。通过执行此基因在不同的单元格背景，在这里我们酵母基因变异各种校正实验中，我们可以检测因子/ s的具体影响，针对含有RNA的寡核苷酸。因此，我们可以揭示细胞如何调节的RNA / DNA杂交的稳定机制。 ，我们可以通过简单的设计含有RNA的寡核苷酸的不同变种确定一个特定的RNA为模板，在DNA修饰的道的可能性，我们可以判断嵌入到DNA的特定RNA序列的稳定性。此外，我们可以找出是什么在体内 RNA / DNA杂交相互作用的因素基板的首选。 虽然几个在体外研究中，主要是利用短RNA寡核苷酸，已进行了表征因素的功能，能够识别DNA的RNA混合，如核糖核酸 酶H酶4在体内核糖核酸酶H功能，以及其他可能影响的RNA / DNA杂交的稳定仍然大多是未知的蛋白质的身份。利用的可能性RNA包含一个相当长（50至80个碱基）和最优的质量的寡核苷酸（如包含的Thermo Scientific Dharmacon的RNA寡核苷酸）打开的方式直接在体内研究的分子过程的广泛细胞的兴趣。此视频，转换，利用RNA寡核苷酸要求基本上只有一个相比，使用DNA分子的改造，以防止RNase的降解一些额外的步骤。因此，改造使用的RNA寡核苷酸不限酵母系统，但可以适用于任何生物体或细胞类型的DNA寡核苷酸的转型是精通。 总之，针对细胞含有RNA寡核苷酸生成的RNA / DNA杂交和嵌入的DNA在细胞体内的核糖提供了机会。的稳定性，功能和在体内的这些后果产生的RNA / DNA杂交，可分析和特点，潜在的发现未知的DNA修复机制，揭示新的战略目标基因。

答:转换试剂和媒体（从Stor​​ici和雷斯尼克修改，2006年<ol><li> YPD（酵母蛋白胨葡萄糖）:1升，葡萄糖20克，20克大豆蛋白胨10克酵母提取物。添加15克琼脂，使YPD固体培养基。使用高压灭菌器前。在室温下储存。 </li><li>解决方案1:0.1的醋酸锂中号。改造前立即准备。解决方案1是一个可行的解决方案，因此，它是准备直接从粉末。没有股票的解决方案是。保持室温。 LiAc增加酵母细胞壁的通透性，DNA。 </li><li>解决方案2:0.1 M的醋酸锂和聚乙二醇4000的50％。 2解决方案是一个可行的解决方案，它是直接从粉末。没有股票的解决方案是准备。保持室温。 PEG的存款到酵母细胞中的寡核苷酸。 </li><li>含RNA寡核苷酸（的Thermo Scientific Dharmacon），50 80个碱基，脱盐，deprotected和非纯化。重悬，以250 pmoles /μL。储存在-80 ° C。 </li><li>唯一的DNA寡核苷酸，50 80个碱基，脱盐和非纯化。到50 pmoles /μL重悬。储存在-20 ° C。 </li><li> SC -色氨酸（合成完整的媒体缺乏色氨酸）固体介质。 </li><li>直径0.5厘米的玻璃珠，高压灭菌消毒。 </li><li>核糖核酸酶小康:核糖核酸酶净化解决方案。 </li><li> DNA酶/无RNase的，无菌的离心管。 </li><li> DNA酶/无RNase，无菌锥形管。 </li><li> DNA酶/无RNase，无菌气溶胶与ZAP枪头:1-200毫升，100〜1000毫升。 </li></ol> B.菌落PCR材料。 <ol><li> DNA引物，脱盐和非纯化。 50 pmoles /μL无菌水溶解。储存在-20 ° C。 </li><li> Taq DNA聚合酶，缓冲液，dNTPs。 </li><li> PCR管。 </li></ol> C. PCR纯化。 <ol><li> PCR纯化试剂盒。 </li></ol> D.凝胶电泳。 <ol><li>琼脂糖。 </li><li> 1 × TBE缓冲液（45毫米的Tris -硼酸盐和1毫米乙二胺四乙酸）从10倍TBE稀释。 </li><li>预染分子量标记。 </li><li> DNA上样染料。 </li></ol> E.酶切。 <ol><li>限制性内切酶，10X缓冲区，BSA。 </li></ol> F.碱含有的RNA寡核苷酸治疗。 <ol><li> 1 M的氢氧化钠溶液。 </li><li> 1.2 M的盐酸溶液。 </li><li> 1米的Tris - HCl，pH 7.4的解决方案。 </li></ol>

generation of rna/dna hybrids in genomic dna by transformation using rna-containing oligonucleotides

合成短核酸聚合物，寡核苷酸（寡核苷酸），是分子生物学的功能和最广泛的工具。寡核苷酸可以生产包含任何所需的DNA或RNA序列，可以准备，包括各种各样的基地和糖修改。此外，寡核苷酸，可以设计模仿特定的核酸改变，因此，可以作为服务的重要工具，探讨DNA损伤修复机制的影响。我们发现的Thermo Scientific Dharmacon RNA含有之间50和80个核苷酸长度的寡核苷酸可以在体内，职能和染色体RNA / DNA杂交的后果，并嵌入到DNA的核苷酸，特别适合于研究。可以很容易地形成的RNA / DNA杂交，在DNA复制，修复和转录，然而，很少已知的RNA / DNA杂交细胞和这些混合动力车可以在何种程度上影响细胞的遗传完整性的稳定。因此，含有RNA的寡核苷酸，代表一个完美的载体，引入染色体DNA的核苷酸，并产生选择长度和碱基组成的RNA / DNA杂交。在这里，我们现在到的真核细胞模型系统酵母/啤酒酵母/基因组的核苷酸掺入协议。然而，我们的实验室已利用的Thermo Scientific Dharmacon的RNA含寡核苷酸在染色体水平在不同的细胞系统产生的RNA / DNA杂交，从细菌到人类细胞。

Watch this Scientific Journal Video about Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides at JoVE.com

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

这项工作表明如何形成RNA / DNA杂交和染色体水平揭示基因组DNA在酵母细胞中的遗传信息从RNA转移。

改造使用含RNA寡核苷酸基因组DNA的RNA / DNA杂交一代

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

generation-rnadna-hybrids-genomic-dna-transformation-using-rna

Research

JoVE Journal

Biology

26.8K 次观看.  Georgia Institute of Technology. 这项工作表明如何形成RNA / DNA杂交和染色体水平揭示基因组DNA在酵母细胞中的遗传信息从RNA转移。

视频: Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

CRISPR/Cas9 和 SsODN 在人细胞中催化点突变修复功能的标准方法研究

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

科研

遗传学

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

发展的细胞类型特异性的抗艾滋病毒gp120基因的siRNA传递适配

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

免疫与感染

High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite coupling reactions with the precision and density of UV light reflected from micrometer-sized mirrors. Photolithography yields microarrays that can accommodate from hundreds of thousands up to several million different DNA sequences, 100-nt or longer, in only a few hours. With this very large sequence space, microarrays are ideal platforms for exploring the mechanisms of nucleic acid&#183;ligand interactions, which are particularly relevant in the case of RNA. We recently reported on the preparation of a new set of RNA phosphoramidites compatible with in situ photolithography and which were subsequently used to grow RNA oligonucleotides, homopolymers as well as mixed-base sequences. Here, we illustrate in detail the process of RNA microarray fabrication, from the experimental design, to instrumental setup, array synthesis, deprotection and final hybridization assay using a template 25mer sequence containing all four bases as an example. In parallel, we go beyond hybridization-based experiments and exploit microarray photolithography as an inexpensive gateway to complex nucleic acid libraries. To do so, high-density DNA microarrays are fabricated on a base-sensitive monomer that allows the DNA to be conveniently cleaved and retrieved after synthesis and deprotection. The fabrication protocol is optimized so as to limit the number of synthetic errors and to that effect, a layer of &#946;-carotene solution is introduced to absorb UV photons that may otherwise reflect back onto the synthesis substrates. We describe in a step-by-step manner the complete process of library preparation, from design to cleavage and quantification.

In this article, we present and discuss new developments in the synthesis and applications of nucleic acid microarrays fabricated in situ. Specifically, we show how the protocols for DNA synthesis can be extended to RNA and how microarrays can be used to create retrievable nucleic acid libraries.

高密度DNA和RNA微阵列.光刻合成、杂交和制备大型核酸库

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

通过显微注射卵母细胞生成转基因小鼠

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

发育生物学

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5&#39;-[CUA CGG AAU CAU]-3&#39; containing zero, one, or two modifications located at the C2&#39;-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2&#39;-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

This article provides a detailed procedure on the solid-phase synthesis, purification, and characterization of dodecamers of RNA modified at the C2&#39;-O-position. UV-vis and circular dichroism photometric analyses are used to quantify and characterize structural aspects, i.e., single-strands or double-strands.

用于固相合成含有2&#39;-<em&gt; 0</em通过圆二色性修饰和表征

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular ...

化学

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an upstream 3&#39; splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

We clone and analyze reporter genes generating circular RNAs. These reporter genes are larger than constructs to analyze linear splicing and contain Alu elements. To investigate the circular RNAs, the constructs are transfected into cells and resulting RNA is analyzed using RT-PCR after removal of linear RNA.

使用含有微型基因的Alu元素来分析圆形RNA

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

生物化学

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is applicable to a wide variety of organisms and is highly efficient in the species-rich order Coleoptera (beetles). Here, we summarize the necessary steps for developing this technique in a novel organism and illustrate its application to the different developmental stages of the aquatic diving beetle Thermonectus marmoratus. Target gene sequences can be obtained cost-effectively through the assembly of transcriptomes against a close relative with known genomics or de novo. Candidate gene cloning utilizes a specific cloning vector (the pCR4-TOPO plasmid), which allows the synthesis of double-stranded RNA (dsRNA) for any gene with the use of a single common primer. The synthesized dsRNA can be injected into either embryos for early developmental processes or larvae for later developmental processes. We then illustrate how RNAi can be injected into aquatic larvae using immobilization in agarose. To demonstrate the technique, we provide several examples of RNAi experiments, generating specific knockdowns with predicted phenotypes. Specifically, RNAi for the tanning gene laccase2 leads to cuticle lightening in both larvae and adults, and RNAi for the eye pigmentation gene white produces a lightening/lack of pigmentation in eye tubes. In addition, the knockdown of a key lens protein leads to larvae with optical deficiencies and a reduced ability to hunt prey. Combined, these results exemplify the power of RNAi as a tool for investigating both morphological patterning and behavioral traits in organisms with only transcriptomic databases.

RNA interference is a widely applicable, powerful technique for manipulating gene expression at specific developmental stages. Here, we describe the necessary steps for implementing this technique in the aquatic diving beetle Thermonectus marmoratus, from the acquisition of gene sequences to the knockdown of genes that affect structure or behavior.

水生甲壳虫中的RNA干扰是特定发育时间点操纵基因表达的有力工具

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is ...

环境科学

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work demonstrates that concepts from DNA nanotechnology can be used to program the enzymatic activity of the phage-derived T7 RNA polymerase (RNAP) and build scalable synthetic gene regulatory networks. First, an oligonucleotide-tethered T7 RNAP is engineered via expression of an N-terminally SNAP-tagged RNAP and subsequent chemical coupling of the SNAP-tag with a benzylguanine (BG)-modified oligonucleotide. Next, nucleic-acid strand displacement is used to program polymerase transcription on-demand. In addition, auxiliary nucleic acid assemblies can be used as &#34;artificial transcription factors&#34; to regulate the interactions between the DNA-programmed T7 RNAP with its DNA templates. This in vitro transcription regulatory mechanism can implement a variety of circuit behaviors such as digital logic, feedback, cascading, and multiplexing. The composability of this gene regulatory architecture facilitates design abstraction, standardization, and scaling. These features will enable the rapid prototyping of in vitro&#160;genetic devices for applications such as bio-sensing, disease detection, and data storage.

We describe the engineering of a novel DNA-tethered T7 RNA polymerase to regulate in vitro transcription reactions. We discuss the steps for protein synthesis and characterization, validate proof-of-concept transcriptional regulation, and discuss its applications in molecular computing, diagnostics, and molecular information processing.

DNA系留RNA聚合酶用于可编程体外转录和分子计算

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work ...

生物工程

Chemical Triphosphorylation of Oligonucleotides

The 5&#8242;-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of oligonucleotides in biotechnology and synthetic biology. Oligonucleotide 5&#8242;-triphosphates have historically been prepared in vitro by enzymatic methods. However, these methods are limited to natural RNA oligonucleotides, have strong sequence preferences, and tend to produce heterogeneous products. New methods of chemical triphosphorylation complement both the reduced cost of automated oligonucleotide synthesis by phosphoramidite chemistry and the diverse range of nucleotide modifications now available. Thus, the synthesis of oligonucleotide triphosphates of arbitrary sequence and length, and optionally containing various nonnatural modifications, is now accessible.
This paper presents the appropriate methods and techniques for chemical triphosphorylation of oligonucleotides using salicyl phosphorochloridite and pyrophosphate. This method uses commercially available reagents, is compatible with most oligonucleotides prepared by standard solid-phase synthesis methods, and can be completed in 2 h following oligonucleotide synthesis, before deprotection and purification. Two uses of chemically triphosphorylated oligonucleotides as substrates for catalytic RNA enzymes are demonstrated, including the synthesis of a mirror-image version of the hammerhead ribozyme from nonbiological L-RNA triphosphates.

Oligonucleotide 5&#8242;-triphosphates are ubiquitous components in essential biological pathways and have seen increasing use in biotechnology applications. Here, we describe techniques for the routine synthesis and purification of oligonucleotide 5&#8242;-triphosphates, starting from oligonucleotides prepared by standard automated synthesis techniques.

寡核苷酸的化学三磷酸化

The 5&#8242;-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em在原位</em&gt;改编为海洋软骨鱼类杂交整装胚胎发育和细胞组织学

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...