Os autores gostariam de agradecer a Lindsey Nash para ajudar a desenvolver a técnica. Este trabalho foi financiado pelo NIH conceder AG028977 a ALF, uma bolsa de projeto piloto da Universidade de Pittsburgh OAIC (AG024827), e fundos de sementes da Universidade de Pittsburgh.

<ol>
	<li>Mello, C., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+transformation.">DNA transformation.</a> Methods Cell Biol. 48, 451-482 (1995).</li><li>Zhang, Y., Nash, L., Fisher, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified,+robust,+and+streamlined+procedure+for+the+production+of+C.+elegans+transgenes+via+recombineering.">A simplified, robust, and streamlined procedure for the production of C. elegans transgenes via recombineering.</a> BMC Dev Biol. 8, 119-119 (2008).</li><li>Antebi, A., Yeh, W. H., Tait, D., Hedgecock, E. M., Riddle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=daf-12+encodes+a+nuclear+receptor+that+regulates+the+dauer+diapause+and+developmental+age+in+C.+elegans.">daf-12 encodes a nuclear receptor that regulates the dauer diapause and developmental age in C. elegans.</a> Genes and Development. 14, 1512-1527 (2000).</li><li>Snow, M. I., Larsen, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+expression+of+daf-12:+a+nuclear+hormone+receptor+with+three+isoforms+that+are+involved+in+development+and+aging+in+Caenorhabditis+elegans.">Structure and expression of daf-12: a nuclear hormone receptor with three isoforms that are involved in development and aging in Caenorhabditis elegans.</a> Biochim. Biophys. Acta. 1494, 104-116 (2000).</li><li>Fisher, A. L., Page, K. E., Lithgow, G. J., Nash, 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+Caenorhabditis+elegans+K10C2.4+Gene+Encodes+a+Member+of+the+Fumarylacetoacetate+Hydrolase+Family.+A+CAENORHABDITIS+ELEGANS+MODEL+OF+TYPE+I+TYROSINEMIA.">The Caenorhabditis elegans K10C2.4 Gene Encodes a Member of the Fumarylacetoacetate Hydrolase Family. A CAENORHABDITIS ELEGANS MODEL OF TYPE I TYROSINEMIA.</a> J Biol.Chem. 283, 9127-9135 (2008).</li><li>Wightman, B., Ha, I., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posttranscriptional+regulation+of+the+heterochronic+gene+lin-14+by+lin-4+mediates+temporal+pattern+formation+in+C.+elegans.">Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans.</a> Cell. 75, 855-862 (1993).</li><li>Lehrbach, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LIN-28+and+the+poly(U)+polymerase+PUP-2+regulate+let-7+microRNA+processing+in+Caenorhabditis+elegans.">LIN-28 and the poly(U) polymerase PUP-2 regulate let-7 microRNA processing in Caenorhabditis elegans.</a> Nat Struct Mol Biol. 16, 1016-1020 (2009).</li><li>Tursun, B., Cochella, L., Carrera, I., Hobert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+toolkit+and+robust+pipeline+for+the+generation+of+fosmid-based+reporter+genes+in+C.+elegans.">A toolkit and robust pipeline for the generation of fosmid-based reporter genes in C. elegans.</a> PLoS One. 4, e4625-e4625 (2009).</li><li>Bamps, S., Hope, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+gene+expression+pattern+analysis,+in+situ,+in+Caenorhabditis+elegans.">Large-scale gene expression pattern analysis, in situ, in Caenorhabditis elegans.</a> Brief. Funct. Genomic. Proteomic. , (2008).</li><li>Dolphin, C. T., Hope, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+reporter+fusion+genes+generated+by+seamless+modification+of+large+genomic+DNA+clones.">Caenorhabditis elegans reporter fusion genes generated by seamless modification of large genomic DNA clones.</a> Nucleic Acids Res. 34, e72-e72 (2006).</li><li>Sarov, 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+recombineering+pipeline+for+functional+genomics+applied+to+Caenorhabditis+elegans.">A recombineering pipeline for functional genomics applied to Caenorhabditis elegans.</a> Nat. Methods. 3, 839-844 (2006).</li><li>Yang, X. W., Model, P., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homologous+recombination+based+modification+in+Escherichia+coli+and+germline+transmission+in+transgenic+mice+of+a+bacterial+artificial+chromosome.">Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome.</a> Nat Biotechnol. 15, 859-865 (1997).</li><li>Court, D. L., Sawitzke, J. A., Thomason, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+engineering+using+homologous+recombination.">Genetic engineering using homologous recombination.</a> Annu.Rev.Genet. 36, 361-388 (2002).</li><li>Westenberg, M., Bamps, S., Soedling, H., Hope, I. A., Dolphin, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+MW005:+lambda+Red-mediated+recombineering+and+copy-number+induction+of+oriV-equipped+constructs+in+a+single+host.">Escherichia coli MW005: lambda Red-mediated recombineering and copy-number induction of oriV-equipped constructs in a single host.</a> BMC Biotechnol. 10, 27-27 (2010).</li><li>Warming, S., Costantino, N., Court, D. L., Jenkins, N. A., Copeland, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+highly+efficient+BAC+recombineering+using+galK+selection.">Simple and highly efficient BAC recombineering using galK selection.</a> Nucleic Acids Res. 33, e36-e36 (2005).</li><li>Penfold, R. J., Pemberton, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+suicide+vector+for+construction+of+chromosomal+insertion+mutations+in+bacteria.">An improved suicide vector for construction of chromosomal insertion mutations in bacteria.</a> Gene. 118, 145-146 (1992).</li><li>Praitis, V., Casey, E., Collar, D., Austin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+low-copy+integrated+transgenic+lines+in+Caenorhabditis+elegans.">Creation of low-copy integrated transgenic lines in Caenorhabditis elegans.</a> Genetics. 157, 1217-1226 (2001).</li><li>Puig, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tandem+affinity+purification+(TAP)+method:+a+general+procedure+of+protein+complex+purification.">The tandem affinity purification (TAP) method: a general procedure of protein complex purification.</a> Methods. 24, 218-229 (2001).</li><li>Rigaut, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+generic+protein+purification+method+for+protein+complex+characterization+and+proteome+exploration.">A generic protein purification method for protein complex characterization and proteome exploration.</a> Nat.Biotechnol. 17, 1030-1032 (1999).</li><li>Achilleos, A., Wehman, A. M., Nance, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAR-3+mediates+the+initial+clustering+and+apical+localization+of+junction+and+polarity+proteins+during+C.+elegans+intestinal+epithelial+cell+polarization.">PAR-3 mediates the initial clustering and apical localization of junction and polarity proteins during C. elegans intestinal epithelial cell polarization.</a> Development. 137, 1833-1842 (2010).</li><li>Maduro, M., Pilgrim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+cloning+of+unc-119,+a+gene+expressed+in+the+Caenorhabditis+elegans+nervous+system.">Identification and cloning of unc-119, a gene expressed in the Caenorhabditis elegans nervous system.</a> Genetics. 141, 977-988 (1995).</li></ol>

Não há conflitos de interesse declarados.

A geração de transgenes de fosmids oferece o benefício de manter todos os elementos promotor nativo, splice variantes, e três elementos &#39;UTR de regulamentação. Isso pode levar à construção de um transgene que é mais um reflexo do padrão de expressão nativa, ou a construção de um transgene funcional quando outras abordagens não 5. Os transgenes resultante pode carregar uma variedade de etiquetas de epítopo incluindo GFP ou um tag TAP. A construção de transgene...

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		<div>
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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>FosmidMAX kit</td>
<td>&nbsp;</td>
<td>Epicentre</td>
<td>FMAX046</td>
<td>&nbsp;</td>
<tr>
<td>GoTaq</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>M7122</td>
<td>&nbsp;</td>
<tr>
<td>MOPS Media</td>
<td>&nbsp;</td>
<td>Teknova</td>
<td>M2120</td>
<td>&nbsp;</td>
<tr>
<td>0.132 M Potassium phosphate solution</td>
<td>&nbsp;</td>
<td>Teknova</td>
<td>M2102</td>
<td>&nbsp;</td>
<tr>
<td>D-galactose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G0750</td>
<td>&nbsp;</td>
<tr>
<td>2-deoxygalactose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D4407</td>
<td>&nbsp;</td>
<tr>
<td>Biotin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B4639</td>
<td>&nbsp;</td>
<tr>
<td>Leucine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L8000</td>
<td>&nbsp;</td>
<tr>
<td>NH4Cl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A9434</td>
<td>&nbsp;</td>
<tr>
<td>Phusion DNA polymerase</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>F-530S</td>
<td>&nbsp;</td>
<tr>
<td>MacConkey agar base</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>281810</td>
<td>&nbsp;</td>
<tr>
<td>Arabinose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A3131</td>
<td>&nbsp;</td>
<tr>
<td>Chloramphenicol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C1919</td>
<td>&nbsp;</td>
<tr>
<td>Sodium phosphate dibasic</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S5136</td>
<td>&nbsp;</td>
<tr>
<td>Potassium phosphate monobasic</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5655</td>
<td>&nbsp;</td>
<tr>
<td>Sodium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S5886</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G2025</td>
<td>&nbsp;</td>
<tr>
<td>Bacto Agar</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>214010</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

the production of c. elegans transgenes via recombineering with the galk selectable marker

A criação de animais transgênicos é amplamente utilizada em C. elegans pesquisa, incluindo o uso de proteínas de fusão GFP para estudar a regulação e padrão de expressão de genes de interesse ou de geração de conjunto de purificação de afinidade (TAP) com a tag versões de genes específicos para facilitar a sua purificação. Tipicamente transgenes são gerados pela colocação de um promotor a montante de um gene repórter GFP ou cDNA de interesse, e isso muitas vezes produz um padrão de expressão representativa. No entanto, elementos críticos da regulação de genes, como elementos de controle na região não traduzida 3 &#39;ou promotores alternativos, podem ser perdidos por esta abordagem. Mais apenas uma variante única emenda pode ser geralmente estudado por este meio. Em contraste, o uso de DNA genômico verme transportado por clones DNA fosmid provavelmente inclui a maioria se não todos os elementos envolvidos na regulação de genes in vivo que permite a maior capacidade de capturar o padrão de expressão genuína e timing. Para facilitar a geração de transgenes usando DNA fosmid, descrevemos um E. coli baseado recombineering procedimento para inserir GFP, a TAP tag, ou outras seqüências de interesse em qualquer local do gene. O procedimento utiliza o gene galK como o marcador de seleção para as duas etapas de seleção positiva e negativa em recombineering o que resulta na obtenção da modificação desejada com alta eficiência. Além disso, plasmídeos contendo o gene galK ladeado por braços homologia com GFP comumente usados ​​e TAP genes de fusão estão disponíveis que reduzem o custo de oligos em 50% ao gerar um GFP, ou proteína de fusão TAP. Estes plasmídeos use a origem de replicação R6K que impede a necessidade de purificação extensa produto da PCR. Finalmente, também demonstram uma técnica para integrar o marcador unc-119 para o backbone fosmid que permite que o fosmid a ser injetado diretamente ou bombardeados em vermes para gerar animais transgênicos. Este vídeo demonstra os procedimentos envolvidos na geração de um transgene através recombineering utilizando este método.

Watch this Scientific Journal Video about The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker at JoVE.com

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

A capacidade de produzir transgenes para<em&gt; Caenorhabditis elegans</em&gt; Usando DNA genômico realizado por fosmids é particularmente atraente como todos os elementos nativos reguladoras são retidos. Descrito é um procedimento simples e robusto para a produção de transgenes via recombineering com o<em&gt; GalK</em&gt; Marcador selecionável.

Produção de C. elegans Transgenes via Recombineering com o GalK Marcador selecionável

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

the-production-c-elegans-transgenes-via-recombineering-with-galk

Research

JoVE Journal

Biology

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

14.5K visualizações.  Beth Israel Deaconess Medical Center, Harvard Medical School. A capacidade de produzir transgenes para Caenorhabditis elegans Usando DNA genômico realizado por fosmids é particularmente atraente como todos os elementos nativos reguladoras são retidos. Descrito é um procedimento simples e robusto para a produção de transgenes via recombineering com o GalK Marcador selecionável.

Vídeo: The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Geração de transgênicos Estável C. elegans Usando microinjeção

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Biologia

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

Dissecação e gravação a partir da junção neuromuscular C. Elegans

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

A Protocol for the Production of KLRG1 Tetramer

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. KLRG1 exists both as a monomer and as a disulfide-linked homodimer. This well-conserved receptor is found on the most mature and recently activated NK cells as well as on a subset of effector/memory T cells.
Using KLRG1 tetramer as well as other methods, E-, N-, and R-cadherins were identified as KLRG1 ligands. These Ca2+-dependent cell-cell adhesion molecules comprises of an extracellular domain containing five cadherin repeats responsible for cell-cell interactions, a transmembrane domain and a cytoplasmic domain that is linked to the actin cytoskeleton. 
Generation of the KLRG1 tetramer was essential to the identification of the KLRG1 ligands. KLRG1 tetramer is also a unique tool to elucidate the roles cadherin and KLRG1 play in regulating the immune response and tissue integrity.

This protocol describes the production of KLRG1 tetramer, which is a powerful tool for the analysis of KLRG1 ligands.

Um protocolo para a Produção de KLRG1 tetrâmero

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

Geração de transgênicos C. elegans Por biobalística Transformação

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

Alto rendimento Triagem e Biossensoriais com Fluorescente C. elegans Cepas

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

Criando ambientes definidos gasosos para estudar os efeitos da hipóxia na<em&gt; C. elegans</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

Usando RNA mediada estratégia de alimentação Interferência em tela para genes envolvidos na regulação do tamanho corporal na Nemátodo<em&gt; C. elegans</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that exhibit partial and variable transmission rates of extrachromosomal arrays to the next generation. In addition, transgenic animals are mosaic and express the transgene in a variable number of cells. Extrachromosomal arrays can be integrated into the C. elegans genome using UV irradiation to establish nonmosaic transgenic strains with 100% transmission rate of the transgene. To that extent, F1 progenies of UV irradiated transgenic animals are screened for animals carrying a heterozygous integration of the transgene, which leads to a 75% Mendelian transmission rate to the F2 progeny. One of the challenges of this method is to distinguish between the percentage of transgene transmission in a population before (X% transgenic animals) and after integration (&#8805;75% transgenic F2 animals). Thus, this method requires choosing a nonintegrated transgenic line with a percentage of transgenic animals that is significantly lower than the Mendelian segregation of 75%. Consequently, nonintegrated transgenic lines with an extrachromosomal array transmission rate to the next generation &#8804;60% are usually preferred for integration, and transgene integration in highly transmitting strains is difficult. Here we show that the efficiency of extrachromosomal arrays integration into the genome is increased when using highly transmitting transgenic lines (&#8805;80%). The described protocol allows for easy selection of several independent lines with homozygous transgene integration into the genome after UV irradiation of transgenic worms exhibiting a high rate of extrachromosomal array transmission. Furthermore, this method is quite fast and low material consuming. The possibility of rapidly generating different lines that express a particular integrated transgene is of great interest for studies focusing on gene expression pattern and regulation, protein localization, and overexpression, as well as for the development of subcellular markers.

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

This protocol describes a rapid and low material consuming procedure for the integration of transgenic extrachromosomal arrays into the Caenorhabditis elegans genome using ultra violet (UV) irradiation. Furthermore, this protocol is particularly well suited for transgenic lines that transmit extrachromosomal arrays at a high rate.

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

Medição dos Efeitos de bactérias em<em&gt; C. Elegans</em&gt; Comportamento Usando um Retenção Ensaio Egg

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...