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><tbody><tr><td>Agar</td><td>Prolabo</td><td>20768.292</td><td>for NGM (nematode growth medium) preparation (6,7)</td></tr><tr><td>NaCl</td><td>Chimie-Plus</td><td>40045</td><td>for NGM preparation</td></tr><tr><td>Bacto-peptone</td><td>BD</td><td>211820</td><td>for NGM preparation</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Roth</td><td>3904-1</td><td>for NGM preparation</td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Prolabo</td><td>26930.293</td><td>for NGM preparation</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Roth</td><td>P027.2</td><td>for NGM preparation</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Prolabo</td><td>22317.297</td><td>for NGM preparation</td></tr><tr><td>Cholesterol</td><td>Rectapur</td><td></td><td>for NGM preparation</td></tr><tr><td>LB-broth</td><td>Sigma</td><td>L3022</td><td>for culture of OP50 bacteria</td></tr><tr><td>EQUIPMENT</td><td></td><td></td><td></td></tr><tr><td>U.V. cross-linker</td><td>Fischer Scientific</td><td></td><td>Wavelength 254 nm</td></tr><tr><td>Microscope SteREO Discovery V8</td><td>Carl Zeiss, Inc.</td><td></td><td></td></tr><tr><td>Petri dishes, 60 mm</td><td>CML</td><td>BPS55E6</td><td></td></tr><tr><td>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 ...

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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 ...

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JoVE Journal

Biology

8.9K Aufrufe.  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.

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

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Optogenetische Zufallsmutagenese Mit Histon-miniSOG in<em&gt; C. elegans</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Forschung

Genetik

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

CRISPR/Cas9-vermittelten gezielte Integration In Vivo mit einer Homologie-vermittelte Ende verbinden-basierte Strategie

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

C. Elegans Excretory Kanal als Modell für die intrazelluläre Lumen Morphogenese und In Vivo polarisiert Membrane Biogenesis in einer einzelnen Zelle: Kennzeichnung von GFP-Fusionen, RNAi Interaktion Bildschirm und Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended ...

Entwicklungsbiologie

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes such as development are affected. The Caenorhabditis elegans germline is a great model system for studying PPIs that are related to the regulation of stem cells, meiosis, and development. There are a variety of well-developed techniques that allow proteins of interest to be tagged for recognition by standard antibodies, making this system advantageous for proximity ligation assay (PLA) reactions. As a result, the PLA is able to show where PPIs occur in a spatial and temporal manner in germlines more effectively than alternative approaches. Described here is a protocol for the application and quantification of this technology to probe PPIs in the C. elegans germline.

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

This protocol demonstrates use of the proximity ligation assay to probe for protein-protein interactions in situ in the C. elegans germline.

In Situ Detektion von Ribonucleoprotein Complex Assembly in der C. elegans Germline mit Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes ...

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.

Proteinextrakt-Präparation und Co-Immunpräzipitation von Caenorhabditis elegans

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

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.

Die Produktion von C. elegans Transgene via Recombineering mit dem GalK Selektionsmarker

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

Biologie

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Imaging C. elegans Embryos mit einem Epifluoreszenz-Mikroskop und Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

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.

Bestimmung genetischer Expressionsprofile in C. elegans Verwenden Microarray-und 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, ...

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.

Kombinierte Nukleotid- und Protein Extraktionen 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 ...

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.

Kleine Extraktion von Caenorhabditis elegans Genomischer DNA

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

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

Zusammenfassung

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