Name: isolation of active caenorhabditis elegans nuclear extract and reconstitution for in vitro transcription
Uploaded: 2021-08-11T14:00:00
Description: Watch this Scientific Journal Video about Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription at JoVE.com

这项工作得到了NIH MIRA赠款（R35GM124678给J. S.）的支持。

1. 媒体准备
<ol><li>按照制造商的说明准备无菌溶菌汤（LB）琼脂平板和液体培养基。</li>
	<li>条纹 大肠杆菌 （大肠杆菌）菌株OP50在LB琼脂平板上。将细菌条纹在37°C下孵育过夜。</li>
	<li>孵育后将 大肠杆菌 OP50条纹板储存在4°C。如果用parafilm包裹， 大肠杆菌 OP50板可以在4°C下安全储存2周，以防止水分流失。</li>
	<li>使用 表1中的配方准备2L线虫生长培养基（...

<ol>
	<li>Corsi, A. K., Wightman, B., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Transparent+window+into+biology:+A+on+Caenorhabditis+elegans.">A Transparent window into biology: A on Caenorhabditis elegans.</a> Genetics. 200 (2), 387-407 (2015).</li><li>Blackwell, T. K., Walker, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+mechanisms.">Transcription mechanisms.</a> WormBook: The Online Review of C. elegans Biology. , 1-16 (2006).</li><li>Page, A. P., Johnstone, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cuticle.">The cuticle.</a> WormBook: The Online Review of C. elegans Biology. , 1-15 (2007).</li><li>Lichtsteiner, S., Tjian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+properties+of+the+Caenorhabditis+elegans+TATA-box-binding+protein.">Cloning and properties of the Caenorhabditis elegans TATA-box-binding protein.</a> Proceedings of the National Academy of Sciences of the United States of America. 90 (20), 9673-9677 (1993).</li><li>Lichtsteiner, S., Tjian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+activation+of+transcription+by+UNC-86+and+MEC-3+in+Caenorhabditis+elegans+embryo+extracts.">Synergistic activation of transcription by UNC-86 and MEC-3 in Caenorhabditis elegans embryo extracts.</a> EMBO Journal. 14 (16), 3937-3945 (1995).</li><li>Shan, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotope-labeled+immunoassays+without+radiation+waste.">Isotope-labeled immunoassays without radiation waste.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (6), 2445-2449 (2000).</li><li>Voss, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel,+non-radioactive+eukaryotic+in+vitro+transcription+assay+for+sensitive+quantification+of+RNA+polymerase+II+activity.">A novel, non-radioactive eukaryotic in vitro transcription assay for sensitive quantification of RNA polymerase II activity.</a> BMC Molecular Biology. 15, 7 (2014).</li><li>Wibisono, P., Liu, Y., Sun, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+in+vitro+Caenorhabditis+elegans+transcription+system.">A novel in vitro Caenorhabditis elegans transcription system.</a> BMC Molecular and Cell Biology. 21 (1), 87 (2020).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook: The Online Review of C. elegans Biology. , 1-11 (2006).</li><li>Zbacnik, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+buffers+in+protein+formulations.">Role of buffers in protein formulations.</a> Journal of Pharmaceutical Sciences. 106 (3), 713-733 (2017).</li></ol>

秀丽隐杆线虫 是一种研究真核转录系统的有吸引力的模式生物，因为它的维护成本低，易于遗传操作。这里描述了从L4 C. elegans 中一致分离功能活性核提取物的方案。虽然该协议侧重于可视化转录活性，但转录后产生的cDNA可以使用RT-qPCR进行定量，以获得更精确的转录活性测量8。这种从 秀丽隐杆线虫 中分离核蛋白的方法可以帮助扩大真核转录机制的研究。?...

小型，自由生活的线虫 秀丽隐杆线虫 是一种简单而强大的模式生物，用于解决广泛的生物学问题。自1963年引入以来，线虫对于回答神经生物学，新陈代谢，衰老，发育，免疫和遗传学方面的问题非常宝贵1。该动物的许多特征使其成为理想的模式生物，包括较短的生成时间，RNA干扰的有效性，透明的身体以及其细胞谱系和神经系统的完整图谱。
虽然线虫对科学的贡献是巨大的，但它们在阐明真核转录系统方面没有得到充分利用，我们对这些机制的大部分理解来自使用酵母，果蝇和哺乳动物细胞培养物的核提取物的研究2。阻止研究人员提取功能性核提取物的最大障碍是线虫坚韧的外角质层。这种外骨骼由交联的胶原蛋白、角质层、糖蛋白和脂质组成，使秀丽隐杆线虫从幼虫期到成年期都耐通过化学或机械力提取蛋白质3。使用秀丽隐杆线虫核提取物的体外转录系统曾经开发出来，但由于系统范围有限，未被广泛采用，使用Dounce均质机制备提取物可能导致蛋白质不稳定4，5。
与先前利用Dounce均质机破坏 秀丽隐杆线虫 的核提取物分离协议不同，该协议使用Balch均质机。Balch均质机由两个主要部件组成：一个碳化钨球和一个不锈钢块，其通道从一端钻到另一端。Balch均质机装有碳化钨球，并在两侧盖上盖子以密封研磨室。注射器可以装载在通往研磨室的两个垂直端口上。当材料通过研磨室从一个注射器传递到另一个注射器时，来自注射器的压力迫使材料通过球和腔室壁之间的狭窄间隙。这种缓慢而恒定的压力会破坏材料，直到它达到能够轻松穿过狭窄间隙的一致尺寸。通过恒定而温和的压力迫使 秀丽隐杆线虫 穿过狭窄的间隙，使动物打开，将它们的内容释放到周围的缓冲区中。切换球的大小进一步收紧间隙，破坏新释放的细胞并将细胞核释放到缓冲液中。多次离心将细胞核与其余的细胞碎片分开，从而可以收集干净的核提取物。Balch均质机优于Dounce均质机，原因如下：该系统可以处理大量动物，从而可以在一次尝试中提取大量活性蛋白;滚珠和钢块的精确加工允许在多个样品之间进行一致的磨削;重型钢块充当散热器，均匀地将热量从研磨室中吸走，防止变性。
分离后，核提取物转录活性必须在用于任何生化实验之前进行验证。传统上，使用放射性标记的核苷酸来测量转录活性，以跟踪和可视化新合成的RNA。然而，放射性标签可能很繁琐，因为它在使用和处置过程中需要采取预防措施6。技术进步使今天的研究人员能够使用危害性更小或麻烦得多的方法，使用定量实时荧光定量PCR（qRT-PCR）7等技术来测量即使是很小的RNA量。在这里，该协议描述了一种从幼虫4期（L4） 秀丽隐杆线虫 中分离活性核提取物并在 体外 系统中可视化转录活性的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Consumables and reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 mL 8-Strip Tubes &#38; Flat Strip Caps, Clear</td>
			<td>Genesee Scientific</td>
			<td>&#160;24-706</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 mL Individual PCR tubes</td>
			<td>Genesee Scientific</td>
			<td>&#160;24-153G</td>
			<td></td>
		</tr>
		<tr>
			<td>1.7 mL sterile microtubes</td>
			<td>Genesee Scientific</td>
			<td>24-282S</td>
			<td></td>
		</tr>
		<tr>
			<td>100% absolute molecular grade ethanol</td>
			<td>Fisher Scientific</td>
			<td>&#160;BP2818</td>
			<td></td>
		</tr>
		<tr>
			<td>100% ethanol, Koptec</td>
			<td>Decon Labs</td>
			<td>&#160;V1001</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL serological pipet</td>
			<td>VWR international</td>
			<td>&#160;89130-898</td>
			<td></td>
		</tr>
		<tr>
			<td>150 mm petri plates</td>
			<td>Tritech Research</td>
			<td>&#160;T3325</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical centrifuge tubes</td>
			<td>Genesee Scientific</td>
			<td>&#160;28-103</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL plastic syringes</td>
			<td>Fisher Scientific</td>
			<td>14955460</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Norm-Ject syringes</td>
			<td>Henke-Sass Wolf GmbH</td>
			<td>4020</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL vacuum filter cup 0.22 &#181;m PES, Stericup Millipore Express Plus</td>
			<td>Millipore Sigma</td>
			<td>SCGPU10RE</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>339652</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL serological pipet</td>
			<td>VWR international</td>
			<td>&#160;89130-902</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL serological pipet</td>
			<td>VWR international</td>
			<td>&#160;89130-896</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar, Criterion</td>
			<td>VWR International</td>
			<td>&#160;C7432</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Denville Scientific</td>
			<td>&#160;CA3510-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol proof marker</td>
			<td>VWR International</td>
			<td>&#160;52877-310</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto peptone</td>
			<td>VWR International</td>
			<td>&#160;90000-264</td>
			<td></td>
		</tr>
		<tr>
			<td>Caenorhabditis elegans</td>
			<td>CGC</td>
			<td>N2</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium dichloride</td>
			<td>Millipore Sigma</td>
			<td>&#160;C4901</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Millipore Sigma</td>
			<td>&#160;C8667</td>
			<td></td>
		</tr>
		<tr>
			<td>Control DNA temple cloning primers, Forward 5&#8217;- ctc atg ttt gac agc tta tcg atc cgg gc -3&#8217;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Control DNA temple cloning primers, Forward 5&#8217;- aca gga cgg gtg tgg tcg cca tga t -3&#8217;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Invitrogen</td>
			<td>&#160;15508-013</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA gel stain, SYBR safe</td>
			<td>Invitrogen</td>
			<td>&#160;S33102</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA ladder mix, O&#8217;gene ruler</td>
			<td>Fisher Scientific</td>
			<td>&#160;SM1173</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Loading Dye, 6x TriTrack</td>
			<td>Fisher Scientific</td>
			<td>&#160;FERR1161</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase, Baseline-ZERO</td>
			<td>Lucigen</td>
			<td>&#160;DB0715K</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli OP50 strain</td>
			<td>CGC</td>
			<td>OP50</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Fisher Scientific</td>
			<td>&#160;A38</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Millipore Sigma</td>
			<td>&#160;G6279</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa nuclear extract in vitro transcription system, HeLaScribe</td>
			<td>Promega</td>
			<td>&#160;E3110</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes Solution, 1 M Gibco</td>
			<td>Millipore Sigma</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid 37%</td>
			<td>Millipore Sigma</td>
			<td>&#160;P0662</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypochlorite bleach</td>
			<td>Clorox</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td>Millipore Sigma</td>
			<td>&#160;L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium dichloride</td>
			<td>Millipore Sigma</td>
			<td>&#160;M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Millipore Sigma</td>
			<td>&#160;M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium weigh dishes</td>
			<td>Fisher Scientific</td>
			<td>&#160;02-202-101</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope slides, Vista vision</td>
			<td>VWR International</td>
			<td>16004-368</td>
			<td></td>
		</tr>
		<tr>
			<td>molecular grade water, Hypure</td>
			<td>Hyclone Laboratories</td>
			<td>&#160;SH30538</td>
			<td></td>
		</tr>
		<tr>
			<td>Nystatin</td>
			<td>Millipore Sigma</td>
			<td>&#160;N1638</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR system, FailSafe with premix A</td>
			<td>Lucigen</td>
			<td>&#160;FS99100</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Millipore Sigma</td>
			<td>&#160;P39111</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Millipore Sigma</td>
			<td>&#160;P3786</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Millipore Sigma</td>
			<td>&#160;P0662</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor, Halt single use cocktail 100x</td>
			<td>ThermoFisher Scientific</td>
			<td>78430</td>
			<td></td>
		</tr>
		<tr>
			<td>protein assay kit, Qubit</td>
			<td>ThermoFisher Scientific</td>
			<td>&#160;Q33211</td>
			<td></td>
		</tr>
		<tr>
			<td>reverse transcription kit, Sensiscript</td>
			<td>Qiagen</td>
			<td>205211</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA extraction kit RNeasy micro kit</td>
			<td>Qiagen</td>
			<td>74004</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase Inhibitor</td>
			<td>Applied Biosystems</td>
			<td>&#160;N8080119</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>VWR International</td>
			<td>&#160;BDH9286-12KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Millipore Sigma</td>
			<td>&#160;1-09137</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile syringe filter with 0.2 &#181;m Polyethersulfone membrane</td>
			<td>VWR international</td>
			<td>28145-501</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>VWR International</td>
			<td>&#160;200-334-9</td>
			<td></td>
		</tr>
		<tr>
			<td>transcription primers, Forward 5&#8217;- gcc ggg cct ctt gcg gga tat -3&#8217;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>transcription primers, Reverse 5&#8217;- cgg cca aag cgg tcg gac agt-3&#8217;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-Base</td>
			<td>Fisher Scientific</td>
			<td>&#160;BP152</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Millipore Sigma</td>
			<td>&#160;P2287</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-20 &#176;C incubator</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#176;C incubator</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176;C incubator</td>
			<td>Forma Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 &#176;C refrigerator</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-80 &#176;C freezer</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>Sanyo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Balch homogenizer, isobiotec cell homogenizer</td>
			<td>Isobiotec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop Vortexer</td>
			<td>Fisher Scientific</td>
			<td>2215365</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge, Eppendorf 5418 R</td>
			<td>Eppendorf</td>
			<td>5401000013</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge, VWR Clinical 50</td>
			<td>VWR International</td>
			<td>82013-800</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope, Leica M80</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorometer, Qubit 2.0</td>
			<td>Invitrogen</td>
			<td>Q32866</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel imaging system, iBright FL1500</td>
			<td>ThermoFisher Scientific</td>
			<td>&#160;A44241</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel system</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heat block</td>
			<td>VWR International</td>
			<td>12621-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuges, Eppendorf 5424</td>
			<td>Eppendorf</td>
			<td>22620401</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPETBOY acu 2</td>
			<td>Integra</td>
			<td>155017</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette L-1000 XLS+, Pipet-Lite LTS</td>
			<td>Rainin</td>
			<td>17014382</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette L-10 XLS+, Pipet-Lite LTS</td>
			<td>Rainin</td>
			<td>17014388</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette L-200 XLS+, Pipet-Lite LTS</td>
			<td>Rainin</td>
			<td>17014391</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette L-20 XLS+, Pipet-Lite LTS</td>
			<td>Rainin</td>
			<td>17014392</td>
			<td></td>
		</tr>
		<tr>
			<td>Rocking platform</td>
			<td>VWR International</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocycler, Eppendorf Mastercycler Pro</td>
			<td>Eppendorf</td>
			<td>950030010</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of active caenorhabditis elegans nuclear extract and reconstitution for in vitro transcription

秀丽隐杆线虫 自1963年推出以来，一直是生物学研究的重要模型系统。然而， 秀丽隐杆线虫 尚未充分利用其核提取物（如 体外 转录和DNA复制）进行生物反应的生化研究。在生化研究中使用 秀丽隐杆线虫 的一个重大障碍是破坏线虫厚厚的外角质层，而不会牺牲核提取物的活性。虽然有几种方法用于破坏角质层，例如Dounce均质化或超声处理，但它们通常会导致蛋白质不稳定。没有建立从幼虫或成年 秀丽隐杆线虫 中分离活性核蛋白用于 体外 反应的方案。在这里，该协议详细描述了使用Balch均质机对幼虫4 C.秀丽隐杆线虫的 均质化。Balch均质机使用压力缓慢地迫使动物通过狭窄的间隙，在此过程中破坏角质层。Balch 均质机的统一设计和精密加工允许在实验之间对动物进行一致的研磨。分馏从Balch均质机获得的匀浆产生功能活性核提取物，可用于 体外 方法测定 秀丽隐杆线虫的转录活性。

按照概述的步骤应产生功能性核提取物 （图1），研磨或洗涤步骤的偏差可能导致活性差或产量低。如果获得功能性 秀丽隐杆线虫 核提取物，当添加到前面描述的 体外测定中 时，它将转录DNA模板上CMV启动子下游的区域。所得RNA转录本可以使用常规方法从核蛋白和DNA模板中纯化。如果没有模板DNA，逆转录和随后的PCR产物只能是核提取物...

Watch this Scientific Journal Video about Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription at JoVE.com

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

在这里，我们描述了从幼虫4 C. elegans 中分离活性核提取物并在 体外 系统中可视化转录活性的详细方案。

活性 秀丽隐杆线 虫核提取物的分离及重建用于 体外 转录

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully ...

该协议填补了秀丽隐杆线虫生化研究的技术空白，并扩大了它们作为模式生物的用途。该技术既简单又稳健，可以一致地分离功能活性秀丽隐杆线虫核提取物。首先将同步的L1动物放在10个含有150毫米线虫生长培养基的板上，这些板中播种了大肠杆菌OP50，并允许动物在20摄氏度下生长48小时，直到它们达到L4阶段。一旦制备了低渗和高渗缓冲液，通过用70%乙醇淹没研磨室来清洁Balch均质机。然后用去离子水冲洗腔室以除去多余的乙醇。将碳化钨球插入磨削室。盖上Balch均质机枪管的两端，并用提供的指旋螺钉固定盖子。然后，按照文本手稿中所述，为每个样品准备五毫升完整的低渗和高渗缓冲液。接下来，用一毫升完全低渗缓冲液填充无菌的两毫升注射器，并轻轻冲洗Balch均质机的研磨室，在腔室中留下约500微升的完整低渗缓冲液。将冲洗过的均质机存放在冰上，冷却30分钟。将用M9缓冲液喂养良好的L4动物收集在15毫升锥形管中，并以1， 000次g离心动物三分钟。除去上清液，并继续洗涤动物沉淀，直到上清液清除。接下来用三毫升冷的低渗缓冲液洗涤动物，并再次离心，如图所示。除去上清液低渗缓冲液后，向动物沉淀中加入一毫升完全低渗缓冲液，并将动物悬浮液转移到新的无菌两毫升注射器中。为了使保持在冰上的动物均质化，轻轻地将动物推过装有钨球的Balch均质机的研磨室。然后将动物收集回注射器中，并重复此过程30次。经过30个均质循环后，从Balch均质机收集最大的动物悬浮液，并将注射器的尖端向下放置在1.7毫升微管内。取出钨球，并用去离子水清洁研磨室。干燥并将球放回相应的标记管中。然后将直径为7.9880毫米，间隙为12微米的钨球插入研磨室，并重新密封均质机。用一毫升冰冷的完全低渗缓冲液再次冲洗研磨室。如图所示，将悬浮液研磨25次。将动物悬浮液转移到干净的1.7毫升微管中，并将悬浮液储存在冰上。通过离心将动物尸体和碎屑沉淀下来。将40微升的上清液移液到标有输入级分的管中，并将该级分储存在冰上。将剩余的上清液转移到新的1.7毫升管中而不干扰沉淀，并离心以沉淀细胞核。然后在不干扰沉淀细胞核的情况下将上清液转移到标记为胞质级分的新的1.7毫升管中。为了洗涤细胞核沉淀，向沉淀中加入500微升完整的低渗缓冲液，悬浮沉淀，并在4摄氏度下以4， 000次g离心5分钟。在离心结束时，将核沉淀重悬于500微升新鲜完全低渗缓冲液中，并再次按照所示离心样品。然后将沉淀溶解在40微升完全高渗缓冲液中。将核悬浮液转移到一个新的1.7毫升管中，标有核馏分，并将其储存在冰上。使用荧光定量试剂盒测定三个组分的蛋白质浓度。将核部分等分到含有六微克核蛋白的一次性管中，并在干冰和乙醇浴中快速冷冻。将样品储存在零下80摄氏度，直至进一步使用。代表性凝胶图像显示了使用CMV启动子DNA模板的秀丽隐杆线虫L4幼虫核提取物的转录产物。活性核蛋白的成功分离导致体外转录后产生132碱基对条带，分离失败导致条带弱或无条带。请记住清楚地标记完整的缓冲区。不适当的缓冲液会损害核蛋白的功能。此外，保持均质机冰冷，以防止蛋白质变性。开发这种技术使研究人员能够在压力条件下测量秀丽隐杆线虫的RNA转录速率，表明动物的转录速率可以根据环境条件而变化。

Research

JoVE Journal

Biochemistry

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

拟南芥线粒体的分离和呼吸测量

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

科研

生物化学

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

光谱分析 TRPV1 的纯化与重构

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

线虫线虫中乳酸和丙酮酸细胞的小尺度比色测定

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

基于板块的秀丽线虫规模化栽培: 糖尿病代谢变化研究的样品制备

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple ...

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

体外支持性脂质双层自组织蛋白模式的重组

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays

生理活性类囊体的分离及其在能量依赖性蛋白转运检测中的应用

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

生物层干涉测量(BLI)在转录中研究蛋白质-蛋白质相互作用的应用

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

Caenorhabditis elegans中 2-阿拉希多诺诺基甘油定量的分解标准合成

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid ...

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

在Vivo羟基基蛋白足迹研究卡诺哈布迪特人的蛋白质相互作用

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, ...

活性 秀丽隐杆线 虫核提取物的分离及重建用于 体外 转录

活性秀丽隐杆线虫核提取物的分离及重建用于体外转录