Name: analysis of rna processing reactions using cell free systems: 3' end cleavage of pre-mrna substrates in vitro
Uploaded: 2014-05-03T15:00:00
Description: Watch this Scientific Journal Video about Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro at JoVE.com

SV是感谢来自美国国立卫生研究院K22AI077353和Landenberger基金会的资助。 KR感激来自美国国立卫生研究院（5SC1GM083754）承认资金。

1，适应贴壁细胞悬浮成 （这是可选步骤。悬浮细胞通常做出更好的核提取物，但是也可以使用在平板上生长的细胞。） <ol><li>适应贴壁细胞使用Joklik改性的MEM补充有5-10％新生小牛血清或胎牛血清和1％L-谷氨酰胺生长在悬浮液：青霉素 - 链霉素。繁殖细胞转瓶在37℃的过滤器盖，用8％的CO 2。 </li><li>当适应细胞，开始用100毫升的500毫升旋转瓶中。保持最小的0.3×10 <su...

<ol>
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体外前体mRNA 3&#39;裂解反应，在HeLa细胞核提取物或从这些提取物分馏裂解因素进行，使识别的核心裂解因素，其主要复合物16-21。许多与这些因素相关的更多的蛋白质最近已确定了22个，并在体外的反应可能会继续阐明这些蛋白是如何有助于反应轻。也许是因为在体内的反应似乎是cotranscriptional 23，或者是因为一些影响因素可能提取和透析过程中丢失时...

最成熟的真核生物的RNA消息（mRNA的）的生物合成需要几个转录后修饰，如封盖，剪接和多聚腺苷酸化。这些修饰通常耦合，以确保正确的处理1，并强烈增加mRNA的稳定性。 由聚（A）聚合酶（PAP）2-4 3&#39;哺乳动物预mRNA的末端形成由新生RNA，随后加入腺苷酸残基到5的内切核苷酸裂解产生&#39;裂解产物。在哺乳动物中，断裂是由多组分蛋白复合物完成约800 kDa的，是组装在特定的预RNA序列。所述聚（A）信号序列，一个高度保守的典型六核苷酸序列AAUAAA，引导切割位点在约10-30个核苷酸的下游。这个网站是专门由切割和多聚腺苷酸化特异性因子（CPSF）及73 kD的亚基确认CSPF包含内切核酸酶的活性。裂解刺激因子（CSTF）结合更加堕落GU-或U丰富的元素序列中的poly（A）位点下游。还需要裂解是哺乳动物裂解因子I（CFIM）和哺乳动物裂解因子II（CFII）。 CFIM结合上游序列中的元素（使用）的具体UGUA（N）的网站，已经被定义为一些基因，似乎参与重要的生理过程5-8。 在体外实验中 ，RNA加工反应通过使用放射性标记的RNA底物9-12常用分析。这些可以通过从噬菌体启动子的T7或SP6失控转录来合成。当研究一种多聚腺苷酸化位点尚未之前其特征在于，它需要使用的基因组DNA，而不是基因产生的RNA底物，作为重要的下游序列中可能不存在于cDNA的。设计底物，包括至少150个核苷酸upstrea米和50从上成熟的mRNA切割位点/结束nt的下游。裂解产物迁移比基材快;然而，因为其他片段可以通过非特异性核酸酶作用而生成的，该反应的特异性具有由其上的正确处理的信号序列依赖性进行验证。因此，RNA底物具有在AAUAAA序列（ 例如，AAGAAA）的点突变可作为阴性对照的裂解反应。 由于少量的放射性标记的RNA被用于裂解反应中，存在于高丰度在大多数核提取核糖核酸酶可以是有问题的，并限制对提取物的制备起始材料的选择范围。 HeLa细胞中往往含有内源性核糖核酸酶水平低，因而在这些测定中表现良好。 在RNA底物在体内和体外的信使核糖核酸是紧接着的poly（A）另外，周四S中的裂解的中间不存在可检测量。因此，研究一个特定的RNA序列或涉及的裂解反应的蛋白质，实验是在防止多聚腺苷酸化的发生条件进行。有乳沟的多聚腺苷酸化无依赖性，反之亦然，所以人能阻止多聚腺苷酸化而不伤害裂解反应。因此，ATP被替换为缺少3&#39;羟基基团，以便只有一个单核苷酸可以在多聚（A）位点被结合并可以被检测到刚切割的RNA链终止类似物。 由于复杂性和这种类型的检测的特殊性程度高，我们描述了一个详细的视频协议由前体mRNA的体外切割/聚（A）机械研究核酸内切裂解。我们描述了如何准备主管核提取物，产生放射性标记的RNA底物，进行裂解反应，并分析和解释结果ING产品。 图1显示了HIV-1的前mRNA的3&#39;末端也可以用于裂解分析法进行编码的RNA底物的例子。艾滋病毒RNA基因组的3&#39;端是由许多重要的调控序列，如聚（A）位点，一个G+ü丰富的地区，以及使用的元素，这些都是必要的病毒mRNA转录13高效成熟。在这个例子中，我们所期望的输入RNA底物为338 nt的，一经裂解237 nt的。如果多聚腺苷酸化被允许发生，产品一抹黑将237和437个核苷酸之间被观察到。

<table border="0" cellpadding="0" cellspacing="0" width="757">
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			<td height="21" style="height:21px;width:216px;">1L Celstir Flask</td>
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			<td style="width:141px;">VWR</td>
			<td style="width:119px;">12621-076</td>
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			<td height="21" style="height:21px;width:216px;">Swinging Bucket Centrifuge</td>
			<td style="width:141px;">Beckman Coulter</td>
			<td style="width:119px;"></td>
			<td style="width:281px;">Allegra x-15R with SX4500 Rotor</td>
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			<td height="21" style="height:21px;width:216px;">Ultra Centrifuge</td>
			<td style="width:141px;">Beckman Coulter</td>
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			<td>Optima L-100 XP Ultra with SW41 Rotor</td>
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			<td height="21" style="height:21px;width:216px;">Table Top Centrifuge 5417R</td>
			<td style="width:141px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td>Refridgerated</td>
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			<td height="21" style="height:21px;width:216px;">Thermomixer incubator</td>
			<td style="width:141px;">Eppendorf</td>
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			<td height="21" style="height:21px;width:216px;">250ml Conicle Tubes</td>
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			<td style="width:119px;">430776</td>
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			<td height="21" style="height:21px;width:216px;">50ml Conicle Tubes</td>
			<td style="width:141px;">BD Falcon</td>
			<td style="width:119px;">352098</td>
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			<td height="21" style="height:21px;width:216px;">Ultra-clear Centrifuge Tubes</td>
			<td style="width:141px;">Beckman Coulter</td>
			<td style="width:119px;">344059</td>
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			<td height="21" style="height:21px;width:216px;">JOKLIK Modified MEM</td>
			<td style="width:141px;">Lonza</td>
			<td style="width:119px;">04-719Q</td>
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			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:141px;">Atlas&#160;</td>
			<td style="width:119px;">F-0500-A</td>
			<td>Heat inactivated</td>
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			<td style="width:141px;">Gemini Bio-Products</td>
			<td style="width:119px;">400-110</td>
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			<td height="21" style="height:21px;width:216px;">1M Tris-HCl pH 8.0</td>
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			<td style="width:119px;">46-031-CM</td>
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			<td height="21" style="height:21px;width:216px;">Magnesium Chloride</td>
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			<td height="21" style="height:21px;width:216px;">HEPES</td>
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			<td height="21" style="height:21px;width:216px;">DTT</td>
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			<td height="21" style="height:21px;width:216px;">Glycerol</td>
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			<td height="21" style="height:21px;width:216px;">5M Sodium Chloride Solution</td>
			<td style="width:141px;">Mediatech</td>
			<td style="width:119px;">46-032-CV</td>
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			<td height="21" style="height:21px;width:216px;">EDTA 0.5M Solution</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">E7889-100ml</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP120-500</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PMSF</td>
			<td style="width:141px;">Thermo Scientific</td>
			<td style="width:119px;">36978</td>
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			<td height="21" style="height:21px;width:216px;">Ammonium Sulfate</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">A702-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">cOmplete EDTA-Free Protease inhibitor cocktail tablets</td>
			<td style="width:141px;">Roche</td>
			<td style="width:119px;">04 693 132 001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Slide-A-Lyzer Dialysis Cassettes Kit</td>
			<td style="width:141px;">Thermo Scientific</td>
			<td style="width:119px;">66372</td>
			<td>MWCO 7000 0.5ml-3ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">15ml Dounce Tissue grinder set</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9938-1SET</td>
			<td>Different sizes available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Expand High Fidelity PCR Kit</td>
			<td style="width:141px;">Roche</td>
			<td style="width:119px;">11 732 650 001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10mM dNTP Mix</td>
			<td style="width:141px;">Invitrogen</td>
			<td style="width:119px;">Y02256</td>
			<td>10mM each nucleotide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MaxiScript SP6/T7 Kit</td>
			<td style="width:141px;">Ambion</td>
			<td style="width:119px;">AM1322</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">m7G(5&#39;)ppp(5&#39;) G RNA Cap</td>
			<td style="width:141px;">New England Biolabs</td>
			<td style="width:119px;">S1404S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Century Marker Template Plus</td>
			<td style="width:141px;">Ambion</td>
			<td style="width:119px;">AM7782</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Easytides UTP [alpha-32p]-250uCi</td>
			<td style="width:141px;">Perkin Elmer</td>
			<td style="width:119px;">BLU507H250UC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel loading buffer II</td>
			<td style="width:141px;">Ambion</td>
			<td style="width:119px;">8546G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DEPC treated water</td>
			<td style="width:141px;">Ambion</td>
			<td style="width:119px;">AM9906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10x TAE</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP13354</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10x TBE</td>
			<td style="width:141px;">Ameresco Life Sciences</td>
			<td style="width:119px;">0658-4L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10x PBS</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP399-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Urea</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP169-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ammonium Persulfate</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;">161-0700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TEMED</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP150-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ammonium Acetate</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">A637-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">40% 19:1 Acrylamide:Bis-acrylamide</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;">161-0144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycogen</td>
			<td style="width:141px;">Roche</td>
			<td style="width:119px;">10 901 393 001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100% Absolute Ethanol 200 Proof</td>
			<td style="width:141px;">Acros</td>
			<td style="width:119px;">61509-0040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acid Phenol-Chloroform</td>
			<td style="width:141px;">Ambion</td>
			<td style="width:119px;">9720</td>
			<td>For RNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scintilation Fluid</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">SX18-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rnase Inhibitor</td>
			<td style="width:141px;">Promega</td>
			<td style="width:119px;">N261B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Poly (Vinyl alcohol) PVA</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8136-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Creatine Phosphate</td>
			<td style="width:141px;">Calbiochem</td>
			<td style="width:119px;">2380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100mM dATP</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP2560-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SDS</td>
			<td style="width:141px;">Acros</td>
			<td style="width:119px;">23042-5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Proteinase K</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP1700-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adjustable Sequencing Unit</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z351881-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Binder Clips</td>
			<td style="width:141px;">Office Depot</td>
			<td style="width:119px;">838-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">20cmx42cm glass plates</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z352543</td>
			<td>1SET</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">20cmx22cm glass plates</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z35252-7</td>
			<td>1SET</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">20cmx42cm Aluminum Cooling Plates</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z352667</td>
			<td>1EA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.4mmx22cm Spacers</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z35230-6</td>
			<td>1SET</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.4mmx42cm Spacers</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z352314-1</td>
			<td>1SET</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">8-well Comb</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z35195-4</td>
			<td>1EA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">16-well Comb</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z351962</td>
			<td>1EA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">32-well Comb</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z351970</td>
			<td>1EA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel Repel Coating</td>
			<td style="width:141px;">C.B.S. Scientific</td>
			<td style="width:119px;">SGR-0401</td>
			<td>or SGR-0101 for individual bottle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel Loading Tips</td>
			<td style="width:141px;">Rainin</td>
			<td style="width:119px;">GT-10-4</td>
			<td>0.1-10uL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sequencing PowerPac HV</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;">PowerPac HV</td>
			<td>5000V/500mA/400W</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel Dryer Model 583</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;">Model 583</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hydrotech Vacuum Pump for gel dryer</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glogos II Glow-in-the-dark Markers</td>
			<td style="width:141px;">Agilent</td>
			<td style="width:119px;">420201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Film 8x10</td>
			<td style="width:141px;">Midsci</td>
			<td style="width:119px;">BX810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Film 14x17</td>
			<td style="width:141px;">Phenix</td>
			<td style="width:119px;">F-BX1417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Autoradiography Cassette</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">FBCA 1417</td>
			<td>8x10 size available</td>
		</tr>
	</tbody>
</table>

analysis of rna processing reactions using cell free systems: 3' end cleavage of pre-mrna substrates in vitro

哺乳动物mRNA的3&#39;末端不被由RNA聚合酶II（RNPII）突然终止转录的形成。相反，RNPII合成前体的mRNA之外的成熟RNA的末端，内切酶活性的主动过程，需要在特定的位点。前体RNA切割的CA二核苷酸后通常会发生10-30个核苷酸下游的共识polyA位点（AAUAAA）。从裂解复合物，约800 kDa的多因子蛋白复合物，蛋白质完成这个特定的核酸酶活性。特定的RNA序列的上游和多聚A位点的下游控制裂解复合物的募集。紧接在切割后，mRNA前由聚腺苷酸聚合酶（PAP）多聚腺苷酸化，以产生成熟的稳定的RNA消息。 一种RNA转录物的3&#39;端的处理可以使用移动的核提取物与特定的放射性标记的RNA底物进行研究。总之，长32 &lt;/ SUP&gt; P-标记的未裂解的前体RNA被孵育体外核提取物和裂解是通过凝胶电泳和放射自显影评估。当适当的切割发生在较短的5&#39;裂解产物进行检测和定量。在这里，我们描述了裂解分析法详细使用，作为一个例子，对HIV-1的mRNA的3&#39;末端加工。

将RNA的poly（A）的HIV-1的部位（ 图2）的裂解试验的代表性结果。我们可以观察到的未裂解的RNA底物，它是最慢迁移带在凝胶的顶部。具体的裂解产物是运行在上面的预期大小的凝胶更快最激烈的较短片段的带，并且是从mutpoly（A）的控制，其中包含多聚腺苷酸序列（mutPolyA）+ RNA的点突变的裂解测定法特别缺席基材。输入基板的降解产物有时可观察到。裂解活性的定量可以通过未切割?...

Watch this Scientific Journal Video about Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro at JoVE.com

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

RNA聚合酶II合成延伸超过所述成熟mRNA的3&#39;末端的前体RNA。的成熟RNA的末端cotranscriptionally生成的，在由RNA序列决定的一个部位，通过裂解复合物的内切核酸酶的活性。在这里，我们详细研究裂解反应在体外的方法。

前体mRNA底物的3&#39;末端切割：用无细胞体系中的RNA加工反应分析<em&gt;在体外</em

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

Research

JoVE Journal

Biology

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

视频人类胚胎干细胞克隆生长的生物信息学分析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

科研

生物学

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

MG53介导的细胞膜使用维修的可视化在体内在体外系统

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

单次读取和配对的最终从10微克总RNA的mRNA - Seq的Illumina的图书馆

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

在体外转录和Gaussia荧光素酶基因的上限由HeLa细胞转染

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

细胞特异性分析<em&gt;拟南芥</em&gt;叶用荧光激活细胞分选

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

CRISPR / CAS系统内切酶的底物生成

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

高分辨率基因表达谱RNA的合成，加工和衰变的新转录RNA在细胞培养的代谢标记

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene ...

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

基于海拉细胞自由表达系统中的表达<em&gt;疟原虫</em&gt;棒状体蛋白

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

无细胞化验，研究染色质解聚在有丝分裂的结束

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA结合蛋白的检测通过<em&gt;体外</em在脂肪细胞培养&gt; RNA下拉

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...