Name: efficient transcriptionally controlled plasmid expression system for investigation of the stability of mrna transcripts in primary alveolar epithelial cells
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Description: Watch this Scientific Journal Video about Efficient Transcriptionally Controlled Plasmid Expression System for Investigation of the Stability of mRNA Transcripts in Primary Alveolar Epithelial Cells a

Francis Migneault得到魁北克呼吸保健网络和加拿大卫生研究所肺活训练方案、法国社会研究中心的学生和苏佩里厄斯学院的学生奖学金的支持。蒙特利尔大学博士后。这项工作得到了戈塞林-拉玛雷临床研究主席和加拿大卫生研究所[YBMOP-79544]的支持。

所有动物程序都是根据加拿大动物护理理事会的指导方针进行的，并经蒙特利尔大学中心研究中心机构动物护理委员会批准。
1. 表达感兴趣的基因的响应质粒的设计与生成（GOI）
<ol><li>使用可诱导的四环素关闭矢量，如 pTRE-Tight。</li>
	<li>分析向量的 GOI 和多个克隆站点 （MCS） 的顺序，以标识在 GOI 内部不存在的 MCS 中的限制站点。</li>
	<li>分离原发性肺泡上皮细胞从雄性斯?...

<ol>
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原培养中奥维拉尔上皮细胞转染率低，是使用Tet-Off系统评估这些细胞mRNA稳定性的严重限制。然而，这一限制通过移液器电穿孔而克服，允许25-30%的转染效率（图1和图3）26。
笔录稳定性的测量对于理解给定mRNA的调制及其对细胞平衡和代谢的影响至关重要。GOI生物利用度的变化可能会改变翻译效率，并直接影响细?...

已经开发出几种技术来确定mRNA的半衰程。脉冲-追逐衰减技术，利用标记的mRNA，允许同时评估一个大池的mRNA，最小的细胞扰动。然而，这种方法不允许直接估计单个基因转录的半衰期，不能实施研究mRNA在刺激后与生长因子，ROS，报警，或炎症1的后转后调制。
使用转录抑制剂，如行为霉素D和β-阿马尼特，是一种相对简单的方法，测量mRNA降解动力学随着时间的推移。与以前的技术（即脉冲追逐）相比，这种方法的一个主要优点依赖于直接估计给定笔录的半衰程并比较不同处理如何影响其降解动力学的能力。然而，转录抑制剂对细胞生理学的显著有害影响是方法2的一个主要缺点。事实上，用这些药物抑制整个细胞转录素具有干扰mRNA稳定性关键元素（如微RNA（miRNA））的合成，以及RNA结合蛋白的表达和合成，这对mRNA降解和稳定性非常重要。"因此，这些药物对基因转录的严重扰动可以实际改变转录器的降解曲线。
启动器诱导系统是测量特定 mRNA 半衰程的第三种方法。此方法测量特定 mRNA 的降解方法与使用转录抑制剂的方法类似。经常使用两种类型的诱导系统：血清诱导的c-fos促进剂3和Tet-Off诱导系统4。使用 c-fos 系统时，无需使用对细胞有毒的转录抑制剂。但是，此方法需要细胞周期同步，从而阻止在相位5期间评估笔录的实际稳定性。相反，Tet-Off系统允许在合成四环素调节促进剂的控制下强烈表达感兴趣的基因（GOI）。此系统需要存在两个元素，必须共同转换到细胞中才能正常工作。第一个质粒（pTet-off）表示调节蛋白tTA-Adv，一种混合合成转录因子，由原核抑制剂TetR（来自大肠杆菌）融合到病毒蛋白HSV VP16的三个转录转活域。GOI在合成启动子（PTight）的控制下克隆成pTRE-Tight质粒，包括微小病毒（CMV）促进剂的最小序列，并融合到ttO运算符序列的七次重复。PTight下游基因的转录取决于TetR与TtO的相互作用。在四环素或其衍生物多西环素的存在下，TetR抑制器失去对ttO运算符的亲和力，导致转录4停止。Tet-Off系统的特点使其成为研究真核细胞中特定mRNA表达的理想模型，同时避免潜在的普列益效应，这种效应与真核细胞6中缺乏原核调控序列是次要的。通常，双稳定Tet-off细胞系（HEK 293，HeLa和PC12）用于集成调节器和响应质粒的副本，以方便获得可控基因表达7，8，9。
培养中的藻菌上皮细胞的几种模型被用来研究阿尔维拉尔上皮细胞的细胞和分子生物学。多年来，研究人员广泛利用了人类或啮齿动物原细胞10、11以及不朽的细胞系，如人类A549或大鼠RLE-6TN细胞12、13。虽然它们通常繁殖较少，更容易培养和转染，但原生培养中的奥维拉尔上皮细胞仍然是研究生理和病理条件下奥维拉尔上皮功能和功能障碍的黄金标准。事实上，不朽的细胞系，如A549细胞，没有表现出原细胞的复杂特性和表型，而原发培养物中的球状上皮细胞重述了食位细胞上皮的主要特性，特别是形成偏振和紧密屏障的能力14，15。不幸的是，这些细胞对传统的转染技术非常耐受，例如使用脂体，使得使用启动器诱导系统（如Tet-Off）非常困难。
mRNA的后转后调制是快速调节转录16基因表达的最有效方法之一。mRNA 3' 未翻译区域 （3' UTR） 在这一机制中起着重要的作用。已经表明，与5'UTR不同，3'UTR的长度与生物体的细胞和形态复杂性之间存在指数相关性。这种相关性表明，3'UTR，像mRNA编码区域一样，一直受到自然选择，以便在整个进化过程中进行越来越复杂的后转后调制17。3' UTR包含几个蛋白质和miRNA结合位点，影响笔录的稳定性和翻译。
在目前的工作中，我们开发了一个工具，用于调查高度保守的域在 GOI 3' UTR 中控制笔录稳定性的作用。我们专注于上皮钠通道，α亚基（#ENaC），在上皮生理学中起着关键作用18。在原发培养中，Alveolar上皮细胞成功地与Tet-Off系统的两个组件进行瞬态转染，这使得可以研究与使用转录抑制剂与其他协议相比，3'UTR在mRNA稳定性中的作用。开发了一种克隆策略，使用非内源表达表皮（V5）将GOI的表达与内源基因的表达区分开来。然后，使用移液器电穿孔技术将反应和调节质粒转移到电体上皮细胞中。随后，通过以不同时间间隔孵育多西环素细胞来测量笔录的表达。RT-qPCR使用经过转染的tTA-Ad mRNA产物对笔录的半衰程进行了评价，并采用经过改造的Cq方法进行规范化。通过我们的协议，我们为研究不同条件下成绩单的后笔调制和更详细地定义未翻译区域的参与提供了一种便捷的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
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			<td>Actinomycin D</td>
			<td>Sigma-Aldrich</td>
			<td>A9415</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma-Aldrich</td>
			<td>A1593</td>
			<td></td>
		</tr>
		<tr>
			<td>Bright-LineHemacytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform - Molecular biology grade</td>
			<td>Sigma-Aldrich</td>
			<td>C2432</td>
			<td></td>
		</tr>
		<tr>
			<td>ClaI</td>
			<td>New England Biolabs</td>
			<td>R0197S</td>
			<td></td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma-Aldrich</td>
			<td>C7698</td>
			<td></td>
		</tr>
		<tr>
			<td>DM IL LED Inverted Microscope with Phase Contrast</td>
			<td>Leica</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I, Amplification Grade</td>
			<td>Invitrogen</td>
			<td>18068015</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycycline hyclate</td>
			<td>Sigma-Aldrich</td>
			<td>D9891-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate-buffered Saline (D-PBS), without calcium and magnesium</td>
			<td>Wisent Bioproducts</td>
			<td>311-425-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol - Molecular biology grade</td>
			<td>Fisher Scientific</td>
			<td>BP2818100</td>
			<td></td>
		</tr>
		<tr>
			<td>Excella E25 ConsoleIncubatorShaker</td>
			<td>Eppendorf</td>
			<td>1220G76</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES pH 7.3</td>
			<td>Sigma-Aldrich</td>
			<td>H3784</td>
			<td></td>
		</tr>
		<tr>
			<td>Heracell 240i</td>
			<td>ThermoFisher Scientific</td>
			<td>51026420</td>
			<td></td>
		</tr>
		<tr>
			<td>iScript cDNA Synthesis Kit</td>
			<td>Bio-Rad Laboratories</td>
			<td>1708890</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol - Molecular biology grade</td>
			<td>Sigma-Aldrich</td>
			<td>I9516</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth (Lennox)</td>
			<td>Sigma-Aldrich</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth with agar (Lennox)</td>
			<td>Sigma-Aldrich</td>
			<td>L2897</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharides fromPseudomonas aeruginosa10</td>
			<td>Sigma-Aldrich</td>
			<td>L9143</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM, powder</td>
			<td>Gibco</td>
			<td>61100103</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp Optical 96-Well Reaction Plate</td>
			<td>Applied Biosystems</td>
			<td>N8010560</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp Optical Adhesive Film</td>
			<td>Applied Biosystems</td>
			<td>4360954</td>
			<td></td>
		</tr>
		<tr>
			<td>MSC-Advantage Class II Biological Safety Cabinets</td>
			<td>ThermoFisher Scientific</td>
			<td>51025413</td>
			<td></td>
		</tr>
		<tr>
			<td>Mupid-exU electrophoresis system</td>
			<td>Takara Bio</td>
			<td>AD140</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000c</td>
			<td>ThermoFisher Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Neon Transfection System 10 &#181;L Kit</td>
			<td>Invitrogen</td>
			<td>MPK1025</td>
			<td></td>
		</tr>
		<tr>
			<td>Neon Transfection System Starter Pack</td>
			<td>Invitrogen</td>
			<td>MPK5000S</td>
			<td></td>
		</tr>
		<tr>
			<td>NheI</td>
			<td>New England Biolabs</td>
			<td>R0131S</td>
			<td></td>
		</tr>
		<tr>
			<td>One Shot OmniMAX 2 T1RChemically CompetentE. coli</td>
			<td>Invitrogen</td>
			<td>C854003</td>
			<td></td>
		</tr>
		<tr>
			<td>pcDNA3 vector</td>
			<td>ThermoFisher Scientific</td>
			<td>V790-20</td>
			<td></td>
		</tr>
		<tr>
			<td>pcDNA3-EGFP plasmid</td>
			<td>Addgene</td>
			<td>13031</td>
			<td></td>
		</tr>
		<tr>
			<td>PlatinumTaqDNA Polymerase High Fidelity</td>
			<td>Invitrogen</td>
			<td>11304011</td>
			<td></td>
		</tr>
		<tr>
			<td>pTet-Off Advanced vector</td>
			<td>Takara Bio</td>
			<td>631070</td>
			<td></td>
		</tr>
		<tr>
			<td>pTRE-Tight vector</td>
			<td>Takara Bio</td>
			<td>631059</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified alveolar epithelial cells</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAEX II Gel Extraction Kit</td>
			<td>QIAGEN</td>
			<td>20021</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAGEN Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td>12162</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>QIAGEN</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantStudio 6 and 7 Flex Real-Time PCR System Software</td>
			<td>Applied Biosystems</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantStudio 6 Flex Real-Time PCR System, 96-well Fast</td>
			<td>Applied Biosystems</td>
			<td>4485697</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Rat TNF-alpha Protein</td>
			<td>R&#38;D Systems</td>
			<td>510-RT-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Septra</td>
			<td>Sigma-Aldrich</td>
			<td>A2487</td>
			<td></td>
		</tr>
		<tr>
			<td>Shrimp Alkaline Phosphatase (rSAP)</td>
			<td>New England Biolabs</td>
			<td>M0371S</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>SsoAdvanced Universal SYBR Green Supermix</td>
			<td>Bio-Rad Laboratories</td>
			<td>1725270</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript IV Reverse Transcriptase</td>
			<td>Invitrogen</td>
			<td>18090010</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>ThermoFisher Scientific</td>
			<td>EL0011</td>
			<td></td>
		</tr>
		<tr>
			<td>Tet System Approved FBS</td>
			<td>Takara Bio</td>
			<td>631367</td>
			<td></td>
		</tr>
		<tr>
			<td>Tobramycin</td>
			<td>Sigma-Aldrich</td>
			<td>T4014</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Invitrogen</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Gibco</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Agarose</td>
			<td>Invitrogen</td>
			<td>16500500</td>
			<td></td>
		</tr>
		<tr>
			<td>Water, Molecular biology Grade</td>
			<td>Wisent Bioproducts</td>
			<td>809-115-EL</td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient transcriptionally controlled plasmid expression system for investigation of the stability of mrna transcripts in primary alveolar epithelial cells

研究转录后调节对于理解给定信使RNA（mRNA）的调制及其对细胞平衡和代谢的影响至关重要。事实上，笔录表达的波动可以改变笔录的翻译效率，并最终改变笔录的细胞活动。已经开发了几种实验方法来研究mRNA的半衰程，尽管其中一些方法有局限性，无法对后笔后调制进行适当的研究。促进剂诱导系统可以在合成四环素调节促进剂的控制下表达感兴趣的基因。该方法允许在任何实验条件下对给定mRNA进行半衰期估计，而不会干扰细胞平衡。这种方法的一个主要缺点是转染细胞的必要性，这限制了在对传统转染技术高度抗性的分离原发细胞中使用这种技术。原发培养中的阿尔维拉尔上皮细胞已广泛用于研究阿尔维拉尔上皮细胞和分子生物学。原性球状细胞的独特特性和表型使得研究这些细胞中感兴趣的基因的转录后调制至关重要。因此，我们的目标是开发一种新的工具，以研究在原发培养中，对阿尔维拉尔上皮细胞感兴趣的mRNA的后笔后调制。我们设计了一种快速高效的瞬态转染方案，将转录控制的质粒表达系统插入原发性上皮细胞。这种克隆策略，使用病毒表位来标记构造，允许从内源性mRNA的构造表达式容易区分。使用经过修改的+量化周期 （Cq） 方法，可以以不同的时间间隔对笔录的表达式进行量化，以测量其半衰。我们的数据证明了这种新方法在研究原发性外皮细胞中各种病理生理条件下的后转后调节方面的效率。

该协议已成功用于生成Tet-off转录转录控制的质粒表达系统，以评估βENaC 3' UTR不同部分在主性外皮细胞的分录稳定性调制中的重要性。
该系统实施的第一步是建立一种快速、简单、高效的转染技术，用于初级培养中难以转染的食管上皮细胞。如图1所示，移液器电穿孔技术允许25-30%的转染效率，如荧光显?...

Watch this Scientific Journal Video about Efficient Transcriptionally Controlled Plasmid Expression System for Investigation of the Stability of mRNA Transcripts in Primary Alveolar Epithelial Cells a

Efficient Transcriptionally Controlled Plasmid Expression System for Investigation of the Stability of mRNA Transcripts in Primary Alveolar Epithelial Cells

在这里，我们提出了一个工具，可以用来研究原脑外皮细胞中笔录的后笔次调制，方法是使用可诱导的表达系统耦合到移液器电穿孔技术。

有效转录控制质粒表达系统，用于研究原发性奥子拉皮细胞中mRNA笔录稳定性

Studying posttranscriptional regulation is fundamental to understanding the modulation of a given messenger RNA (mRNA) and its impact on cell homeostasis ...

因此，我们的模型允许研究原培养中起白细胞中特定转录的转录后调制及其生理和病理生理条件。因此，原发性alvelar上皮细胞的瞬态转染对细胞生理和代谢的影响最小，与使用转录抑制剂的经典方案不同，具有明显的优势。为了产生表达感兴趣基因的响应质粒，必须分析该基因的序列和可受骗载体的多个克隆位点，以确定基因内部不存在的多个克隆位点中的识别序列。使用高保真 taq 聚合酶和标准重叠 PCR 技术，使用设计底像使用两个选定的限制性酶识别位点对感兴趣的基因进行侧翼处理。必须包括编码感兴趣基因上游V5表位序列，以区分转染基因与内源性表达的表达。突变体可以通过顺序删除来研究不同三个未翻译的正向区域对基因mRNA稳定性的影响，使用编码聚亚亚二聚亚化站点的反向底像，逐渐删除未翻译区域的三个主要端。最终，通过限制分析可以确认感兴趣的基因的插入。基因取向和在rtPCR期间可能引入的突变的缺失可以通过测序得到确认。对于原发性大鼠肺二型肺上皮细胞的转染，在100毫米培养皿中，以每平方厘米10至6个细胞的1倍至6个细胞在完全最低基本介质的情况下播种细胞。培养24小时在37摄氏度的5%二氧化碳与湿度。第二天，在一个新的12井板的每个井中加入500微升无抗生素的完全介质，并在37摄氏度下预热板30分钟。在孵育过程中，将一微克的反应质粒和一微克的调节载体添加到每井1.5毫升管中。在不加钙或镁的情况下，用每井10毫升的上皮细胞清洗，不带钙或镁，用每井5毫升0.05%的三头肌在细胞培养箱中治疗5毫升，每井0.05%的三头肌素2至4分钟。当细胞分离后，用每井10毫升的滴答声中和没有抗生素的，并收集细胞到一个新的50毫升管。用四毫升新鲜培养剂清洗盘子，收集任何剩余的细胞，通过离心沉淀细胞。将颗粒重新在一毫升 PBS 中，用于计数并再次离心细胞。将颗粒以每毫升再增缓冲液的四倍10的密度重新填充至第七细胞，并将四乘以十十的颗粒添加到每管质粒和载体中，并轻轻混合。将一根管子放在电穿孔装置中，用3.5毫升电解缓冲液填充管子。完全按下活塞，将镀金电极尖端插入移液器中，然后轻轻混合管内侧。小心地用移液器吸气细胞，并将移液器插入电穿孔站，直到听到咔嗒声。选择对起白体上皮细胞的适当电穿孔方案，然后按触摸屏上的"开始"。转染后立即取出移液器，将细胞转移到预热的12个井板的一个井中。当所有细胞都经过电分和镀，将板放在细胞培养培养箱中，在两天后用抗生素取代每井中的上经剂和完整的培养剂。通过荧光显微镜或流式细胞学使用对照载体观察到的EGFP的表达可以证实转染的成功。为了抑制感兴趣的基因的转录，转染后72小时用全米每井一毫升代替上流水，并辅以每毫升新鲜准备的副氧环素一微克。要评估感兴趣的基因的mRNA半寿命，将板返回细胞培养箱，从15分钟到6小时。用一毫升冰冷PBS洗好一口，然后用500微升的解液缓冲液从市售的酚氯仿RNA提取试剂盒中解压细胞，并在每个实验时间点轻轻摇动。然后，根据试剂盒指令分离RNA，通过230、260和280纳米的分光光度测量确定RNA的屈服度和纯度。要确定分离的RNA的稳定性，首先用无RNase无DNase一个来治疗每个样品中总RNA的一微克，以去除任何质粒DNA痕迹。使用市售的cDNA合成试剂盒，混合寡聚dT和随机六分位体底转，提高逆转录效率，根据制造商的说明将DNA耗尽的总RNA反向转录到cDNA中。将180微升分子生物级水加入反应混合物的20微升，以每微升浓度5南克稀释cDNA反应，并立即用新鲜分子生物学级水稀释cDNA混合物，达到每微升浓度1.25纳克。将网络绿色染料主混合5微升与分子生物学级水0.1微升、0.45微升7.5微摩尔前底、0.45微升7.5微摩尔反向底头、4微升（每微升cDNA）1.25纳米微升相结合，获得10微升的总反应量。将样品放在 qPCR 热循环器中，并使用指示的 qPCR 条件放大感兴趣的 V5 标记基因和四环素转活器推进安普利子，并生成高分辨率熔化曲线，以评估所需安普利翁的特定熔化温度，并确保没有噪声放大器峰值。这种移液器电穿孔技术可提高25-30%的转染效率，如荧光显微镜和流式细胞学检测的EGFP细胞的比例。在治疗期间的任何时间点，用每毫升多西环素一微克治疗的alvelar上皮细胞对内源性α Α RNA MRNA的表达没有显著影响。使用所演示的转录控制质粒表达系统，V5 alpha ENaC信号可以根据四环素转活器高级信号进行规范化，使用双增量定量循环方法确定转染效率。在四环素离系统的情况下，α ENaC mRNA 的半寿命比在四环素离系统存在时短七倍，这证实了阿提诺霉素 D 导致伪阿尔法 ENaC mRNA 稳定。此外，环异酰胺和肿瘤坏死因子α治疗显著降低了转录本的稳定性，而脂质多糖则不然。V5 alpha ENaC mRNA 稳定性的调制可根据三个未翻译区域的删除和包含区域而诱发显著变化。此外，与空PCDNA三质粒的转染相比，特定RNA结合蛋白的过表达会降低5-αENaC mRNA的稳定性。此工具将有助于查询对参与起维骨上皮功能及其生理或病理状况的关键基因的转录后调控的新见解。

Research

Education

JoVE Journal

JoVE Core

Molecular Biology

Genetics

Unit 1

Additional Roles of RNA

SCIENTISTS IN ACTION

RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing

角膜上皮伤口愈合过程中的热休克蛋白27的功能为基础的RNA干扰研究

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a ...

科研

遗传学

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

在人原代细胞培养生物钟基因表达及荷尔蒙分泌的并行测量

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

可诱导T7 RNA聚合酶介导的多基因表达系统，pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

Controllable Ion Channel Expression through Inducible Transient Transfection

通过诱导瞬时转染可控离子通道表达

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

体外生物荧光法对乳腺上皮细胞昼夜节律的表征

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to ...

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

昆虫细胞 (sf9) 外源基因在蛋白质功能检测中的瞬态表达

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

斑马斑马模型系统中 CRISPR/Cas9-generated 基因挖空的高效生产与鉴定

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

CRISPR 的染色质环结构重组

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Purification of Low-abundant Cells in the Drosophila Visual System

果蝇视觉系统中低丰细胞的纯化

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small ...