The authors are very grateful to RJ Sommer and X Wang for assistance with microinjection, as well as insightful comments from the anonymous reviewers. This work is supported by NIH grant SC2GM089602.

1. Transgenesis: DNA preparation
<ol>
 <li>Dominant co-injection marker: pRL3 [Ppa-prl-1(tu92)] 
 The pRL3 plasmid is a dominant co-injection marker for visually identifying successful transformation events. This plasmid encodes for a dominant mutant allele (tu92) of the Ppa-prl-1 gene closely related to the sqt-1 collagen gene in C. elegans and transforms the wildtype sinusoidal locomotion into clockwise twisting motions along the worm's body axis1,5. The transformed animal is very similar to the popular dominant selection marker rol-6 (su1006) used in C. elegans for the past 20 years.</li>
 <li>Target reporter transgene: Ppa-egl-4p::gfp 
 A gene of interest can be linked transcriptionally or translationally to a GFP coding sequence9. In this study, the Ppa-egl-4promoter::gfp (egl-4, cGMP dependent protein kinase) was used to determine if a 2 kb fragment upstream of the start of translation can confer GFP expression in P. pacificus PS312. This GFP vector contains a GFP coding region with modified 
 introns and the multipurpose Ppa-rpl-23 3' UTR terminator5 kindly provided by Xiaoyue Wang and Ralf J. Sommer (Max-Planck Institute, Tuebingen, Germany, EU).</li>
 <li>Genomic DNA: PS312 
 The addition of wildtype P. pacificus PS312 genomic DNA is required to obtain stable transgene inheritance, particularly from transgenic F1 animals to their F2 progeny5. It is not yet certain if the genomic DNA forms complex extra chromosomal arrays with the two transgenes (the co-injection marker pRL-3 and the Ppa-egl-4p::gfp reporter) similar to those observed in stable F2 transgenic lines in C. elegans3. However, the requirement to have the genomic DNA and transgenes to share identical cohesive overhangs strongly suggests the formation of complex extra-chromosomal arrays the host cells can recognize for proper DNA transmission (gDNA prepared using Sigma G1N10 kit).</li>
 <li>DNA digestion 
 Restriction enzymes are used to linearize the Ppa-egl-4p::gfp vector DNA and to produce sticky overhangs compatible with the co-injection marker pRL3 and PS312 gDNA. Mix by tapping, not vortexing. Incubate for one hour at 37&deg;C.</li>
</ol>
<table border="1">
 <tbody>
 <tr>
 <td>pRL3</td>
 <td>4 &mu;g</td>
 </tr>
 <tr>
 <td>10xbuffer</td>
 <td>10 &mu;l</td>
 </tr>
 <tr>
 <td>PstI (10 U/&mu;l)</td>
 <td>4 &mu;l</td>
 </tr>
 <tr>
 <td>dH2O</td>
 <td>~</td>
 </tr>
 <tr>
 <td>Total:</td>
 <td>100 &mu;L</td>
 </tr>
 <tr>
 <td>Ppa-egl-4p::gfp</td>
 <td>4 &mu;g</td>
 </tr>
 <tr>
 <td>10x buffer</td>
 <td>10 &mu;l</td>
 </tr>
 <tr>
 <td>SalI (10 U/&mu;l)</td>
 <td>4 &mu;l</td>
 </tr>
 <tr>
 <td>dH2O</td>
 <td>~</td>
 </tr>
 <tr>
 <td>Total:</td>
 <td>100 &mu;l</td>
 </tr>
 <tr>
 <td>gDNA PS312</td>
 <td>10 &mu;g</td>
 </tr>
 <tr>
 <td>10x buffer</td>
 <td>10 &mu;l</td>
 </tr>
 <tr>
 <td>Pst I and Sal I (10 U/&mu;l)</td>
 <td>8 &mu;l</td>
 </tr>
 <tr>
 <td>dH2O</td>
 <td>~</td>
 </tr>
 <tr>
 <td>Total:</td>
 <td>100 &mu;l</td>
 </tr>
 </tbody>
</table>
Table 1. Mixture for Restriction Enzyme Digest.
<ol start="5">
 <li>DNA precipitation and preparation
 <ol>
 <li>Add 1:10 sodium acetate (3 M NaCOOCH3 pH 5.2) and 2.5X the volume of 100% ethanol to the to digested product; add 20 ng/&mu;l of glycogen to see pellet easier.</li>
 <li>Centrifuge at 14,000 rpm for 15 minutes at 4&deg;C.</li>
 <li>Decant the supernatant by tipping slowly the centrifuge tube upside down and then tapping it on a tissue paper, making sure that the pellet is secure at the bottom corner of the tube and free of ethanol.</li>
 <li>Add 1 ml of 75% ethanol.</li>
 <li>Immediately spin at 14,000 rpm for 5 minutes at 4&deg;C.</li>
 <li>Decant the supernatant by slowly tipping the centrifuge tube upside down and then tapping it on a tissue paper making sure that the pellet it secure at the bottom corner of the tube and free of ethanol.</li>
 <li>Dry pellet in vacuum centrifuge at 14,000 rpm for 5 minutes at 25-30&deg;C, until dry and completely free of ethanol.</li>
 <li>Add 30 &mu;l of TE or sterile dH2O and leave overnight at 4&deg;C before mixing briefly with a vortex.</li>
 <li>Table 2 describes how to create the injection mixture from each purified DNA for injection.</li>
 <li>Store at -20&deg;C. After thawing the mixture for injection, spin down the tube at 14,000 rpm for 5 minutes to rid debris particles that may clog the injection needle.</li>
 </ol>
 </li>
</ol>
<table border="1">
 <tbody>
 <tr>
 <td>Genetic material</td>
 <td>Concentration</td>
 </tr>
 <tr>
 <td>Ppa-egl-4p::gfp (SalI)</td>
 <td>&gt;5 ng/&mu;l (0.1-10ng/&mu;l)</td>
 </tr>
 <tr>
 <td>pRL3 (PstI)</td>
 <td>&gt;1 ng/&mu;l</td>
 </tr>
 <tr>
 <td>gDNA PS312 (PstI + SalI)</td>
 <td>&gt;60 ng/&mu;l</td>
 </tr>
 <tr>
 <td>dH2O</td>
 <td>~</td>
 </tr>
 <tr>
 <td>Total:</td>
 <td>30 &mu;l</td>
 </tr>
 </tbody>
</table>
Table 2. Ppa-prl-1 injection mixture
2. RNA Interference: In vitro double stranded RNA synthesis
<ol>
 <li>Use PCR to amplify the gene of interest. RHL091 5'- agtggatccGAAGGTCCATACGGGAGC-3' (BamHI site) and RHL092 5'- tatctgcagGTGAGGAGTACCAGGAGAG-3' (PstI site) were used to amplify a 464 bp genomic fragment (position 3-466) from the pRL3 vector containing the Ppa-prl-1(tu92) allele.</li>
 <li>Digest the PCR product with BamHI and PstI.</li>
 <li>Ligate the fragment to BamHI/PstI digested pL4440 RNAi feeding vector2.</li>
 <li>Transform the inserted vector into competent cells and grow them on Ampicillin LB media plates (50 &mu;g/ml).</li>
 <li>Select the successfully inserted vector by PCR using the T7 primers.</li>
 <li>Use the T7 flanked PCR product containing the prl-1 gene to make double-stranded RNA dsRNA) using the BLOCK-IT RNAi synthesis kit (Invitrogen).</li>
 <li>The dsRNA concentration is best if &gt;200 ng/&mu;l, up to 1 &mu;g/&mu;l for injection.</li>
 <li>Store at -20&deg;C. After thawing the mixture for injection, spin down the tube at 14,000 rpm for 5 minutes to rid of debris particles that may clog the injection needle.</li>
</ol>
3. Microinjection: Protocol for injection of transgenes and/or dsRNA
<ol>
 <li>Injection Needles
 <ol>
 <li>Set the Narishige needle puller (model #: PC-10) temperature:
 <ol>
 <li>Turn bottom knob to "No.2 Heater."</li>
 <li>Turn the "No.2 Heater Adj." knob until panel reads 55.0-60.0&deg;C for tapered needle tip of varying lengths.</li>
 </ol>
 </li>
 <li>Turn the bottom knob back to "Step 1" (Pulls needle in one step).</li>
 <li>Load needle into the chamber and make sure it is secure.</li>
 <li>Press the "Start" button (red button) and allow needle to be pulled with all the weights (this should make two identical needles in opposite directions).</li>
 <li>Remove needle from the chamber and store in a plastic culture plate secured in place by Play-Doh to prevent dust from accumulating on the needles.</li>
 </ol>
 </li>
 <li>Mounting Pads
 <ol>
 <li>In a 1.5 ml centrifuge tube, make a 2% Noble Agar solution by adding 0.02 g of Noble Agar to 1 ml of H2O. The agar can be stored at 4&deg;C for up to a year.</li>
 <li>Mix thoroughly and melt agar in a heat block set to &gt;88&deg;C.</li>
 <li>Using a 1 ml micropipette, place a drop of liquid 2% Noble Agar to the middle of the glass slide (Fisher, 12-544-F).</li>
 <li>Flatten by placing another glass slide on top of the drop.</li>
 <li>Repeat "Drop Step" until there is a layer of five-glass slide.</li>
 <li>Remove each glass slide by sliding the glass slides off each other.</li>
 <li>Dry flatten agar pad on glass slides in a vacuum oven at 70&deg;C for 4 hours to overnight (or at room temperature overnight).</li>
 <li>Make multiple Injection pads and store for future use.</li>
 </ol>
 </li>
 <li>Microinjection into hermaphroditic gonads</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/3270/3270fig1.jpg" /> 
Figure 1. A schematic drawing of the P. pacificus anatomy. The clear nucleated cells are female germ cells (oocytes) and the gray shaded part is the intestine. Only one distal anterior gonad is shown but notice the two distal gonads cross each other dorsally near mid-body.
<img alt="Figure 2" src="/files/ftp_upload/3270/3270fig2.jpg" /> 
Figure 2. Images of microinjection. (A) The injection needle tip is in focus with the far right line of the pulled capillary piece. By lightly touching the tip of the needle to that edge, the needle tip should break while still retaining a sharp point. (B) The needle inserts into the top gonad just above the gut curvature for the first injection. (C) The needle inserts into the bottom gonad just below the gut curvature for the second injection.
<ol start="3">
 <li>
 <ol>
 <li>Make sure the contrast and DIC shearing setting is ideal for viewing the hermaphrodite gonads.</li>
 <li>Load 1.0-2.0 &mu;l of injection mixture into the pulled capillary needle.</li>
 <li>Place the needle in the microinjection manipulator; pressure gauges of the nitrogen tank should be set to ideal conditions (10-20 psi for 0.5-1 sec).</li>
 <li>Break the needle tip by touching it slightly to edge of a thinly pulled capillary tube placed on a slide (figure 2A).
 <ol>
 <li>Needle breaker slide: the thinnest part of a pulled capillary placed on a glass slide in a drop of oil</li>
 </ol>
 </li>
 <li>Once the needle is broken it can be place at an idle position while you pick your worm to the pad (P. pacificus have a 5 minutes window for injection before they dry-out and die).</li>
 <li>Using a plate full of J4-young adult worms, pour enough paraffin oil (heavy) to just cover all the worms in OP50 bacterial lawn.</li>
 <li>Pick a worm and place it on the injection pad.</li>
 <li>Place the glass slide with the worm on the microscope; position the worm's gonad in direction of the needle and the vulva away from the needle (Figure 2B, 2C).</li>
 <li>Lower the needle in to position of the microscope view.</li>
 <li>Position the top gonad and the needle in the same focal plane (Figure 2B).</li>
 <li>Poke the worm gently and pump in the injection mixture; ooctyes separating towards the distal tip indicate a successful injection (To optimize transgene or dsRNA delivery, you should upon injection see a wave of expansion in the gonad that continues until it is close to the distal tip).</li>
 <li>Reposition the needle to inject to the lower gonad and repeat injection; however since this gonad is under the gut the spreading of the oocytes will not be seen and is thus more difficult to achieve (Figure 2C).</li>
 <li>Normal looking worm indicates a successful injection after the injection; there should not be any damaged gonads (protruding out from the vulva or popped inside the body cavity).</li>
 <li>Place the needle back in idle position.</li>
 <li>Prepare to rescue the worm.</li>
 <li>Add a drop (0.5 &mu;l) of M9 buffer onto the pad where the injected worm is placed.</li>
 <li>Using some OP50 bacteria on your pick, touch the worm with the OP50, and it should stick. We do not use mouth pipette.</li>
 <li>Place the worm onto plate seeded with 50 &mu;l spots of OP50, 2-4 worms can be rescued on such plates.</li>
 </ol>
 </li>
 <li>Post-injection Storage &amp; Screening for Transgenesis
 <ol>
 <li>Store injected worms (P0) at 20&deg;C for ~4 days to lay eggs and grow up F1.</li>
 <li>Screen worms on the fourth day for selected phenotype (To screen for transgenesis, search for rolling animals expressing the Ppa-prl-1 dominant marker).</li>
 <li>Pick single independent F1's animals with the observed phenotype to new seeded plate and store at 25&deg;C; this temperature encourages the formation of stable lines from F1's.</li>
 <li>Single F2 from independent F1 lines. Individual F2 lines have similar transgene transmission rates. We often obtain &gt;15 F2 lines per transformed F1 animal.</li>
 <li>Store subsequent generations at 25&deg;C.</li>
 <li>The transmission rate of rollers usually remains constant after the F4 generation, thus it is necessary at this time to determine the expression level of the target transgene (Ppa-egl-4p::gfp) for each individual line. GFP expression may not correlate with the extent of penetrance of the dominant roller phenotype.</li>
 </ol>
 </li>
 <li>Post-injection Storage &amp; Screening for RNAi phenotypes
 <ol>
 <li>Store injected worms (P0) at 20&deg;C for ~4 days to lay and grow F1.</li>
 <li>Screen F1 worms on the fourth day for selected phenotype (To screen for RNAi knockdown phenotype, search for penetrant phenotypes that may be associated with loss of target gene activity, in our case the loss of the Ppa-prl-1 phenotype).</li>
 <li>In our experience, the non-roller knockdown phenotype is lost in &gt;97% of the F2.</li>
 </ol>
 </li>
</ol>
4. Representative results:
1. Result of Transgenesis
<table border="1">
 <tbody>
 <tr>
 <td>Session</td>
 <td>Injected P0</td>
 <td>F1 Rollers</td>
 <td>% transgenic F2</td>
 <td>Average % transmission after F2</td>
 </tr>
 <tr>
 <td>1</td>
 <td>60</td>
 <td>2</td>
 <td>(line 1) 0% 
 (line 2) 13%</td>
 <td>NA 
 13%&plusmn;10</td>
 </tr>
 <tr>
 <td>2</td>
 <td>40</td>
 <td>1*</td>
 <td>(line 3) 0%</td>
 <td>NA</td>
 </tr>
 <tr>
 <td>3</td>
 <td>40</td>
 <td>0</td>
 <td>0%</td>
 <td>NA</td>
 </tr>
 <tr>
 <td>4</td>
 <td>40</td>
 <td>1</td>
 <td>(line 4) 0%</td>
 <td>NA</td>
 </tr>
 <tr>
 <td>5</td>
 <td>20</td>
 <td>0</td>
 <td>0%</td>
 <td>NA</td>
 </tr>
 <tr>
 <td>6</td>
 <td>40</td>
 <td>1</td>
 <td>(line 5) 26%</td>
 <td>22%&plusmn;10</td>
 </tr>
 </tbody>
</table>
* a male roller that did not cross; NA: Not applicable
Table 3. Results of injected PS312 with Ppa-egl-4p::gfp (Sal I digested), pRL3(roller) (Pst I digested) , and gDNA PS312 (PstI and SalI digested).
<img alt="Figure 3" src="/files/ftp_upload/3270/3270fig3.jpg" /> 
Figure 3. (A) wildtype (B, C) A stable F4 pRL3; Ppa-egl-4p::gfp transgenic line showing head neuron GFP expression.
2. Result of RNA interference
<img alt="Figure 4" src="/files/ftp_upload/3270/3270fig4.jpg" /> 
Figure 4. RNA injected prl-1 mutants show complete knockdown of "rolling" locomotion. (A) prl-1 roller gain-of-function mutant tu92. (B) "knocked down" prl-1 mutant exhibit normal wildtype posture and locomotion. (C) Injected prl-1 (bottom) also has longer body than prl-1 mutant (top). (D) The longer body of injected prl-1 (bottom) is also longer than the wildtype PS312 (top). The longer body phenotype was also observed in the rol-5 (sqt-1) RNAi knockdown of C. elegans N2 (data not shown).
<table border="1">
 <tbody>
 <tr>
 <td>&nbsp;</td>
 <td>roll</td>
 <td>non-roll</td>
 </tr>
 <tr>
 <td>A prl-1 dsRNA (200 ng/&mu;l)</td>
 <td>13</td>
 <td>21</td>
 </tr>
 <tr>
 <td>B prl-1 dsRNA (1000 ng/&mu;l)</td>
 <td>9</td>
 <td>16</td>
 </tr>
 <tr>
 <td>control</td>
 <td>20</td>
 <td>2</td>
 </tr>
 </tbody>
</table>
Table 4. Summary of Ppa-prl-1 dsRNA injections. (A) [dsRNA] = 200 ng/&mu;l; (B) [dsRNA] = 1000 ng/&mu;l. P = 0.0019 and 0.041 by Fisher's Exact Test, two-tailed, for 200 and 1000 ng/&mu;l injections, respectively.

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	<li>Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., Mello, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391, 806-811 (1998).</li><li>Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+Genetics.">Reverse Genetics.</a> Wormbook. , (2006).</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>Junio, A. J., Li, X., Massery, H. C., Nolan, T. J., Lamitina, S. T., Sundaram, M. V., Lok, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strongyloides+stercoralis:+cell-+and+tissue-specific+transgene+expression+and+co-transformation+with+vector+constructs+incorporating+a+common+multifunctional+3'+UTR.">Strongyloides stercoralis: cell- and tissue-specific transgene expression and co-transformation with vector constructs incorporating a common multifunctional 3' UTR.</a> Exp. Parasitology. 118, 253-265 (2008).</li><li>Schlager, B., Wang, X., Braach, G., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+a+dominant+roller+mutant+and+establishment+of+DNA-mediated+transformation+in+the+nematode+Pristionchus+pacificus.">Molecular cloning of a dominant roller mutant and establishment of DNA-mediated transformation in the nematode Pristionchus pacificus.</a> Genesis. 47, 300-304 (2009).</li><li>Hong, R. L., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pristionchus+pacificus:+a+well-rounded+nematode.">Pristionchus pacificus: a well-rounded nematode.</a> Bioessays. 28, 651-659 (2006).</li><li>Hong, R. L., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoattraction+in+Pristionchus+nematodes+and+implications+for+insect+recognition.">Chemoattraction in Pristionchus nematodes and implications for insect recognition.</a> Curr. Biol. 16, 2359-2365 (2006).</li><li>Rudel, D., Riebesell, M., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gonadogenesis+in+Pristionchus+pacificus+and+organ+evolution:+development,+adult+morphology+and+cell-cell+interactions+in+the+hermaphrodite+gonad.">Gonadogenesis in Pristionchus pacificus and organ evolution: development, adult morphology and cell-cell interactions in the hermaphrodite gonad.</a> Dev. Biol. 277, 200-221 (2005).</li><li>Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., Prasher, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein+as+a+marker+for+gene+expression.">Green fluorescent protein as a marker for gene expression.</a> Science. 11, 802-805 (1994).</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-e2293 (2011).</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></ol>

No conflicts of interest declared.

P. pacificus populations are found in close association with various scarab beetle species worldwide and is a model nematode intermediate between free living and parasitic nematodes. The strength of the P. pacificus as an emerging model organism lay in the integration of its genetic and physical maps that promote positional mapping of mutants isolated from unbiased forward genetic screens (i.e. not just for candidate genes previously characterized in C. elegans)6,10. However...

<table border="1">
<tbody>
<tr>
	<th>Product:</th>
	<th>Catalog #:</th>
	<th>Supplier:</th>
</tr>
<tr>
	<td>DMI3000 Injection microscope		</td>
	<td>DMI3000</td>
	<td>Leica</td>
</tr>
<tr>
	<td>Microinjector manipulator (Direct drive)		</td>
	<td>Narashige BC-3 Ball joint</td>
	<td>Tritech Research</td>
</tr>
<tr>
	<td>Needle Puller		</td>
	<td>Narashige PC-10</td>
	<td>Tritech Research</td>
</tr>
<tr>
	<td>MicroInjector&trade; All-Digital Multi-pressure System		</td>
	<td>Narashige MINJ-D</td>
	<td>Tritech Research</td>
</tr>
<tr>
	<td>GeneElute&trade;Mammalian Genomic DNA Miniprep Kit 		</td>
	<td>G1N70</td>
	<td>Sigma</td>
</tr>
<tr>
	<td>pJet Cloning Jet kit		</td>
	<td>K1231</td>
	<td>Fermentas</td>
</tr>
<tr>
	<td>GeneJET plasmid Miniprep kit		</td>
	<td>K0502</td>
	<td>Fermentas</td>
</tr>
<tr>
	<td>DNA Clean &amp; Concentrator&trade;		</td>
	<td>D4005</td>
	<td>ZYMO Research</td>
</tr>
<tr>
	<td>BLOCK-IT&trade; RNAi TOPO&reg; transcription kit		</td>
	<td>K3500-01 &amp; K3650-01</td>
	<td>Invitrogen</td>
</tr>
<tr>
	<td>Difco&trade;Agar Noble	</td>
	<td>DF0142-15-2	</td>
	<td>Fisher Scientifi</td>
</tr>
<tr>
	<td>Microscope cover glass (1.5 - 0.16 to 0.19mm thick; Size: 50 x 45mm)</td>
	<td>12-554-F</td>
	<td>Fisher Scientific</td>
</tr>
<tr>
	<td>Glass capillaries (filament)</td>
	<td>615000</td>
	<td>A-M systems</td>
</tr>
<tr>
	<td>Paraffin Oil (Heavy)		</td>
	<td>O122-1</td>
	<td>Fisher Scientific</td>
</tr>
<tr>
	<td>KH2PO4 	</td>
	<td>P386-500</td>
	<td>Fisher Scientific</td>
</tr>
<tr>
	<td>Na2HPO4	</td>
	<td>AC20651-5000</td>
	<td>Fisher Scientific</td>
</tr>
<tr>
	<td>NaCl		</td>
	<td>BP3581</td>
	<td>Fisher Scientific</td>
</tr>
<tr>
	<td>MgSO4		</td>
	<td>M80-500</td>
	<td>Fisher Scientific</td>
</tr>
</tbody>
</table>

rnai mediated gene knockdown and transgenesis by microinjection in the necromenic nematode pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

Watch this Scientific Journal Video about RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus at JoVE.com

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

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JoVE Journal

Biology

16.4K Views.  California State University. In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

Video: RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls.
The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of ~50-150 &#181;m and extends over ~15 &#181;m around the mean penetration depth, which is adjustable between 0 and 50 &#181;m. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches.
For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery.
When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.

In this video, we show a procedure for an accurate biolistic delivery of reagents into live tissue with a novel miniature gene gun. We are knocking down the expression of the axon guidance molecule Netrin in leech embryos by delivering molecules of dsRNA into the ventral body wall and ganglia of single segments.

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to ...

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

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

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

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

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

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