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/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>

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

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...

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

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.

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 ...

isolation-active-caenorhabditis-elegans-nuclear-extract

Research

JoVE Journal

Biochemistry

2.5K Views.  Washington State University. Dit protocol vult een technische leemte op voor de biochemische studie van C.elegans en breidt hun nut als modelorganisme uit. Deze techniek is zowel eenvoudig als robuust, waardoor een consistente isolatie van functioneel actief C.elegans nucleair extract mogelijk is. Begin met het plaatsen van de gesynchroniseerde L1-dieren op 10 150 millimeter nematodengroeimedia-bevattende platen gezaaid met Escherichia coli OP50, en laat de dieren 48 uur groeien bij 20 graden Celsius totdat ze het L4-sta...

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

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

Genetics

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein complexes is essential to understanding mRNA regulation inferred by interacting proteins or non-coding RNAs. Herein, we describe a tandem RNA isolation procedure (TRIP) that enables the purification of endogenously formed mRNA-protein complexes from cellular extracts. The two-step protocol involves the isolation of polyadenylated mRNAs with antisense oligo(dT) beads and subsequent capture of an mRNA of interest with 3&#39;-biotinylated 2&#39;-O-methylated antisense RNA oligonucleotides, which can then be isolated with streptavidin beads. TRIP was used to recover in vivo crosslinked mRNA-ribonucleoprotein (mRNP) complexes from yeast, nematodes and human cells for further RNA and protein analysis. Thus, TRIP is a versatile approach that can be adapted to all types of polyadenylated RNAs across organisms to study the dynamic re-arrangement of mRNPs imposed by intracellular or environmental cues.

A tandem RNA isolation procedure (TRIP) for recovery of endogenously formed mRNA-protein complexes is described. Specifically, RNA-protein complexes are crosslinked in vivo, polyadenylated RNAs are isolated from extracts with oligo(dT) beads, and particular mRNAs are captured with modified RNA antisense oligonucleotides. Proteins bound to mRNAs are detected by immunoblot analysis.

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Developmental Biology

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological conditions. Post-mitotic neurons represent a major cell type affected by aging. Although multiple mammalian models of neuronal aging exist, they are challenging and expensive to establish. The roundworm Caenorhabditis elegans is a powerful model to study neuronal aging, as these animals have short lifespan, an available robust genetic toolbox, and well-cataloged nervous system. The method presented herein allows for seamless isolation of specific cells based on the expression of a transgenic green fluorescent protein (GFP). Transgenic animal lines expressing GFP under distinct, cell type-specific promoters are digested to remove the outer cuticle and gently mechanically disrupted to produce slurry containing various cell types. The cells of interest are then separated from non-target cells through fluorescence-activated cell sorting, or by anti-GFP-coupled magnetic beads. The isolated cells can then be cultured for a limited time or immediately used for cell-specific ex vivo analyses such as transcriptional analysis by real time quantitative PCR. Thus, this protocol allows for rapid and robust analysis of cell-specific responses within different neuronal populations in C. elegans.

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

Here, we present a protocol for simple isolation of specific groups of live neuronal cells expressing green fluorescent protein from transgenic Caenorhabditis elegans lines. This method enables a variety of ex vivo studies focused on specific neurons and has the capacity to isolate cells for further short-term culturing.

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological ...

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or identification of new ones can provide invaluable information about the function of a protein of interest. Some of the traditional methods for extract preparation frequently require labor-intensive and time-consuming techniques. Here, a modified extract preparation protocol using a bead mill homogenizer and metal beads is described as a rapid alternative to traditional protein preparation methods. This extract preparation method is compatible with downstream co-immunoprecipitation studies. As an example, the method was used to successfully co-immunoprecipitate&#160;C. elegans microRNA Argonaute ALG-1 and two known ALG-1 interactors: AIN-1, and HRPK-1. This protocol includes descriptions of animal sample collection, extract preparation, extract clarification, and protein immunoprecipitation. The described protocol can be adapted to test for interactions between any two or more endogenous, endogenously tagged, or overexpressed C. elegans proteins in a variety of genetic backgrounds.

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

This method describes a protocol for high-throughput protein extract preparation from Caenorhabditis elegans samples and subsequent co-immunoprecipitation.

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Biology

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and sequencing. The traditional proteinase K-based methods for genomic DNA extraction from C. elegans take several hours. Commercial extraction kits that effectively break open the C. elegans cuticle and extract genomic DNA are limited. An easy, faster (~15 min), and cost-efficient method of extracting C. elegans genomic DNA that works well for classroom and research applications is reported here. This DNA extraction method is optimized to use single or a few late-larval (L4) or adult nematodes as starting material for obtaining a reliable template to perform PCR. The results indicate that the DNA quality is suitable for amplifying gene targets of different sizes by PCR, permitting genotyping of single or a few animals even at dilutions to one-fiftieth of the genomic DNA from a single adult per reaction. The reported protocols can be reliably used to quickly produce DNA template from a single or a small sample of C. elegans for PCR-based applications.

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Presented here is a description of the straightforward and relatively rapid isolation of Caenorhabditis elegans genomic DNA from one or a few animals using a commercially available tissue kit. The resulting gDNA preparation is a suitable template for PCR.

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and ...

Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans

Aging is a complex process characterized by progressive physiological changes resulting from both environmental and genetic contributions. Lipids are crucial in constituting structural components of cell membranes, storing energy, and as signaling molecules. Regulation of lipid metabolism and signaling is essential to activate distinct longevity pathways. The roundworm Caenorhabditis elegans is an excellent and powerful organism to dissect the contribution of lipid metabolism and signaling in longevity regulation. Multiple research studies have described how diet supplementation of specific lipid molecules can extend C. elegans lifespan; however, minor differences in the supplementation conditions can cause reproducibility issues among scientists in different labs. Here, two detailed supplementation methods for C. elegans are reported employing lipid supplementation either with bacteria seeded on plates or bacterial suspension in liquid culture. Also provided herein are the details to perform lifespan assays with lifelong lipid supplementation and qRT-PCR analysis using a whole worm lysate or dissected tissues derived from a few worms. Using a combination of longitudinal studies and transcriptional investigations upon lipid supplementation, the feeding assays provide dependable approaches to dissect how lipids influence longevity and healthy aging. This methodology can also be adapted for various nutritional screening approaches to assess changes in a subset of transcripts using either a small number of dissected tissues or a few animals.

Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans

The present protocol describes lipid supplementation methods in liquid and on-plate cultures for Caenorhabditis elegans, coupled with longitudinal studies and gene transcriptional analysis from bulk or a few worms and worm tissues.

Aging is a complex process characterized by progressive physiological changes resulting from both environmental and genetic contributions. Lipids are ...

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

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