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Pristionchus pacificus genetic protocols*

Andre Pires da Silva§
Department of Biology, University of Texas at Arlington, Arlington, TX 76019-0498 USA

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Table of Contents

1. Introduction
2. Protocols
2.1. Part A: Freezing worms
2.2. Part B: Mutagenesis
2.3. Part C: Construction of deletion libraries to generate P. pacificus gene knock-outs
2.4. Part D: RNAi and morpholino by injection
3. Acknowledgements
4. References

1. Introduction

The diplogastrid nematode Pristionchus pacificus has been recently established as a new genetic model system for evolutionary studies. Forward and reverse genetics tools have been developed to allow detailed comparisons to the rhabditid nematode C. elegans. P. pacificus has distinctive body morphology and diverges in development both at the cellular and molecular level when compared to C. elegans (see Evolution of development in nematodes related to C. elegans). Despite these differences, P. pacificus shares many traits with C. elegans that are of advantage for genetic analyses: it is hermaphroditic, has a small genome size (160 Mb), small number of chromosomes (haploid set with 5 autosomes and 1 sex chromosome), short life-cycle (4 days at 20°C), and produces large brood sizes (150–200 eggs). Similarly to C. elegans, P. pacificus has 4 larval stages (J1-J4). However, the first larval stage molts within the eggshell (Sudhaus et al., 2003).

2. Protocols

2.1. Part A: Freezing worms

P. pacificus can be frozen in liquid nitrogen, although with lower efficiency than C. elegans. It was observed that the addition of calcium to the M9 dramatically increased P. pacificus freezing efficiency. As for C. elegans, P. pacificus early hatched larvae are most likely to survive freezing and thawing. Some modifications were added to the common C. elegans freezing protocol to increase efficiency (Protocol 1).

2.2. Part B: Mutagenesis

Ethyl methanesulfonate (EMS) has been widely used in P. pacificus to isolate a wide variety of mutants with defects in the egg-laying system, muscle, sex determination, dauer formation, behaviour, and gonad formation (Protocol 2). The protocol for EMS mutagenesis used is the same as for C. elegans (Brenner, 1974). For mutagenizing P. pacificus with psoralen, however, some modifications were introduced (Protocol 3).

2.3. Part C: Construction of deletion libraries to generate P. pacificus gene knock-outs

A powerful method to isolate C. elegans mutants in a gene of interest for which sequence information is available is by chemical mutagenesis followed by polymerase chain reaction (PCR). The generation of knockouts in a high throughput manner by using deletions in a library of worms has proven very successful for C. elegans. The protocol adopted for P. pacificus does not include the freezing of the library, since the efficiency of recovery following freezing of P. pacificus is too low (Protocol 4). The library comprises 12 96-well microtiter plates and includes the arrayed progeny of nearly 5 × 105 F1 animals representing 106 mutagenized genomes. Worms are grown in plates until the food is exhausted, generating, on the average, 100 F2 progeny per F1 animal.

2.3.1. Designing primers for the gene of interest

Target a region of around 1.5 kbp (1.0-2.5 kbp), with nested oligonucleotides of around 20 bp (annealing temperature of 60°C). The larger the region targeted, the more difficult it is to detect small mutations in agarose gels. The smaller the region, the more difficult it is to find a deletion that happens to have occurred between the primers.

2.3.2. Growth synchronization

  1. Transfer freshly starved larvae from 10 6 cm diameter plates to 15 10 cm diameter plates seeded with 1 ml of OP50 per plate. Incubate at 25°C (or 20°C) until plates are full of eggs.

  2. Transfer worms and eggs to a 50 ml Falcon tube with M9 and centrifuge for 5 minutes at 1300 X g. Discard the supernatant. Add 30 ml of basic hypochlorite solution and incubate at room temperature for about 4 minutes. Collect the eggs by centrifugation (2000 × g, 5 min).

  3. Wash the eggs 2 times with 30 ml of water and 1 time with 30 ml of M9 buffer. Leave the worms to hatch overnight in the buffer or in a 10 cm unseeded NGM plate.

  4. The next day, pipet 10 μl of the suspension onto each of three plates and count to determine titer. The total number of worms in 50 ml should be around 100 000 worms (20 worms/10μl). Distribute about 2000 worms per plate onto 50 seeded 10 cm NGM plates.

2.3.3. Mutagenesis

  1. Culture synchronized culture of worms for about 52 hours at 20°C. Follow protocol 3 for TMP/UV mutagenesis. After mutagenesis, plate the worms on 50 fresh seeded 10 cm NGM plates, with 2000 worms/plate.

2.3.4. Plating the library

  1. After a couple of days, when P0s have laid the F1 eggs, bleach the 50 10 cm plates (as in part B, 2) and leave the eggs in 50 ml of M9 buffer overnight.

  2. The following day, count the number of L1 worms. Distribute about 400 F1s/small plate (total 1200 6 cm plates).

  3. Wait one generation, until the F2 eggs have hatched. Wash plates by adding 800 μl of water and transferring 150 μl of the L1 suspension into a 1.2 ml 96-well plate (to avoid mistakes, leave the yellow tip inside the well). This step will take about half an hour/plate; in total it will result in 12 × 96-well plates. Leave the washed plates at RT until they dry. Then transfer them to 12°C.

  4. Add 150 μl of lysis buffer + proteinase K (120 μg/ml) to each well of the 1.2 ml 96-well plates, seal them with MicroAmp Clear Adhesive films and incubate them first at 80°C for 2 hours and then at 65°C overnight with shaking.

  5. The following morning, centrifuge the 96-well plates for 10 min at 4000 rpm.

  6. Transfer 150 μl of the suspension into a 200 μl 96-well plate and inactivate the proteinase K at 95°C for 10 minutes. The remaining 150 μl should be stored at 80°C.

  7. Pool the rows and columns from each of the 12 plates by combining 75 μl of each of the wells into a single 1.2 ml 96-well plate. For example, pool wells A1 of all 12 plates into the position A1 of the 1.2 ml 96-well plate. Repeat this procedure for each well, making a total of 96 pools. This is the master plate with pooled DNA.

2.3.5. Screening the library by PCR

  1. Take 5 μl as template for a 20 μl PCR reaction. It is necessary to perform nested PCR. For the 2° round, use 1 μl of the 1° round.

a. Example for PCR mix:

1st PCR round: [final Volume 10 μl]
H2O 1.7 μl
dNTP (10 mM each of dATP, dCTP, dGTP, dTTP) 0.2 μl
forward primer (10 μM) 0.5 μl
reverse primer (10 μM) 0.5 μl
Buffer 5X 2.0 μl
Taq (5 U/μl) 0.1 μl
DNA (pooled Library) 5.0 μl

94° C for 2 min.

94° C for 30 sec.

58° C for 20 sec. 35 cycles

72° C for 3 min.

72° C for 6 min.

add 20 μl H2O to 1° round.

2nd PCR round: [final Volume 10 μl]
H2O 3.7 μl
dNTP (10 mM each of dATP, dCTP, dGTP, dTTP) 0.2 μl
forward primer (10 μM) 0.5 μl
reverse primer (10 μM) 0.5 μl
Buffer 5X 2.0 μl
Taq (5 U/μl) 0.1 μl
yellow Loading dye 2.0 μl
DNA (1:3 diluted) 1.0 μl

Same cycling conditions as 1st round.

  1. Select samples which show bands that are smaller than the wild-type for further tests. Determine the precise address of the candidate sample by repeating the PCR using 5 μl of the corresponding well of each of the twelve 96-well plates (200 μl 96-well plates). This will give the number of the plate (2-A5, for example).

2.3.6. Sib selection

  1. Resuspend the original plate in 50 ml water or M9 (50 ml Falcon tube). Count the number of worms/μl, by pippeting 3 times 10 μl into a plate. Calculate the average of worm/μl.

  2. Dispense 50 worms into the wells of 8, 12-well plates. The remaining worms (which were not dispensed) should be centrifuged and put back in the original plate, without bacteria. Keep this plate at 12°C.

  3. Culture the worms at 20°C for 5 days. Harvest a portion of each well by washing with 300 μl of water. Transfer 75 μl of the suspension into a 1.2 ml 96-well plate, plus 75 μl lysis buffer with ptnK. Incubate overnight at 65°C with shaking. The agar plates with worms should be kept at 12°C.

  4. Transfer 100 μl to a 200 μl 96-well plate.

  5. Inactivate the ptnK by incubating the 96-well plate at 95°C for 15 minutes.

  6. Screen the plate by PCR (5 μl as template), identify the plate and repeat procedure by dispensing 96 × 15 worms/plate.

  7. Repeat procedure above with 5 worms/plate and later 1 worm/plate.

2.4. Part D: RNAi and morpholino by injection

The possibility of rapidly knocking-down genes by RNA interference (RNAi), together with the availability of the C. elegans genome sequence, stimulated the study of gene function in a global scale (see Reverse genetics). Currently there are three different ways of delivering double-stranded RNA (dsRNA) into C. elegans: injection, soaking and feeding. Preliminary experiments with P. pacificus indicate that RNAi works when injecting dsRNA into the gonad (personal observation). The conditions used for dsRNA preparation and injection were basically the same as described for C. elegans (see Reverse genetics). The injection of a 1081 bp Ppa-tra-1 dsRNA, for instance, resulted in 20% (n=346) progeny with the transformer phenotype. For genes involved in vulva development, however, the efficiency of the RNAi seems to be much lower (Zheng and Sommer, personal communication). Similar observations have been made with vulva genes in C. elegans (Chen et al., 2004). Another knockdown technology, using morpholino oligonucleotides, has proven robust for P. pacificus (Protocol 5) (Pires-daSilva et al., 2004; Zheng et al., 2005). Morpholino-oligonucleotides, widely used for knocking down genes in other model systems (e.g., sea urchin, zebrafish), are modified oligonucleotides that efficiently block the translation or splicing of specific mRNAs. The properties of morpholino oligonucleotides, which combine nuclease-resistance, water-solutility and high specificity, are of major advantage. However, the injection of morpholinos causes a high rate of lethality in the P. pacificus F1 progeny (about 50%).When selecting oligo sequences, it is recommended to design at least two non-overlapping oligos. In this way, the consistency of the resulting phenotype can be tested. A good target sequence has minimal mRNA secondary structure and includes the translational start codon and/or the 5UTR region.

3. Acknowledgements

Andre Pires da Silva is funded by NSF grant IOB#0615996.

4. References

Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71–94. Abstract

Chen, N., and Greenwald, I. (2004). The lateral signal for LIN-12/Notch in C. elegans vulval development comprises redundant secreted and transmembrane DSL proteins. Dev. Cell 6, 183–192. Abstract Article

Pires-daSilva, A., and Sommer, R.J. (2004). Conservation of the global sex determination gene tra-1 in distantly related nematodes. Genes Dev. 18, 1198–1208. Abstract Article

Sudhaus, W., and Furst von Lieven, A. (2003). A phylogenetic classification and catalogue of the Diplogastridae (Secernentea). J. Nemat. Morphol. Syst. 6, 43–90.

Yandell, M.D., Edgar, L.G. et al. (1994). Trimethylpsoralen induces small deletion mutations in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 91, 1381–1385. Abstract

Zheng, M., Messerschmidt, D. et al. (2005). Conservation and diversification of Wnt signaling function during the evolution of nematode vulva development. Nat. Genet. 37, 300–304. Abstract Article

*Edited by Ralf J. Sommer. WormMethods editor, Victor Ambros. Last revised June 29, 2006. Published July 17, 2006. This chapter should be cited as: Pires da Silva, A. Pristionchus pacificus genetic protocols (July 17, 2006), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.114.1, http://www.wormbook.org.

Copyright: © 2006 Andre Pires da Silva. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

§To whom correspondence should be addressed. E-mail: apires@uta.edu

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