该项目是在巴登 - 符腾堡州，德国资助的欧洲社会基金。

1.增强质粒含有所需的编码DNA序列（CDS） <ol><li>预暖的SOC培养基，其中包括在转化试剂盒，室温并含有100μg/ ml氨苄青霉素至37℃的LB琼脂平板上。平衡的水浴中以42℃。 </li><li>解冻的化学组分E的一个小瓶大肠杆菌在冰上。 </li><li>添加1-5微升质粒（第10至100毫微克）含有CDS成化学成分E.小瓶大肠杆菌 ，轻轻混匀。不要吹打混合细胞。加入质粒后，通过敲击管轻轻拌匀。 </li><li>在冰上孵育混合物30分钟。 </li><li>热休克E.大肠杆菌 30秒在42℃的水浴中不摇动。 </li><li>放置在冰上小瓶2分钟。 </li><li>添加250μl的预热SOC培养基对大肠杆菌大肠杆菌和水平摇晃细菌在300rpm下进行1小时，在37℃的细菌培养箱中培养。 </li><li><html>散布加入100μl和150μl的从转化混合物在预热的LB琼脂平板上，倒置平板孵育过夜，在37℃。 </li><li>后菌落变得可见，接种从各板单菌落到含有5ml的LB培养基中以100μg/ ml的氨苄青霉素的15毫升的培养管中。过夜孵育它们在37℃下，以300rpm振荡培养，直到培养是在晚对数或固定相。 </li><li>对于长期储存的转化的大肠杆菌的大肠杆菌，移液管将75μl100％的甘油，放入离心管。加入225微升的细菌培养（冷冻股票将包含25％甘油）中，拌匀和离心管储存在-80℃。 </li><li>分离含有使用根据制造商的说明书的质粒纯化试剂盒所需的CDS的质粒。 </li><li>测定使用分光光度计质粒浓度。 </li><li>制备的20微升等分试样，并将其存储在-20℃下延长的时间。 </li></ol>插入质粒和_title“&gt; 2。扩增聚合酶链反应加聚T型尾翼（PCR） <ol><li>注：为了获得用于体外转录（IVT）的，感兴趣的CDS的，这里的eGFP，用PCR扩增的DNA模板。同时，聚T型尾翼120胸腺嘧啶（T），通过使用反向引物使用T 120分机加入到插入物。因此，所产生的mRNA得到聚A尾与IVT之后，定义的长度。 </li><li>制备PCR反应混合物中如表1中详述。 </li><li>调匀反应混合物。注：PCR产物可以在4-5，IVT反应中使用。以增加DNA模板量为IVT，PCR混合物的总体积可以增加。几个PCR试管各自含有100微升的PCR混合物可用于获得足够的产率的PCR产物。 </li><li>将PCR管中的热循环仪和用PCR循环方案（ 表2）中运行的PCR。 </li><li>清理P使用根据制造商的说明书和洗脱，用20微升不含核酸酶的水将DNA中的PCR纯化试剂盒CR反应。 </li><li>测量用分光光度计中的DNA的浓度。注：检测DNA浓度是约300微克/毫升。因此，预期的总量应为〜6微克。的DNA的其它蛋白质的量可以根据CDS的长度不同时，用于PCR的退火温度的引物，以及循环数质粒的量。 </li><li>冷冻在-20℃下将DNA很长一段时间，或者直接将其用于IVT。 </li></ol> 3.控制步骤：PCR产物的质量<ol><li>检查PCR产物经DNA凝胶电泳的质量。 </li><li>混合琼脂糖所需量与在烧瓶1×TBE。为1％的琼脂糖凝胶中，添加0.5克琼脂糖至50ml 1×TBE的。 </li><li>微波直到琼脂糖溶解。不要让它沸腾。确保琼脂糖完全在溶液中。 </li><li>添加5μl的核酸凝胶染料至50ml琼脂糖凝胶。 </li><li>倒胶至琼脂糖凝胶中，设置了梳理，并等待大约1小时，直到凝胶固化。 </li><li>取出梳子，并将凝胶中的凝胶托盘。 </li><li>混合的80-10,000 bp的DNA梯2微升（200毫微克）与200纳克的PCR产物的每一个与2μl的6×加样缓冲液中的12微升的总体积。 </li><li>谨慎地装载DNA样品放入琼脂糖凝胶的孔中。 </li><li>使用1×TBE为电泳缓冲液，运行在100伏的琼脂糖凝胶1小时。 </li><li>可视化的成像系统（ 图2）上的DNA条带。 </li></ol> 4. 体外转录（IVT） <ol><li>注：在PCR后，将质粒插入物扩增和聚T形尾序被添加。在IVT中，遗传信息转录的DNA的RNA。所产生的mRNA转录物被用于产生在细胞中的蛋白质。 </li><li>清洁工作区和移液器与核糖核酸酶ðecontamination解决方案。利用PCR洁净和无菌双过滤器枪头，并经常更换手套，以减少污染与RNA酶反应的混合。 </li><li>准备在NTP /帽类似物的混合物，如表3所述。 </li><li>涡旋调匀的NTP /帽模拟混合后短暂离心。 </li><li>组装IVT反应混合物，如表4所述。 </li><li>轻轻上下吹打调匀的IVT反应混合物。然后离心PCR管短暂地在管的底部，收集混合物。 </li><li>在37℃下进行3小时的恒温。 注：最佳孵育时间取决于刀片和定模板的转录效率的长度。对于短插入物（&lt;500 nt）的，较长的温育时间可以是有利的。的时间 - 过程实验可以做到的，以确定最佳孵育时间为最大的产率。因此，设立IVT反应，对于为例即，对1，2，3，4，6小时，并温育过夜，并测定mRNA的量。 在体外转录为代的eGFP基因的动力学显示在图3。 </li><li>要删除的模板DNA，加入1微升的DNA酶（2 U /μL）到IVT反应混合物中，搅拌均匀，孵育15分钟，在37℃。 </li><li>纯化使用根据生产厂家的说明书一RNA纯化试剂盒的反应混合物中。从所述旋转柱的膜用40微升不含核酸酶的水洗脱修饰的mRNA的两倍。注：检测RNA浓度是大约1500微克/毫升。因此，预期的总量应为〜120微克。合成的mRNA的其它蛋白质的量可以不同，取决于CDS的长度和PCR产物的用于IVT的量。 </li></ol> 5.治疗纯化的mRNA与南极磷酸酶<ol><li>注：生成的mRNA与磷酸酶处理，除去5&#39;三磷酸盐，其可以通过RIG-I识别并导致免疫激活。另外，磷酸酶处理防止再环化中的自连接反应。 </li><li>加入9微升10倍南极磷酸酶反应缓冲液79微升纯化的mRNA的解决方案。随后，加入2微升南极磷酸酶（5 U /μL），轻轻混匀。孵育在37℃下将反应混合物搅拌30分钟。 </li><li>纯化使用根据生产厂家的说明书一RNA纯化试剂盒的反应混合物中。从旋转柱膜用50μl无核酸酶的水洗脱修饰的mRNA的两倍。 </li><li>测量用分光光度计改性mRNA的浓度。检查吸收在260nm / 280nm的比值（A 260 / A 280）为≥1.8的260比/ A 230是2.0，这表明纯度。 注：预期总收率应该是大约90微克，这是sufficie新台币约36基因转染。 </li><li>通过加入不含核酸酶的水调整的mRNA至100毫微克/微升的浓度。 </li><li>分装修饰的mRNA成所需的转染，并储存于-80℃下单次使用的等分试样。避免反复冻融循环。正确制备和储存的mRNA稳定多年。在工作​​过程中，始终保持修饰的mRNA在冰上。 </li></ol> 6.控制步骤：合成修饰mRNA的质量<ol><li>查改性mRNA的RNA电泳凝胶的质量。 </li><li>混合琼脂糖所需量与在烧瓶1×TBE。为1％的琼脂糖凝胶中，添加0.5克琼脂糖至50ml 1×TBE的。 </li><li>微波直到琼脂糖溶解。不要让它沸腾。确保琼脂糖完全在溶液中。 </li><li>添加5μl的核酸凝胶染料至50ml琼脂糖凝胶。 </li><li>倒胶成琼脂糖凝胶中，设置了梳理，并等待大约1小时，直到凝胶索利dified。 </li><li>取出梳子，并将凝胶中的凝胶托盘。 </li><li>混合3微升0.5-10 kb的RNA的梯子（3微克）与200纳克的合成的修饰mRNA的每个组中的10微升的总体积7微升加样缓冲液。加样缓冲液的组成在表5中描述。 </li><li>变性梯和mRNA样品在70℃下进行10分钟。 </li><li>谨慎地装载梯并在1％琼脂糖凝胶的mRNA样品。 </li><li> 1小时用1×TBE为电泳缓冲液进行电泳，在100伏。 </li><li>直观上的UV透射和照片使用凝胶记录系统（ 图4）的阶梯和mRNA条带。 </li></ol> 7.制备细胞用于转染<ol><li>板2×10 5个细胞（HEK293细胞）每12孔板井。 </li><li>在细胞培养箱中过夜培养的细胞，在37℃。注：在转染当天，将细胞应具有REAC建置80-90％汇合。 </li></ol> 8.执行细胞基因转染<ol><li>解冻修改后的表达。 </li><li>生成脂质复合物转染。 </li><li>添加25μl的修饰的mRNA（2.5微克）和2微升阳离子脂质转染试剂，以473微升的Opti-MEM（最低必需培养基）予还原的血清培养基中，以产生转染混合物的12孔板的一个孔中的转染。根据要转染井的数量扩展的卷。作为阴性对照，不表达制备转染混合物。 </li><li>通过移液轻轻混合组分。在室温下孵育转染混合物20分钟，以产生脂质复合物进行转染。 注：转染混合物不含抗生素。因此，一定要确保无菌处理，同时细胞。 </li><li>用500微升DPBS洗涤细胞/孔。 </li><li>微升转染混合物加入500一个良好12孔解放军TE。 </li><li>孵育细胞4小时，在37℃和5％的CO 2。 </li><li>吸去转染混合物，并加入1 ml的完整的细胞培养基的细胞中。 </li><li>孵育细胞24小时，在细胞培养箱中培养。 </li><li>进行荧光显微分析使用荧光显微镜（ 图5）。注意：为了检测在细胞中的其它蛋白质，其通过转染产生的与其他的mRNA比的eGFP编码mRNA的表达，该细胞可以培养在盖玻片上。然后将转染能进行和所述细胞可被染色的合适的抗体和通过荧光显微镜进行分析。 </li></ol>在细胞EGFP表达9.流式细胞仪分析<ol><li>从孔中除去细胞培养基，并用1毫升DPBS井洗各（无钙和镁）。加入500μl胰蛋白酶/ EDTA，每孔以从细胞培养板的表面分离的细胞。随后，加入500TNS微升每孔灭活胰蛋白酶。 </li><li>离心分离的细胞5分钟，在300×g离心室温。除去上清液只留下沉淀。重悬在150μl的细胞固定溶液中的细胞团块和细胞悬浮液转移到一个流式细胞管。 </li><li>分析eGFP的表达细胞的百分比，并使用几何平均值（荧光强度的几何平均值）（ 图6）的荧光强度。 </li></ol> 10.测量蛋白表达随着时间的推移<ol><li>作为另外在步骤8中所述的方法进行细胞的3个孔的12孔平板中的mRNA的转染，不加入mRNA的孵育3个孔的HEK293细胞与转染混合物。 </li><li>测量eGFP的1，2的表达，并且在转染后3天，以评估蛋白质表达（ 图7）的持续时间。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

mRNA的疗法在再生医学，治疗的疾病和免疫接种领域的巨大潜力。在这段视频中，我们展示了生产稳定，修饰诱导的细胞蛋白表达的mRNA。使用该协议，其他期望的mRNA可被生成。 在体外合成修饰mRNA的允许细胞的转染具有所需的mRNA诱导靶蛋白的表达。由此，所需的蛋白瞬时表达的生理条件下，直到外源递送的mRNA被完全降解。 在此视频中，绿色荧光蛋白的表达，证实了3?...

在细胞中，转录出信使RNA（mRNA）的与mRNA的下面翻译成所需蛋白质确保细胞的正常功能。遗传性或获得性遗传性疾病可导致合成蛋白质的不足，功能失调而引起严重的疾病。因此，一种新的治疗方法来诱导产生缺失或有缺陷的蛋白质是外源递送合成的修饰基因导入细胞，对于所期望的蛋白质编码。因此，细胞触发合成功能蛋白，它们通常不能生产或自然不会需要。使用这种方法，遗传性疾病可以通过引入基因的被纠正，对于有缺陷或缺失的蛋白1码。 mRNA的治疗也可以用于疫苗接种到synthetize蛋白质抗原，其是由肿瘤细胞或病原体表达的。从而，宿主的免疫系统可被激活，有效地消除肿瘤细胞或防止INFEctions 2,3。此外，近年来，基因被成功地用于产生诱导的多能干细胞（iPS细胞）。为了这个目的，成纤维细胞，转染的mRNA诱导重编程的表达因子4-6，并将其与在再生医学中的巨大潜力转化中的iPSC。 先前，使用常规的mRNA的低稳定性和强免疫原性有关。因此，常规的mRNA的临床应用受到限制。然而，胞苷和尿苷的取代5-甲基胞苷和假尿嘧啶mRNA分子通过Kariko和同事内呈现的mRNA分子稳定的生物流体中，并极大地降低了免疫激活7-10，它现在允许修饰mRNA的临床适用性。 使用合成产生的改性的mRNA，所需基因转录物可以暂时在体内 11交付或体外诱导蛋白表达。所引入的基因是由蜂窝翻译机制在生理条件下转化。由于缺乏整合到宿主细胞基因组相比，病毒的基因治疗载体，肿瘤发生的风险，防止12,13。因此，使用改性合成基因疗法将会得到更好的临床认可的未来。 在这里，我们描述了用于生产改性的mRNA（ 图1），转染细胞的mRNA和蛋白表达的转染细胞中的评价的详细协议。

<table border="0" cellpadding="0" cellspacing="0" width="1058">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Name of Material/ Equipment</td>
			<td style="width:157px;">Company</td>
			<td style="width:172px;">Catalog Number</td>
			<td style="width:435px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Cell culture</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, high glucose</td>
			<td>PAA</td>
			<td>E15-009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Life Technologies</td>
			<td>10500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin&#160;</td>
			<td style="width:157px;">PAA</td>
			<td>P11-010</td>
			<td>100x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine&#160;</td>
			<td>PAA</td>
			<td>M11-004</td>
			<td>200 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS without calcium and magnesium&#160;</td>
			<td>PAA</td>
			<td>E15-002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.04% Trypsin / 0.03% EDTA&#160;</td>
			<td>Promocell</td>
			<td>C-41020</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">TNS</td>
			<td>Promocell</td>
			<td>C-41120</td>
			<td style="width:435px;">Trypsin Neutralizing Solution, 0.05% trypsin inhibitor in 0.1% BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEK-293 cells</td>
			<td style="width:157px;">ATCC</td>
			<td>CRL-1573</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Consumables</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue culture plates, 12-well&#160;</td>
			<td>Corning</td>
			<td>3512</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Cell culture flask (75 cm2)&#160;</td>
			<td>Corning</td>
			<td>430641</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">DNase-and RNase-free 1.5 ml sterile microcentrifuge tubes&#160;</td>
			<td>Eppendorf</td>
			<td>0030 121.589</td>
			<td>Safe-Lock, Biopur</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 ml conical tubes&#160;</td>
			<td>greiner bio-one</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:295px;">PCR clean and sterile epT.I.P.S. dualfilter pipette tips&#160;</td>
			<td>Eppendorf</td>
			<td style="width:172px;">10 &#181;l: 022491202,&#160;&#160;&#160; 100 &#181;l: 022491237, 1000 &#181;l: 022491253</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryovial</td>
			<td>greiner bio-one</td>
			<td>122279-128</td>
			<td>Cryo.s&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">14 ml polypropylene round bottom tube for bacterial culture&#160;</td>
			<td>BD Falcon</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasmid amplification and purification</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pcDNA 3.3_eGFP Plasmid&#160;</td>
			<td style="width:157px;">Addgene</td>
			<td>26822</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">One Shot Top10 chemically component Escherichia coli&#160;</td>
			<td>Invitrogen</td>
			<td>C4040-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Sterile water (Ampuwa)</td>
			<td>Fresenius Kabi</td>
			<td>1636071</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">LB medium (Luria/Miller)&#160;</td>
			<td>Carl Roth</td>
			<td>X968.1</td>
			<td>Dissolve 25 g l-1 in sterile water.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">LB agar (Luria/Miller)&#160;</td>
			<td>Carl Roth</td>
			<td>X969.1</td>
			<td>Dissolve 40 g l-1 in sterile water.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin Ready Made Solution</td>
			<td>Sigma Aldrich</td>
			<td>A5354</td>
			<td>100 mg/ml&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Sigma Aldrich</td>
			<td>G2025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAprep Spin Miniprep Kit&#160;</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mRNA production</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HotStar HiFidelity Polymerase Kit&#160;</td>
			<td>Qiagen</td>
			<td>202602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick PCR Purification Kit&#160;</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEGAscript T7 Kit&#160;</td>
			<td>Life Technologies</td>
			<td>AM1334</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-Methylcytidine-5&#180;-triphosphate&#160;</td>
			<td>Trilink</td>
			<td>N1014</td>
			<td>5-Methyl-CTP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pseudouridine-5&#180;-triphosphate&#160;</td>
			<td>Trilink</td>
			<td>N1019</td>
			<td>Pseudo-UTP</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:295px;">3&#180;-O-Me-m7G(5&#180;)ppp(5`)G RNA cap structure analog&#160;</td>
			<td>New England Biolabs</td>
			<td>S1411L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RiboLock RNase Inhibitor</td>
			<td>Thermo Scientific</td>
			<td>EO0381</td>
			<td>40 U/&#181;l&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TURBO DNase</td>
			<td>Life Technologies</td>
			<td>AM1334</td>
			<td>2 U/&#181;l (from MEGAscript T7 Kit)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antarctic phosphatase&#160;</td>
			<td>New England Biolabs</td>
			<td>MO289S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNeasy mini kit&#160;</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNaseZap solution&#160;</td>
			<td>Life Technologies</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfection</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine 2000 Transfection Reagent&#160;</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td>Cationic lipid transfection reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Opti-MEM I Reduced Serum Media&#160;</td>
			<td>Invitrogen</td>
			<td>11058-021</td>
			<td style="width:435px;">Improved Minimal Essential Medium (MEM) with&#160; reduced Fetal Bovine Serum (FBS) supplementation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel electrophoresis</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelred Nucleic Acid Gel Stain</td>
			<td>Biotium</td>
			<td>41003</td>
			<td>10,000x in water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">peqGOLD Range Mix DNA-Ladder&#160;</td>
			<td>Peqlab</td>
			<td>25-2210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5-10 kb RNA Ladder&#160;</td>
			<td>Invitrogen</td>
			<td>15623-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1x TBE buffer</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow cytometry analyses</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellFIX (1x)</td>
			<td>BD Biosciences</td>
			<td>340181</td>
			<td>10x concentrate&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Primer for insert amplification and&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; poly (T) tail PCR</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forward Primer (HPLC-grade) 10 &#181;M</td>
			<td style="width:157px;">Ella Biotech</td>
			<td style="width:172px;"></td>
			<td>5&#180;-TTGGACCCTCGTACAGAAGCTAATACG-3&#180;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Reverse Primer (HPLC-grade) 10 &#181;M</td>
			<td style="width:157px;">Ella Biotech</td>
			<td style="width:172px;"></td>
			<td>5&#180;- T120-CTTCCTACTCAGGCTTTATTCAAAGACCA-3&#180;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;"></td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Cell incubator</td>
			<td>Binder</td>
			<td style="width:172px;"></td>
			<td>CO2 (5%) and O2 (20%)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CASY cell counter&#160;</td>
			<td>Sch&#228;rfe System</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;">Sterile workbench&#160;</td>
			<td style="width:157px;">BDK Luft-und Reinraumtecknik GmbH</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacterial incubator&#160;</td>
			<td>Incutec</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water bath</td>
			<td style="width:157px;"></td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ScanDrop spectrophotometer&#160;</td>
			<td>Analytic Jena</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR thermocycler&#160;</td>
			<td>Eppendorf</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge</td>
			<td>Eppendorf</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex</td>
			<td>peqlab</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermomixer</td>
			<td>Eppendorf</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel apparatus for electrophoresis&#160;</td>
			<td>Bio-Rad</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel documentation system&#160;</td>
			<td>Bio-Rad</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACScan System&#160;</td>
			<td>BD Biosciences</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescence microscope&#160;</td>
			<td>Nikon</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phase-contrast microscope&#160;</td>
			<td>Zeiss</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro synthesis of modified mrna for induction of protein expression in human cells

外源递送的期望的细胞编码合成的信使RNA（mRNA），用于诱导蛋白质合成的具有在再生医学，基础细胞生物学，治疗疾病，和细胞的重编程领域的巨大潜力。在这里，我们描述了一步步的协议，用于生成修改的表达与诱导蛋白表达的减少的免疫激活潜力和增加的稳定性，产生的mRNA的质量控制，细胞的转染mRNA和验证通过流式细胞术。达用的eGFP基因的单一转染后3天，在转染的HEK293细胞中产生绿色荧光蛋白。在这个视频文章中，绿色荧光蛋白的mRNA的合成被描述为一个例子。但是，该过程可以适用于生产其它所需的基因。采用改良的mRNA的合成，细胞可被诱导以瞬时表达所需的蛋白，它们通常不会表达。

使用含有的eGFP的CDS的一个的pCDNA 3.3质粒的修饰的eGFP基因的合成成立（ 图1）。通过PCR扩增插入物和聚T形拖尾之后，以大约1100碱基对长度的清晰条带被检测到（ 图2）。增加了IVT时间增加的mRNA（ 图3）的产量。 IVT之后，一个清晰的mRNA条带大约1100碱基对的长度被检测，对应于绿色荧光蛋白的mRNA的要生产的长度（ 图4）。 所产?...

Watch this Scientific Journal Video about In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells at JoVE.com

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

在这个视频文章中，我们描述了诱导细胞蛋白表达的体外合成修饰的mRNA。

在体外合成在人体细胞修饰蛋白表达的mRNA的感应

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

in-vitro-synthesis-modified-mrna-for-induction-protein-expression

Research

JoVE Journal

Biology

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

24.8K 次观看.  University Hospital Tuebingen. 在这个视频文章中，我们描述了诱导细胞蛋白表达的体外合成修饰的mRNA。 

视频: In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

在人类胚胎干细胞体外培养的标签用于磁共振成像

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

科研

生物学

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

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 ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

分析单个人类胚胎干细胞通过定量RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...