Name: Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription (Video) | JoVE Journal
Uploaded: 2021-08-11T14:00:00.000+00:00
Duration: 6 min 9 s
Description: Learn to isolate active Caenorhabditis elegans nuclear extract using a Balch homogenizer for in vitro transcription studies. This robust biochemical method enables visualization of transcription activity and expands C. elegans utility.

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This work was supported by an NIH MIRA grant (R35GM124678 to J. S.).

<ol><li>Corsi, A. K., Wightman, B., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Transparent+window+into+biology%3A+A+on+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">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/pubmed?term=((Transcription+mechanisms%5BTitle%5D)+AND+%22WormBook%3A+The+Online+Review+of+C.+elegans+Biology%22%5BJournal%5D)">Transcription mechanisms.</a> WormBook: The Online Review of C. elegans Biology. , Pasadena, CA. 1-16 (2006).</li>
<li>Page, A. P., Johnstone, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+cuticle%5BTitle%5D)+AND+%22WormBook%3A+The+Online+Review+of+C.+elegans+Biology%22%5BJournal%5D)">The cuticle.</a> WormBook: The Online Review of C. elegans Biology. , Pasadena, CA. 1-15 (2007).</li>
<li>Lichtsteiner, S., Tjian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cloning+and+properties+of+the+Caenorhabditis+elegans+TATA-box-binding+protein%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((Synergistic+activation+of+transcription+by+UNC-86+and+MEC-3+in+Caenorhabditis+elegans+embryo+extracts%5BTitle%5D)+AND+%22EMBO+Journal%22%5BJournal%5D)">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/pubmed?term=((Isotope-labeled+immunoassays+without+radiation+waste%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((A+novel%2C+non-radioactive+eukaryotic+in+vitro+transcription+assay+for+sensitive+quantification+of+RNA+polymerase+II+activity%5BTitle%5D)+AND+%22BMC+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((A+novel+in+vitro+Caenorhabditis+elegans+transcription+system%5BTitle%5D)+AND+%22BMC+Molecular+and+Cell+Biology%22%5BJournal%5D)">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/pubmed?term=((Maintenance+of+C.+elegans%5BTitle%5D)+AND+%22WormBook%3A+The+Online+Review+of+C.+elegans+Biology%22%5BJournal%5D)">Maintenance of C. elegans.</a> WormBook: The Online Review of C. elegans Biology. , Pasadena, CA. 1-11 (2006).</li>
<li>Zbacnik, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+buffers+in+protein+formulations%5BTitle%5D)+AND+%22Journal+of+Pharmaceutical+Sciences%22%5BJournal%5D)">Role of buffers in protein formulations.</a> Journal of Pharmaceutical Sciences. 106 (3), 713-733 (2017).</li>
</ol>

The authors have no competing interests to disclose.

C. elegans is an appealing model organism to study the eukaryotic transcription system because of its low-cost maintenance and the ease of genetic manipulation. Here a protocol for consistent isolation of functionally active nuclear extract from L4 C. elegans is described. Although this protocol focused on visualizing transcription activity, the cDNA produced post-transcriptionally can be quantified using RT-qPCR to obtain a more precise measurement of the transcription activity8...

The small, free-living nematode Caenorhabditis elegans is a simple yet powerful model organism for addressing a wide range of biological questions. Since its introduction in 1963, the nematodes have been invaluable to answering questions in neurobiology, metabolism, aging, development, immunity, and genetics1. Some of the animal&#39;s many characteristics that make it an ideal model organism include short generation time, the effectiveness of RNA interference, transparent body, and the completed maps of both its cellular lineage and nervous system.
While the nematode&#39;s contributions to science are vast, they have been under-utilized to elucidate the eukaryotic transcription system, with most of our understanding about these mechanisms coming from studies using nuclear extract from yeast, fruit fly, and mammalian cell culture2. The biggest hurdle that dissuades researchers from extracting functional nuclear extract is the nematode&#39;s tough outer cuticle. This exoskeleton comprises cross-linked collagens, cuticlins, glycoproteins, and lipids, making C. elegans from larval stage to adulthood resistant to protein extraction via chemical or mechanical forces3. An in vitro transcription system using C. elegans nuclear extract was once developed but not widely adopted due to the system&#39;s limited scope, and the use of Dounce homogenizer for preparing the extract could lead to protein instability4,5.
Unlike the previous protocol for nuclear extract isolation which utilized a Dounce homogenizer to break C. elegans, this protocol uses a Balch homogenizer. The Balch homogenizer consists of two main components: a tungsten carbide ball and a stainless-steel block with a channel bored through from one end to another. The Balch homogenizer is loaded with the tungsten carbide ball and capped on either side to seal the grinding chamber. Syringes can be loaded on the two vertical ports leading into the grinding chamber. As the material is passed from one syringe to the other through the grinding chamber, the pressure from the syringes forces the material through a narrow gap between the ball and the wall of the chamber. This slow and constant pressure breaks the material until it reaches a consistent size that is able to pass through the narrow gap easily. Forcing C. elegans through the narrow gap via a constant yet gentle pressure breaks the animals open, releasing their content into the surrounding buffer. Switching ball sizes further tightens the gap, breaking the newly released cells and frees the nuclei into the buffer. Multiple instances of centrifugation separate the nuclei from the rest of the cell debris, allowing for the collection of a clean nuclear extract. The Balch homogenizer is preferred over the Dounce homogenizer for several reasons: the system can handle a large number of animals, making it possible to extract a high amount of active proteins in a single attempt; the precise machining of the balls and the steel block allows for consistent grinding between multiple samples; the heavy steel block act as a heat sink, uniformly drawing heat away from the grinding chamber, preventing denaturing.
After isolation, the nuclear extract transcriptional activity must be verified before being used in any biochemical experiments. Traditionally, transcription activity was measured using radiolabeled nucleotides to track and visualize the newly synthesized RNA. However, radioactive labeling can be burdensome as it requires precaution during use and disposal6. Technological advancements allow researchers today to use much less harmful or troublesome methods to measure even small RNA quantities using techniques such as quantitative real-time PCR (qRT-PCR)7. Here, the protocol describes a method to isolate active nuclear extract from larval stage 4 (L4) C. elegans and visualize transcription activity in an in vitro system.

<table><tbody><tr><td>&lt;strong&gt;Consumables and reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.2 mL 8-Strip Tubes &amp;amp; Flat Strip Caps, Clear</td><td>Genesee Scientific</td><td>&amp;nbsp;24-706</td><td></td></tr><tr><td>0.2 mL Individual PCR tubes</td><td>Genesee Scientific</td><td>&amp;nbsp;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>&amp;nbsp;BP2818</td><td></td></tr><tr><td>100% ethanol, Koptec</td><td>Decon Labs</td><td>&amp;nbsp;V1001</td><td></td></tr><tr><td>10 mL serological pipet</td><td>VWR international</td><td>&amp;nbsp;89130-898</td><td></td></tr><tr><td>150 mm petri plates</td><td>Tritech Research</td><td>&amp;nbsp;T3325</td><td></td></tr><tr><td>15 mL conical centrifuge tubes</td><td>Genesee Scientific</td><td>&amp;nbsp;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 &amp;micro;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>&amp;nbsp;89130-902</td><td></td></tr><tr><td>5 mL serological pipet</td><td>VWR international</td><td>&amp;nbsp;89130-896</td><td></td></tr><tr><td>Agar, Criterion</td><td>VWR International</td><td>&amp;nbsp;C7432</td><td></td></tr><tr><td>Agarose</td><td>Denville Scientific</td><td>&amp;nbsp;CA3510-6</td><td></td></tr><tr><td>Alcohol proof marker</td><td>VWR International</td><td>&amp;nbsp;52877-310</td><td></td></tr><tr><td>Bacto peptone</td><td>VWR International</td><td>&amp;nbsp;90000-264</td><td></td></tr><tr><td>&lt;em&gt;Caenorhabditis elegans&lt;/em&gt;</td><td>CGC</td><td>N2</td><td></td></tr><tr><td>Calcium dichloride</td><td>Millipore Sigma</td><td>&amp;nbsp;C4901</td><td></td></tr><tr><td>Cholesterol</td><td>Millipore Sigma</td><td>&amp;nbsp;C8667</td><td></td></tr><tr><td>Control DNA temple cloning primers, Forward 5&amp;rsquo;- ctc atg ttt gac agc tta tcg atc cgg gc -3&amp;rsquo;</td><td></td><td></td><td></td></tr><tr><td>Control DNA temple cloning primers, Forward 5&amp;rsquo;- aca gga cgg gtg tgg tcg cca tga t -3&amp;rsquo;</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>&amp;nbsp;15508-013</td><td></td></tr><tr><td>DNA gel stain, SYBR safe</td><td>Invitrogen</td><td>&amp;nbsp;S33102</td><td></td></tr><tr><td>DNA ladder mix, O&amp;rsquo;gene ruler</td><td>Fisher Scientific</td><td>&amp;nbsp;SM1173</td><td></td></tr><tr><td>DNA Loading Dye, 6x TriTrack</td><td>Fisher Scientific</td><td>&amp;nbsp;FERR1161</td><td></td></tr><tr><td>DNase, Baseline-ZERO</td><td>Lucigen</td><td>&amp;nbsp;DB0715K</td><td></td></tr><tr><td>Dry ice</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt; OP50 strain</td><td>CGC</td><td>OP50</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Fisher Scientific</td><td>&amp;nbsp;A38</td><td></td></tr><tr><td>Glycerol</td><td>Millipore Sigma</td><td>&amp;nbsp;G6279</td><td></td></tr><tr><td>HeLa nuclear extract in vitro transcription system, HeLaScribe</td><td>Promega</td><td>&amp;nbsp;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>&amp;nbsp;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>&amp;nbsp;L3022</td><td></td></tr><tr><td>Magnesium dichloride</td><td>Millipore Sigma</td><td>&amp;nbsp;M8266</td><td></td></tr><tr><td>Magnesium Sulfate</td><td>Millipore Sigma</td><td>&amp;nbsp;M7506</td><td></td></tr><tr><td>Medium weigh dishes</td><td>Fisher Scientific</td><td>&amp;nbsp;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>&amp;nbsp;SH30538</td><td></td></tr><tr><td>Nystatin</td><td>Millipore Sigma</td><td>&amp;nbsp;N1638</td><td></td></tr><tr><td>PCR system, FailSafe with premix A</td><td>Lucigen</td><td>&amp;nbsp;FS99100</td><td></td></tr><tr><td>Potassium chloride</td><td>Millipore Sigma</td><td>&amp;nbsp;P39111</td><td></td></tr><tr><td>Potassium phosphate dibasic</td><td>Millipore Sigma</td><td>&amp;nbsp;P3786</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Millipore Sigma</td><td>&amp;nbsp;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>&amp;nbsp;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>&amp;nbsp;N8080119</td><td></td></tr><tr><td>Sodium Chloride</td><td>VWR International</td><td>&amp;nbsp;BDH9286-12KG</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Millipore Sigma</td><td>&amp;nbsp;1-09137</td><td></td></tr><tr><td>Sterile syringe filter with 0.2 &amp;micro;m Polyethersulfone membrane</td><td>VWR international</td><td>28145-501</td><td></td></tr><tr><td>Sucrose</td><td>VWR International</td><td>&amp;nbsp;200-334-9</td><td></td></tr><tr><td>transcription primers, Forward 5&amp;rsquo;- gcc ggg cct ctt gcg gga tat -3&amp;rsquo;</td><td></td><td></td><td></td></tr><tr><td>transcription primers, Reverse 5&amp;rsquo;- cgg cca aag cgg tcg gac agt-3&amp;rsquo;</td><td></td><td></td><td></td></tr><tr><td>Tris-Base</td><td>Fisher Scientific</td><td>&amp;nbsp;BP152</td><td></td></tr><tr><td>Tween20</td><td>Millipore Sigma</td><td>&amp;nbsp;P2287</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>-20 &amp;deg;C incubator</td><td>ThermoFisher Scientific</td><td></td><td></td></tr><tr><td>20 &amp;deg;C incubator</td><td>ThermoFisher Scientific</td><td></td><td></td></tr><tr><td>37 &amp;deg;C incubator</td><td>Forma Scientific</td><td></td><td></td></tr><tr><td>4 &amp;deg;C refrigerator</td><td>ThermoFisher Scientific</td><td></td><td></td></tr><tr><td>-80 &amp;deg;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>&amp;nbsp;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

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

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully utilized in the biochemical study of biological reactions using its nuclear extracts such as in vitro transcription and DNA replication. A significant hurdle for using C. elegans in biochemical studies is disrupting the nematode&#39;s thick outer cuticle without sacrificing the activity of the nuclear extract. While several methods are used to break the cuticle, such as Dounce homogenization or sonication, they often lead to protein instability. There are no established protocols for isolating active nuclear proteins from larva or adult C. elegans for in vitro reactions. Here, the protocol describes in detail the homogenization of larval stage 4 C. elegans using a Balch homogenizer. The Balch homogenizer uses pressure to slowly force the animals through a narrow gap breaking the cuticle in the process. The uniform design and precise machining of the Balch homogenizer allow for consistent grinding of animals between experiments. Fractionating the homogenate obtained from the Balch homogenizer yields functionally active nuclear extract that can be used in an in vitro method for assaying transcription activity of C. elegans.

Following the outlined steps should yield functional nuclear extract (Figure 1), deviation in the grinding or wash steps can lead to poor activity or low yields. If functional C. elegans nuclear extract is obtained, it will transcribe the region downstream of the CMV promoter on the DNA template when added to the previously described in vitro assay. The resulting RNA transcript can be purified from the nuclear proteins and DNA template usin...

Here, we describe a detailed protocol for isolating active nuclear extract from larval stage 4 C. elegans and visualizing transcription activity in an in vitro system.

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

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Learn to isolate active Caenorhabditis elegans nuclear extract using a Balch homogenizer for in vitro transcription studies. This robust biochemical method enables visualization of transcription activity and expands C. elegans utility.

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

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