This work was supported by seed funds from the University of Pittsburgh and NIH grant AG028977 to A.L.F.

Overview
Traditionally transgenic worms were generated by microinjecting transgene DNA into the C. elegans germline 1,2,3,4. While successful, this approach required specialized equipment and the development of experience with worm anatomy and the microinjection technique. More recently biolistic bombardment has been developed as an alternate approach which uses DNA-coated gold particles to introduce foreign DNA into the germline 5,6. This approach requires less time investment in terms of practice to become successful.
The biolistic bombardment of C. elegans to generate transgenic worms has low efficiency at the level of the individual worm so success requires a large quantity of animals. These can be grown in several ways, but we use egg plates to reduce the work and number of plates involved. Our protocol starts with the preparation of egg plates and worms and then focuses on the bombardment protocol using the Bio-Rad PDS-1000/He Hepta System. We adapted our protocol in part from work of Berezikov et al 7.
With bombardment, the unc-119 gene is typically used as a marker for transgenic animals. Worm strains carrying this mutation are severely uncoordinated and barely move, are dumpy in appearance, and are unable to form dauer larvae 8. The dauer is an alternate larval stage which is used to delay development to a reproductive adult under adverse conditions, and the entry into dauer is regulated by multiple genes 9. Several strains carrying the unc-119 gene are available from the C. elegans Genetics Center (CGC, Minneapolis, MN). The original DP38 strain (unc-119(ed3)) also carries an unrelated dauer-formation constitutive (daf-c) mutation which could affect downstream experiments. Several labs have outcrossed this mutation, and the HT1593 strain is available from the CGC. We find that transgenic animals are somewhat easier to identify with the DP38 strain and tend to use this strain for creating transgenic animals. When necessary, we use HT1593 to outcross the transgenic worms to remove the daf-c mutation. Getting the DP38 worms to grow is one of the slowest steps in the protocol so we maintain a stock of growing plates while preparing the transgenes.
Expression of the unc-119 marker along with the gene of interest allows easy identification of the animals expressing the transgene based on rescue of normal motility and body size 5. The unc-119 gene can be obtained from several sources, and vectors using the unc-119 genomic DNA, unc-119 promoter and cDNA, and the genomic DNA for the smaller C. briggsiae gene are used 8,10. We recently described a simple protocol to add an unc-119 cassette to any plasmid containing an ampicillin resistance gene by homologous recombination 11 and a method to modify worm fosmids to carry the unc-119 gene by Cre-lox recombination to generate transgenic animals 12. The plasmids are available from Addgene Inc. (Cambridge, MA).
While the effort involved in performing a single bombardment is significant but manageable, the additional work involved in performing multiple bombardments on a single day is minimal. Consequently, we routinely cluster the production of transgenic worms to decrease the work involved in generating each.
1. Egg Plate Preparation
The unc-119 mutant worms are difficult to grow on standard NGM plates because with their impaired mobility they tend to starve on parts of a plate while other parts of the plate still contain food. Egg plates solve this problem as the thick food layer on egg plates allows unc-119 worms to crawl more easily and to take over all the plate 7. The egg plates also have the benefit of supporting the growth of a large number of worms so fewer plates are needed 13. Usually 5 egg plates are sufficient to grow worms for one bombardment. The recipe below makes roughly 50 plates but can be halved to make fewer if desired.
The egg plates can become easily contaminated so we use both antibiotics and antifungal drugs in the plates and only use eggs generated by hypochlorite treatment for seeding the plates.
Day 1:
<ol>
	<li>Prepare LB (400 mL) in a 500mL bottle. Autoclave for 20min.</li>
	<li>Pour ~50 10 cm NGA plates using 1 liter NGA with 2% agar. We add nystatin 10 mL per liter and streptomycin to 200 &mu;g/mL 13.</li>
	<li>Grow a 40 mL overnight culture of OP50-1 in LB with 200 &mu;g/mL streptomycin. This strain is streptomycin resistant and available from CGC.</li>
	<li>Autoclave a 500mL bottle for next day.</li>
</ol>
Day 2:
<ol>
	<li>Separate yolks of 10 chicken eggs into the sterile bottle. We use a yolk separator (available at household stores, including Target). Shake.</li>
	<li>Bring volume to 400mL with LB. Shake</li>
	<li>Incubate at 60&deg;C for 1 hour.</li>
	<li>Cool to room temperature.</li>
	<li>Add 40mL of OP50-1. Shake</li>
	<li>Distribute 5-8 mL of mixture per plate.</li>
	<li>Allow plates to settle at room temperature overnight.</li>
</ol>
Day 3:
<ol>
	<li>Gently pour off remaining liquid from plates. Allow to dry with lids on at room temperature overnight.</li>
</ol>
Day 4:
<ol>
	<li>Put plates into a box or plastic bag and store at 4&deg;C until needed. These plates appear to be OK to use for 1-2 months.</li>
</ol>
2. Seeding Egg Plates
<ol>
	<li>Expand the DP38 worms on 6 cm NGA plates by adding concentrated OP50 to recently starved plates. For a single bombardment, we use ~6 small plates to seed 5 egg plates. For multiple bombardments, we use the small plates to instead seed 2 enriched peptone plates before seeding the egg plates.</li>
	<li>Isolate eggs from the DP38 worms via the use of hypochlorite 13,14. Resuspend the eggs in sterile S-basal or M9 buffers 13,14.</li>
	<li>Add the eggs (&gt;10,000 per plate) to the appropriate number of egg plates. Grow the worms on egg plates at 20&deg;C for 7-10 days to use for bombardment. The plates will be ready to use once the worms have begun to clear the food from the plates. These plates are really hard to starve and growing for longer durations will improve worm yield.</li>
</ol>
Bombardment
We prepare the gold stock ahead of time and keep it stored at 4&deg;C for use. Also needed are unspotted and spotted 10 cm NGA plates. The unspotted plates need to be poured well in advance and allowed to thoroughly dry to facilitate absorption of the liquid added with the worms.
On the day of the bombardment, we wash off the worms first then start preparing the DNA-coated gold particles. We then add the worms to the unspotted NGA plates and finish washing the gold particles and transferring them to the macrocarriers.
Gold particle preparation:
<ol>
	<li>Weigh 60mg of gold particles in a 1.7mL tube and soak in 70% ethanol for 15min. Spin briefly to precipitate the gold particles.</li>
	<li>Wash 3 times in sterile water and spin briefly each time to collect the gold.</li>
	<li>Resuspend the gold in 1mL of 50% sterile glycerol. This is the stock gold particle solution and can be stored for months at 4&deg;C.</li>
</ol>
Worms:
<ol>
	<li>Float the DP38 worms off the egg plates with S-basal or M9 buffer and transfer them to a 50mL conical tube.</li>
	<li>Keep the tubes on the bench until the worms are in the bottom. Then aspirate the buffer and add fresh S-basal or M9. Wash 2 or 3 times, until buffer is relatively clear of debris. Keep the worms at this step until the DNA coating is completed.</li>
	<li>Aspirate and leave approx 2-3 mL of worms in buffer per bombardment. Transfer to a 10 cm unspotted NGA plate on ice. It is important minimize the transferred volume as the NGA plate will need to dry completely before bombardment. Be sure to cover the entire surface of the plate with the worms.</li>
	<li>Allow plate to dry on ice. The ice will prevent the worms from clumping while drying.</li>
</ol>
This step is important: the plate should be dry and the worms evenly dispersed on the NGA plate. Keeping the worms on ice prevents them from moving on the plate and aggregating into piles.
DNA preparation (for one bombardment):
<ol>
	<li>Mix in a 1.7mL tube:
	<ul>
		<li>50&mu;l gold particles. Resuspend thoroughly before adding.</li>
		<li>50&mu;l DNA (10-15ug). We see little difference between circular or linear DNA. Usually we use Qiagen midiprep cartridges for DNA purification (Qiagen Inc., Valencia, CA).</li>
		<li>50&mu;l 2.5M CaCl2. This solution is stable for several weeks at room temperature.</li>
		<li>20&mu;l 0.1M spermidine. 
		 
		The spermidine is unstable in aqueous solution. We make 16&mu;l spermidine aliquots and store at -20&deg;C. We then add 1mL of water right before use to obtain a 0.1M solution. This solution must be prepared fresh for each bombardment. 
		 
		We usually vortex the gold particles in an 1.7mL tube at a low speed and then add the CaCl2, DNA and spermidine/protamine dropwise while vortexing. For the people that have transfected with calcium phosphate this technique is familiar. 
		 
		We have also used protamine instead of spermidine with very good results 15. We prepare a fresh solution (1mg/mL) in water and use 50&mu;l instead of the 20&mu;l of spermidine. If we use protamine, we usually incubate the mixture at room temperature for 10 minutes instead of placing on ice. Protamine does not need to be kept frozen and is a powder instead of a liquid.</li>
	</ul>
	</li>
	<li>Incubate the mixture on ice for 30min, and periodically mix to keep the gold particles in suspension. Then spin briefly and aspirate the supernatant.</li>
	<li>Wash with 300&mu;l 70% ethanol. Spin briefly.</li>
	<li>Wash with 1mL 100% ethanol. Spin briefly.</li>
	<li>Resuspend in 170&mu;l 100% ethanol.</li>
</ol>
Preparation of macrocarriers:
<ol>
	<li>Rinse 7 macrocarriers (Bio-Rad Laboratories, Hercules, CA) in 2-propanol. Put them on tissue paper and let them dry at room temperature.</li>
	<li>Add 20&mu;l of DNA/gold mixture at the center of each macrocarrier. Be sure to vortex right before pipetting to keep the gold in suspension.</li>
	<li>Let the ethanol evaporate.</li>
</ol>
3. Bombardment
Be sure to perform a blank bombardment before the experiment to flush helium through the system.
<ol>
	<li>Turn on vacuum pump and close vacuum pump trap. We use an oil-free vacuum pump.</li>
	<li>Turn on helium tank. Check that pressure is &ge;2200psi.</li>
	<li>Wet rupture disk (1350 psi) in 2-propanol, placing in retaining cap for hepta adapter.</li>
	<li>Screw adapter into PDS-1000 system and tighten with the supplied torque wrench.</li>
	<li>Place the 7 macrocarriers into the holder and seat them with the supplied tool. Then put a hepta stop screen and the bottom on to the holder. Flip the holder over and place on the second shelf from the top. The gold spotted side of the macrocarriers must be facing down at this point.</li>
	<li>Align the holes in the top of the macrocarrier holder with the outlets of the hepta adapter.</li>
	<li>Place the NGA plate coated with worms (lid off) on the lowest shelf in bombardment chamber.</li>
	<li>Completely open the vacuum flow-rate knob on the PDS-1000. Press vac button until chamber reaches 26 In Hg. Then switch to hold position to maintain the vacuum.</li>
	<li>Press and hold the fire button until disk ruptures. This will happen when pressure reaches 1350psi. Sometimes this takes 5-20 seconds to occur. Release the button.</li>
	<li>Release vacuum from chamber (vent position), and remove plate.</li>
	<li>Turn vacuum and helium off.</li>
	<li>Fire several times until the line does not contain helium (helium gauge should mark 0 psi).</li>
	<li>Turn off the PDS-1000 system.</li>
</ol>
Worm recovery after bombardment:
<ol>
	<li>Leave the bombarded plate at 20&deg;C for 20 minutes.</li>
	<li>Float the worms from the plate with 10 mL S-basal or M9 buffer.</li>
	<li>Put 0.5mL worms on 20 - 10 cm spotted NGA plates.</li>
	<li>Leave at 20&deg;C or room temperature (22-24&deg;C) for about 2 weeks before checking for rescued worms.</li>
	<li>Screen for worms with the unc-119(+) phenotype with a dissecting microscope. The optimal time for screening is after the plates have starved since the rescued worms have survival advantage over non-rescued worms.</li>
	<li>Generate one individual line from each 10 cm plate that contained rescued worms.</li>
</ol>
4. Representative Results
The success of the protocol with regards to obtaining transgenic animals depends on the particular transgene. For promoter:GFP reporter transgenes we have obtained up to 20 lines. A more typical result is 3-10 lines. Up to 30% of the transgenic lines are integrated lines, but this is random and we will perform multiple bombardments on a single day if an integrated line is particularly desired.

<ol>
	<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=.">.</a> Wormbook. , 1-15 (2006).</li><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. , (2008).</li><li>Mello, C., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+transformation.">DNA transformation.</a> Methods Cell Biol. 48, 451-482 (1995).</li><li>Mello, C. C., Kramer, J. M., Stinchcomb, 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, 3959-3970 (1991).</li><li>Praitis, V., Casey, E., Collar, D., Austin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+low-copy+integrated+transgenic+lines+in+Caenorhabditis+elegans.">Creation of low-copy integrated transgenic lines in Caenorhabditis elegans.</a> Genetics. 157, 1217-1226 (2001).</li><li>Wilm, T., Demel, P., Koop, H. U., Schnabel, H., Schnabel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ballistic+transformation+of+Caenorhabditis+elegans.">Ballistic transformation of Caenorhabditis elegans.</a> Gene. 229, 31-35 (1999).</li><li>Berezikov, E., Bargmann, C. I., Plasterk, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homologous+gene+targeting+in+Caenorhabditis+elegans+by+biolistic+transformation.">Homologous gene targeting in Caenorhabditis elegans by biolistic transformation.</a> Nucleic Acids Res. 32, e40-e40 (2004).</li><li>Maduro, M., Pilgrim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+cloning+of+unc-119,+a+gene+expressed+in+the+Caenorhabditis+elegans+nervous+system.">Identification and cloning of unc-119, a gene expressed in the Caenorhabditis elegans nervous system.</a> Genetics. 141, 977-988 (1995).</li><li>Riddle, D. L., Swanson, M. M., Albert, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interacting+genes+in+nematode+dauer+larva+formation.">Interacting genes in nematode dauer larva formation.</a> Nature. 290, 668-671 (1981).</li><li>Maduro, M., Pilgrim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+of+function+and+expression+of+unc-119+from+two+Caenorhabditis+species+despite+divergence+of+non-coding+DNA.">Conservation of function and expression of unc-119 from two Caenorhabditis species despite divergence of non-coding DNA.</a> Gene. 183, 77-85 (1996).</li><li>Ferguson, A. A., Fisher, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrofitting+ampicillin+resistant+vectors+by+recombination+for+use+in+generating+C.+elegans+transgenic+animals+by+bombardment.">Retrofitting ampicillin resistant vectors by recombination for use in generating C. elegans transgenic animals by bombardment.</a> Plasmid. 62, 140-145 (2009).</li><li>Zhang, Y., Nash, L., Fisher, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified,+robust,+and+streamlined+procedure+for+the+production+of+C.+elegans+transgenes+via+recombineering.">A simplified, robust, and streamlined procedure for the production of C. elegans transgenes via recombineering.</a> BMC Dev Biol. 8, 119-119 (2008).</li><li>Lewis, J. A., Fleming, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+culture+methods.">Basic culture methods.</a> Methods Cell Biol. 48, 3-30 (1995).</li><li>Sulston, J. E., Horvitz, H. R., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The nematode, Caenorhabditis elegans. , 587-606 (1988).</li><li>Sivamani, E., DeLong, R. K., Qu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protamine-mediated+DNA+coating+remarkably+improves+bombardment+transformation+efficiency+in+plant+cells.">Protamine-mediated DNA coating remarkably improves bombardment transformation efficiency in plant cells.</a> Plant Cell Rep. 28, 213-221 (2009).</li><li>Johnston, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biolistic+transformation:+microbes+to+mice.">Biolistic transformation: microbes to mice.</a> Nature. 346, 776-777 (1990).</li></ol>

No conflicts of interest declared.

Biolistic bombardment is a simple method to introduce foreign DNA into many organisms, including C. elegans 1,5,6,7,16. It relies upon gold particles forming a complex with DNA in the presence of CaCl2. Cationic polyamines, such as spermidine, protect DNA from nuclease degradation in vivo. Since spermidine is a labile molecule, it is important to store it in small aliquots at -20&deg;C, and make the solution right before performing the bombardment. As we described in the bombardment protoco...

The volume for a solution in, <a href="http://www.jove.com/video/2090">Generation of Transgenic C. elegans by Biolistic Transformation</a>, was incorrect.

Erratum: Generation of Transgenic C. elegans by Biolistic Transformation

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Gold particles</td>
<td>&nbsp;</td>
<td>Inbio gold</td>
<td>BD021</td>
<td>1micron</td>
</tr>
<tr>
<td>Midiprep kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12143</td>
<td>&nbsp;</td>
<tr>
<td>Spermidine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S4139</td>
<td>&nbsp;</td>
<tr>
<td>Protamine</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td> P4505</td>
<td>&nbsp;</td>
<tr>
<td>Macrocarriers</td>
<td>&nbsp;</td>
<td>Biorad</td>
<td>165-2335</td>
<td>&nbsp;</td>
<tr>
<td>PDS-1000/He Hepta System</td>
<td>&nbsp;</td>
<td>Biorad</td>
<td>165-2257</td>
<td>&nbsp;</td>
<tr>
<td>Rupture disk</td>
<td>&nbsp;</td>
<td>Biorad</td>
<td>165-2330</td>
<td>&nbsp;</td>
<tr>
<td>Nystatin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>N1638</td>
<td>&nbsp;</td>
<tr>
<td>Streptomycin </td>
<td>&nbsp;</td>
<td>MP</td>
<td>100556</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Generation of Transgenic C. elegans by Biolistic Transformation at JoVE.com

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.

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-of-transgenic-c-elegans-by-biolistic-transformation

Research

JoVE Journal

Biology

21.8K vues.  University of Pittsburgh. Des vers transgéniques sont couramment utilisés dans C. elegans Recherche. Décrite est un protocole simple, mais efficace, d'introduire des transgènes dans les vers utilisant bombardement biolistique avec des particules d'or enrobées d'ADN. L'effort et les résultats des bombardements se comparent favorablement avec microinjection pour la génération d'animaux transgéniques.

Vidéo: Generation of Transgenic C. elegans by Biolistic Transformation

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.

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

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.

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

Dissecting and Recording from The C. Elegans Neuromuscular Junction

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

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

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

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

C. elegans Chemotaxis Assay

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

C. elegans Chemotaxis Assay

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

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

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.