Authors would like to acknowledge Claire Lecroisey and Laurent S&#233;galat for generating nonintegrated Pdyc-1::dyc-1::gfp; Pmyo-2::dsRed and Pmyo-3::gfp transgenic strains. We thank Andy Fire for the gift of the Pmyo-2::dsRed plasmid and Pamela Hoppe for the gift of the Pmyo-3::gfp plasmid. This work was founded by the French National Center of Scientific Research (CNRS) and the Claude Bernard University of Lyon.

1. Culturing Transgenic Nematodes Prior to&#160;Integration
Transgenic lines can be generated as described in1. These lines usually contain a coinjection marker plasmid leading either to the expression of a fluorescent protein or to a specific behavioral or morphological phenotype at a given development stage1,3. This integration protocol can be applied to strains expressing fluorescent or nonfluorescent coinjection markers. In the example to follow, a strain with a fluorescent coinjection marker was used.
<ol>
	<li>Prepare culture plates:
		<ol>
			<li>Prepare about 500 60 mm&#160;large Petri dishes poured aseptically with 10 ml each of Nematode Growth Medium (NGM)6,7.</li>
			<li>Seed each plate with 0.3 ml of saturated Escherichia coli OP50 culture following standard procedure6,7.</li>
		</ol>
	</li>
	<li>Evaluate the transmission rate of the transgenic line to be integrated:
		<ol>
			<li>For each transgenic line, pick ten fluorescent gravid adults onto ten separate culture plates under a fluorescent stereoscope using a worm pick as described in7.</li>
			<li>Culture the animals in a worm incubator at 15-25 &#176;C (depending on the genetic background) to allow the worms to reproduce.</li>
			<li>Observe progeny each day to evaluate at which developmental stage the coinjection marker is expressed and can be best observed. The expression of most of the currently used coinjection markers is observed at the L4 larval stage or at adulthood.</li>
			<li>Evaluate the transmission rate of the progeny by determining the percentage of fluorescent progeny by observation under a fluorescent stereoscope.</li>
		</ol>
	</li>
	<li>Choose a transgenic line for transgene integration:
		<ol>
			<li>Select the transgenic line with the highest transmission rate as possible (ideally &#8805;80%).</li>
		</ol>
	</li>
	<li>Obtain a population of transgenic animals synchronized at the L4 larval stage for integration:
	<ol>
		<li>Pick 30 fluorescent gravid adults onto five culture plates (six animals by plate).</li>
		<li>Let the worms lay eggs for 3-4 hr at 15 &#176;C.</li>
		<li>Eliminate the adults from the plates after checking for the presence of at least 30 eggs by plate.</li>
		<li>Culture the animals in a worm incubator at 15-25 &#176;C until the progeny reaches the L4 larval stage (96 hr at 15 &#176;C or 36 hr at 25 &#176;C).</li>
	</ol>
	</li>
</ol>
2. UV Irradiation and Recovery of Transgenic Worms
<ol>
	<li>Worm irradiation:
		<ol>
			<li>Pick 30-100 fluorescent transgenic L4 animals onto separate culture plates (15-20 animals by plate).</li>
			<li>Place the plates, with the lids removed, in a UV cross-linker. Irradiate the worms at 0.012 J/cm2. Note that strains are more or less sensitive to UV and that results can be variable depending on the UV irradiator used. A dose range between 0.010-0.015 J/cm2 can be tested.</li>
		</ol>
	</li>
	<li>Worm recovery after irradiation:
	<ol>
		<li>Place plates with irradiated worms overnight at 15 &#176;C for recovery.</li>
		<li>Check for the number animals that are alive. In our hands, a survival rate of around 80-90% is adapted for an efficient irradiation.</li>
		<li>Grow the irradiated animals at 15-25 &#176;C until the progeny has reached the developmental stage allowing for the observation of the coinjection marker expression, as established in step 1.2.</li>
	</ol>
	</li>
</ol>
3. Isolation of Integrated Transgenic Lines
<ol>
	<li>Selection of F1 animals:
		<ol>
			<li>Single 150-200 fluorescent F1 animals onto separate culture plates. For highly transmitting lines (&#8805;80%), 100 F1 animals are enough.</li>
			<li>Keep these F1 plates at 15-25 &#176;C until the progeny reaches an appropriate stage for the observation of fluorescent F2 animals.</li>
			<li>Discard all F1 plates exhibiting either i) no progeny, indicating that the F1 animal was sterile or died, or ii) no or only few fluorescent F2 animals, indicating that the F1 animal did not transmit the transgene at the expected rate. This step is quick and represents a real advantage when compared to the standard protocol that requires an estimation of the percentage of fluorescent F2 worms in order to select F1 plates exhibiting &#8805;75% fluorescent progeny (Table 1).</li>
		</ol>
	</li>
	<li>Selection of F2 animals:
		<ol>
			<li>Single four fluorescent transgenic F2 animals from each selected F1 plate. When using fluorescent transgenes (for example mCherry8, gfp9, or dsRed10), pick F2 worms with a high level of fluorescence as this could indicate that these animals are homozygous for the integrated array. Note that when picking F2 animals it is critical not to carry along eggs or larvae, in order to avoid false negatives in the next step.</li>
			<li>Grow F2 animals at 15-25 &#176;C.</li>
		</ol>
	</li>
	<li>Isolation and validation of integrated transgenic strains:
		<ol>
			<li>Screen F2 plates for 100% fluorescent transgenic F3 worms. The screen is quite rapid as the presence of a single nonfluorescent worm indicates that the plate should be thrown away. A F2 animal homozygous for the integrated transgene will give 100% homozygous fluorescent progeny.</li>
			<li>Single eight fluorescent F3 animals from selected plates to confirm the 100% inheritance of the transgene. In theory, picking one animal is enough at this step. However, picking eight fluorescent F3 animals may be necessary to avoid selecting unfertile or unhealthy animals as irradiation can randomly induce mutations that affect genes important for worm physiology.</li>
			<li>If possible keep several independent integrated transgenic strains, i.e. strains recovered from different F1 animals.</li>
		</ol>
	</li>
</ol>

<ol>
	<li>Berkowitz, L. A., Knight, A. L., Caldwell, G. A., Caldwell, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+stable+transgenic+C.+elegans+using+microinjection.">Generation of stable transgenic C. elegans using microinjection.</a> J. Vis. Exp. (18), e833 (2008).</li><li>Mello, C., Kramer, J. M., Stinchlomb, D., Ambros, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+in+C.elegans:+extrachromosomal+maintenance+and+integration+of+transforming+sequences.">Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences.</a> EMBO J. 10 (12), 3959-3970 (1991).</li><li>Evans, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+and+microinjection.">Transformation and microinjection.</a> WormBook. , (2006).</li><li>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=PCR+Fusion-Based+Approach+to+Create+Reporter+Gene+Constructs+for+Expression+Analysis+in+Transgenic+C.+elegans.">PCR Fusion-Based Approach to Create Reporter Gene Constructs for Expression Analysis in Transgenic C. elegans.</a> BioTechniques. 32, 728-730 (2002).</li><li>Stinchcomb, D. T., Shaw, J. E., Carr, S. H., Hirsh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrachromosomal+DNA+transformation+of+Caenorhabditis+elegans.">Extrachromosomal DNA transformation of Caenorhabditis elegans.</a> Mol. Cell. Biol. 5, 3484-3496 (1985).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77, 71-94 (1974).</li><li>Chaudhuri, J., Parihar, M., Pires-daSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Introduction+to+Worm+Lab:+from+Culturing+Worms+to+Mutagenesis.">An Introduction to Worm Lab: from Culturing Worms to Mutagenesis.</a> J. Vis. Exp. (47), e2293 (2011).</li><li>Merritt, C., Rasoloson, D., Gallo, C., Voronina, E., Ko, D., Seydoux, 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+modular+vector+kit+for+transgenesis+in+C.+elegans.">A modular vector kit for transgenesis in C. elegans.</a> International Worm Meeting. , (2007).</li><li>Chalfie, M., Tu, Y., Euskirchen, G., Ward, W., Green Prasher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+protein+as+a+marker+for+gene+expression.">Fluorescent protein as a marker for gene expression.</a> Science. 263, 802-805 (1994).</li><li>Fleenor, J., 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=Discosoma+sp.+Red+Fluorescent+Protein+(dsRED)+as+a+reporter+in+C.+elegans.">Discosoma sp. Red Fluorescent Protein (dsRED) as a reporter in C. elegans.</a> Worm Breeder's Gazette. 16 (3), 16 (2000).</li><li>Lecroisey, 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=ZYX-1,+the+unique+zyxin+protein+of+Caenorhabditis+elegans,+is+involved+in+dystrophin-dependent+muscle+degeneration.">ZYX-1, the unique zyxin protein of Caenorhabditis elegans, is involved in dystrophin-dependent muscle degeneration.</a> Mol. Biol. Cell. , (2013).</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 (3), 1217-1226 (2001).</li><li>Fr&#248;kjaer-Jensen, 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=Single-copy+insertion+of+transgenes+in+Caenorhabditis+elegans.">Single-copy insertion of transgenes in Caenorhabditis elegans.</a> Nat. Genet. 40 (11), 1375-1383 (2008).</li><li>Dickinson, D. J., Ward, J. D., Reiner, D. J., Goldstein, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+the+Caenorhabditis+elegans+genome+using+Cas9-triggered+homologous+recombination.">Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination.</a> Nat. Methods. 10 (10), 1028-1034 (2013).</li></ol>

The authors declare that they have no competing financial interest.

The protocol described here provides an efficient method to integrate extrachromosomal transgenic arrays into the genome of C. elegans. Using different nonintegrated lines, which transmitted the same transgene Pdyc-1::dyc-1::gfp (pKG45) at different rates (10-15%, 50-60%, and &#62;80%), we showed that the number of integrated strains recovered after UV irradiation is dependent of the initial level of transmission of the nonintegrated line (Table 2). Therefore, transgenes are easier to i...

Transgenes are extensively used in C. elegans for a large range of applications. Transgenic strains are typically generated by DNA injection into the syncytial hermaphrodite gonad1,2. The DNA carrying the transgene of interest is coinjected along with plasmids encoding coinjection markers1,3,4. Coinjection markers are established transgenes leading either to the expression of a fluorescent protein or to a specific behavioral or morphological phenotype. The injected DNAs rearrange to form multi copy extrachromosomal arrays; thus the transgene of interest and the coinjection marker are transmitted together5,4. Transgenic lines are selected in the F2 generation of injected animals by following the phenotype induced by the expression of the coinjection marker (fluorescence or specific phenotype)1. Transgenic lines exhibit partial and variable transmission rates of the extrachromosomal arrays to the next generation. Depending on the strain, 10-100% (100% being a rare event) of the animals inherit extrachromosomal arrays. In addition, transgenic animals are mosaic and express the transgene in a variable number of cells. This mosaicism is likely to be correlated to the transmission rate to the next generation. Indeed, in both germ line and somatic cells the transmission rate depends on the segregation of the extrachromosomal arrays during cell divisions. One way to avoid this issue is to integrate the transgene into the genome. Classically, the integration of a transgene into a chromosome relies on irradiation (ultraviolet or gamma) of transgenic worms carrying extrachromosomal arrays3. Briefly, 50-100 hermaphrodite animals are irradiated at the L4 larval stage. In the F1 generation 200-800 transgenic animals are selected and individually cultured onto plates. According to Mendelian segregation, transgenic F1 animals carrying a heterozygous integration of the transgene into the genome would produce 1/2 of transgenic descendants that are heterozygous for the integrated array, 1/4 of transgenic descendants being homozygous for the integrated array, and 1/4 of descendants that have not integrated the array (Figure 1). Thus, plates containing the F2 generation are visually screened for &#8805;75% transgenic animals as observed by the expression of the coinjection marker. About 1/3 of the F2 animals selected for being integrant candidates are assumed to be homozygous for the integrated array and to produce 100% of homozygous F3 animals. Hence, from each selected plate, three to eight transgenic F2 animals are individually cultured and plates with 100% of transgenic descendants are selected. Next, eight F3 animals are individually cultured to confirm the 100% inheritance of the transgene.
The main disadvantages of this method are that
<ol type="i">
	<li>it requires a visual screen of several hundred F1 plates for &#8805;75% transgenic F2 progeny, which is time consuming due to the variable and unpredictable percentage of transmission of nonintegrated extrachromosomal arrays. Alternatively, to avoid the screening of F1 plates several subsequent starvation steps can be performed after irradiation of P0 animals; followed by picking 100 transgenic animals and screening for 100% transgenic progeny3.</li>
	<li>it is not adapted for strains exhibiting a percentage of extrachromosomal array transmission higher than 60% as this percentage is difficult to distinguish this percentage from 75%.</li>
	<li>as only strains with a relatively low transmission rate can be used, a high number of irradiated transgenic P0 animals is necessary as well as the observation of numerous F1 and F2 animals, which is highly time consuming and requires a considerable number of plates.</li>
</ol>
The irradiation of worms presumably induces double-strand breaks in the DNA and integration of extrachromosomal arrays into chromosomes occurs during DNA repair. Thus, it is likely that chances to obtain successful irradiation mediated integration are directly proportional to the number of germ line cells carrying an extrachromosomal array. Strains with a high transmission rate may contain more transgenic DNA arrays than strains with a low transmission rate as suggested in2, and exhibit a higher number of germ line cells carrying extrachromosomal arrays. Thus, we reasoned that in highly transmitting lines, transgenes might be easier to integrate than in lines with a low transmission rate. However, the standard protocol excludes the possibility of integrating transgenes with high transmission frequency as the percentage of transgenic animals with a nonintegrated extrachromosomal array can hardly be distinguished from that of the progeny of a F1 animal carrying a heterozygous integration of the transgene into the genome (Figure 1).
Here we show that for a particular transgene, the number of integrated lines recovered after irradiation with ultraviolet (UV) light is dependent on the initial percentage of transmission of the nonintegrated array. We present an improved protocol for UV irradiation-mediated transgene integration that is particularly relevant to lines exhibiting a high rate of transgene array transmission.

<table border="0" cellpadding="0" cellspacing="0" width="865">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:167px;">Agar</td>
			<td style="width:173px;">Prolabo</td>
			<td style="width:181px;">20768.292</td>
			<td style="width:344px;">for NGM (nematode growth medium) preparation (6,7)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td style="width:173px;">Chimie-Plus</td>
			<td style="width:181px;">40045</td>
			<td style="width:344px;">for NGM preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto-peptone</td>
			<td style="width:173px;">BD</td>
			<td style="width:181px;">211820</td>
			<td style="width:344px;">for NGM preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4</td>
			<td style="width:173px;">Roth</td>
			<td style="width:181px;">3904-1</td>
			<td style="width:344px;">for NGM preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">K2HPO4</td>
			<td style="width:173px;">Prolabo</td>
			<td style="width:181px;">26930.293</td>
			<td style="width:344px;">for NGM preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4</td>
			<td style="width:173px;">Roth</td>
			<td style="width:181px;">P027.2</td>
			<td style="width:344px;">for NGM preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2</td>
			<td style="width:173px;">Prolabo</td>
			<td style="width:181px;">22317.297</td>
			<td style="width:344px;">for NGM preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cholesterol</td>
			<td style="width:173px;">Rectapur</td>
			<td style="width:181px;"></td>
			<td style="width:344px;">for NGM preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LB-broth</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:181px;">L3022</td>
			<td style="width:344px;">for culture of OP50 bacteria</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name of Equipment</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">U.V. cross-linker</td>
			<td>Fischer Scientific</td>
			<td></td>
			<td>Wavelength 254 nm</td>
		</tr>
		<tr height="54">
			<td height="54" style="height:55px;width:167px;">Microscope SteREO Discovery V8</td>
			<td>Carl Zeiss, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dishes, 60 mm</td>
			<td>CML</td>
			<td>BPS55E6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platinium wire 0.25 mm</td>
			<td>Alfa Aesar</td>
			<td>7440-06-4</td>
			<td>To make a pick for worms (7)</td>
		</tr>
	</tbody>
</table>

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.

Integrations of transgenes in two different lines transmitting extrachromosomal arrays at about the same frequency (50-60%) were performed using the standard and the improved protocols (Table 1). The standard protocol allowed the recovery of three independent integrated lines after UV irradiation of 91 transgenic L4 animals, while the improved protocol allowed the recovery of one integrated line after UV irradiation of 30 transgenic L4 animals. This suggests that the efficiency of integration is similar ...

Watch this Scientific Journal Video about A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome at JoVE.com

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.

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 ...

a-rapid-protocol-for-integrating-extrachromosomal-arrays-with-high

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Biology

8.9K visualizações.  Université Claude Bernard Lyon. 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.

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

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

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson&#039;s Disease Genes

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

Aplicação de um elegans dopamina C. Ensaio degeneração dos neurônios de Validação do Potencial Doença de Parkinson Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the anatomy and connectivity of its nervous system has been determined from electron micrographs and refined by pharmacological assays. In this video, we describe how two complementary neural stimulants, an acetylcholinesterase inhibitor, called aldicarb, and a gamma-aminobutyric acid (GABA) receptor antagonist, called pentylenetetrazole (PTZ), may be employed to specifically characterize signaling at C. elegans neuromuscular junctions (NMJs) and facilitate our understanding of antagonistic neural circuits.
Of 302 C. elegans neurons, nineteen GABAergic D-type motor neurons innervate body wall muscles (BWMs), while four GABAergic neurons, called RMEs, innervate head muscles. Conversely, thirty-nine motor neurons express the excitatory neurotransmitter, acetylcholine (ACh), and antagonize GABA transmission at BWMs to coordinate locomotion. The antagonistic nature of GABAergic and cholinergic motor neurons at body wall NMJs was initially determined by laser ablation and later buttressed by aldicarb exposure. Acute aldicarb exposure results in a time-course or dose-responsive paralysis in wild-type worms. Yet, loss of excitatory ACh transmission confers resistance to aldicarb, as less ACh accumulates at worm NMJs, leading to less stimulation of BWMs. Resistance to aldicarb may be observed with ACh-specific or general synaptic function mutants. Consistent with antagonistic GABA and ACh transmission, loss of GABA transmission, or a failure to negatively regulate ACh release, confers hypersensitivity to aldicarb. Although aldicarb exposure has led to the isolation of numerous worm homologs of neurotransmission genes, aldicarb exposure alone cannot efficiently determine prevailing roles for genes and pathways in specific C. elegans motor neurons. For this purpose, we have introduced a complementary experimental approach, which uses PTZ.
Neurotransmission mutants display clear phenotypes, distinct from aldicarb-induced paralysis, in response to PTZ. Wild-type worms, as well as mutants with specific inabilities to release or receive ACh, do not show apparent sensitivity to PTZ. However, GABA mutants, as well as general synaptic function mutants, display anterior convulsions in a time-course or dose-responsive manner. Mutants that cannot negatively regulate general neurotransmitter release and, thus, secrete excessive amounts of ACh onto BWMs, become paralyzed on PTZ. The PTZ-induced phenotypes of discrete mutant classes indicate that a complementary approach with aldicarb and PTZ exposure paradigms in C. elegans may accelerate our understanding of neurotransmission. Moreover, videos demonstrating how we perform pharmacological assays should establish consistent methods for C. elegans research.

This video demonstrates how to employ two neural stimulants, aldicarb and pentylenetetrazole (PTZ), in complementary ways to study synaptic function in the nematode, C. elegans. This complementary approach may also be used to shed light on evolutionarily conserved mechanisms for modulating neuronal synchrony and has implications for epilepsy and seizures.

Paradigmas para Caracterização farmacológica de C. elegans Mutants Transmissão Sináptica

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the ...

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 ...

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

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

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

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

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 ...

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 ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

Rastreamento Deformações Tissue Morfogenéticas no embrião de galinha precoce

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

Quantitativos e automatizada de alto rendimento de todo o genoma Telas de RNAi em C. elegans</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

Um protocolo simples para extrair Hemócitos de Lagartas selvagens

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...