Embryo series courtesy of Einhard Schierenberg
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Table of Contents
Through genetic analyses, the function of genes is investigated by studying organisms where gene function is altered. In classical forward genetic screening, individuals are treated with mutagens to induce DNA lesions and mutants with a phenotype of interest are sought. After a mutant is found, the gene mutated is identified through standard molecular techniques. Detailed studies of the mutant phenotype coupled with molecular analyses of the gene allows elucidation of the gene's function. Forward genetics has been responsible for our understanding of many biological processes and is an excellent method for identifying genes that function in a particular process.
In reverse genetics, the functional study of a gene starts with the gene sequence rather than a mutant phenotype. Using various techniques, a gene's function is altered and the effect on the development or behaviour of the organism is analysed. Reverse genetics is an important complement to forward genetics. For example, using reverse genetics, one can investigate the function of all genes in a gene family, something not easily done with forward genetics. Further, one can study the function of a gene found to be involved in a process of interest in another organism, but for which no forward genetic mutants have yet been identified. Finally, the vast majority of genes have not yet been mutated in most organisms and reverse genetics allows their study. The availability of complete genome sequences combined with reverse genetics can allow every gene to be studied.
This chapter gives detailed protocols for the two main methods of perturbing gene function in C. elegans: RNA interference and the creation of deletion mutants. Either technique can be applied to the study of individual genes. With less than a day of actual work, RNAi creates a knockdown of gene function without altering the organism's DNA (see below). In contrast, with about a month of work, a deletion mutation permanently removes all gene function. Deciding which technique to use will depend on the nature of the experiment. The techniques can also be combined, where RNAi is used for rapid screening of loss of function phenotypes and then deletion mutants are made to study genes of particular interest. RNAi can also be carried out on a global scale, where knockdown of (nearly) every gene is tested for inducing a phenotype of interest. In this case, the reverse genetics technique of RNAi can be thought of as a forward genetic screening tool.
The Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK
In 1998, Fire and Mello discovered that injection of double stranded RNA (dsRNA) into worms leads to specific degradation of the corresponding mRNA, a process termed RNA interference (RNAi; Fire et al., 1998). Soon afterwards, it was found that either soaking worms in dsRNA solution or feeding worms bacteria engineered to produce dsRNA also could induce a robust RNAi response (Tabara et al., 1998; Timmons and Fire, 1998). The technique of RNAi, coupled with the availability of the complete genomic sequence of C. elegans (Consortium, 1998), has made possible the rapid study of gene function, both on a single gene level and at a global scale. This section discusses general features to consider when using this technique. Sections following this give detailed protocols for RNAi by injection, soaking and feeding.
There are three ways to carry out RNAi in C. elegans: injection (Fire et al., 1998), soaking (Tabara et al., 1998), and feeding (Timmons and Fire, 1998). All three can produce efficient gene knockdowns. Which method to use will depend on your particular experiment.
RNAi by injection: dsRNA produced in vitro is injected into young adult hermaphrotides and the progeny scored for mutant phenotypes. RNAi by injection gives very reliable gene inhibition from worm to worm, but is more labor intensive than other methods. A small transcription reaction produces 5–10 μg dsRNA, which is sufficient to conduct many experiments.
RNAi by soaking: worms are soaked in a high concentration dsRNA solution and then subsequently they or their progeny scored for phenotypes. RNAi by soaking is useful for treating a moderately large number of animals (e.g., tens to hundreds) or for high throughput screening in 96 well format. Worms of any stage can be soaked. More dsRNA is needed for soaking than for injection (5–10 μg per experiment).
RNAi by feeding: bacteria producing the desired dsRNA are fed to worms and either they or their progeny are scored. RNAi by feeding is the least labor intensive and most inexpensive method, but produces slightly more variable results than RNAi by soaking or injection. RNAi by feeding can be used to treat a large number of animals at once or for high throughput screening, both on agar plates and in liquid culture. Worms of any stage can be subjected to RNAi by feeding.
This will depend on the nature of the experiment. Worm strains and tissues differ in their sensitivity to RNAi. For many phenotypes and assays, using wild type worms will give good results. However, in some cases, phenotypes are seen in RNAi supersensitive strains [e.g., rrf-3 (Simmer et al., 2002) eri-1 (Kennedy et al., 2004) or eri-1; lin-15B (Wang et al., 2005)] but not in wild-type, or are stronger in RNAi supersensitive strains, so it is a good idea to try these as well. rrf-3 and eri-1 have lower brood sizes than wild-type and are sterile at 25°C, so they require a bit more attention. The sterility can be overcome by mating with males.
Again, this will depend on your experiment and assay. RNAi by injection is usually carried out on adult hermaphrodites, and the progeny assayed. Injecting younger worms is possible but more difficult. Any stage of worms can be fed or soaked, but one of two stages are usually used: L3/L4s or starved L1s. For feeding, it is desirable to allow the worms to feed for 2–3 days before they or their progeny are assayed. In general, feeding or soaking L3/L4 worms is used if the assay will be carried out on the progeny. If the assay will be on the RNAi treated worms, then starved L1s or later stages can be used. It is also possible to assay the progeny of RNAi treated L1s. However, it is important to keep in mind that phenotypes observed can differ depending on which stage is used. For example, RNAi of some genes causes sterility of the fed worm if L1s are used whereas embryonic lethality is induced if L4s are used. (S. Woods, D. Rivers, and J. Ahringer, unpublished). Therefore, if the assay will be on embryos, then it may not be a good idea to use L1s.
Induction of a phenotype is a reliable indication of a positive RNAi result, as the level of false positives is extremely low (<1%, Kamath et al., 2003). However, it is more difficult to draw conclusions from a negative RNAi experiment; the false negative rate is about 30% over all genes, and varies depending on the tissue (Kamath et al., 2003). Using RT-PCR, you can test whether the mRNA has been knocked down. However, the protein product could still be present even if the mRNA is undetectable. Some proteins are stable over many days. An antibody can be used to measure the level of protein reduction. It is important to be cautious when drawing conclusions following a negative RNAi experiment, as it is possible that a small amount of residual protein is sufficient for gene activity.
When targeting more than one gene by RNAi, the efficiency of knockdown may be reduced when compared to targeting each gene separately (e.g., see Table 1 in Gonczy et al., 2000). This may not be a problem for some experiments. For example, if two genes each have no detectable RNAi phenotype, but targeting them together causes a phenotype, then this would show some overlap in function. However, if you are carrying out an epistasis experiment, reduced RNAi efficiency may lead to an inaccurate result, if only one of the two genes is efficiently knocked down. Therefore, it is ideal to target one gene by RNAi in the mutant background of the other gene. If it is not possible to use a mutant, then use antibodies to test that the level of protein knockdown is the same in the double and single RNAi experiments for both genes. Use of rrf-3 or eri-1 RNAi supersensitive strains (Simmer et al., 2002; Kennedy et al., 2004; Wang et al., 2005) enhances the effectiveness of targeting multiple genes (S. Woods, D. Rivers, and J. Ahringer, unpublished; A. Fraser, pers. comm.).
It is essential to optimize screening conditions before embarking on a large-scale screen. If at all possible, assemble a set of control genes that you expect to be positive in your screen, and a set of genes you expect to be negative. Test these under different conditions (e.g., different temperature, stage of worm fed, background, plate configuration, etc) to find the highest hit rate and fastest screening time. Carrying out these controls will save time and make your final list of positive candidates more specific. Positive hits should be rescreened to ensure reproducibility. If possible, is it a good idea to design and carry out a different, second assay on your positive candidate genes to ensure they are involved in the process you are studying.
There are currently two RNAi feeding libraries for C. elegans. One is from the Ahringer lab and has 16,757 clones, made by cloning gene-specific genomic fragments between two inverted T7 promoters (Fraser et al., 2000; Kamath et al., 2003). The inserts contain exons and introns and sizes vary from 500 bp to 2.5 kb. The other is from the Vidal lab and has 11,511 clones, made by the Gateway cloning of full-length open reading frame (ORF) cDNAs into a double T7 vector (Rual et al., 2004). Both libraries use the HT115 bacterial strain as a host for the plasmid RNAi clones; HT115 has IPTG inducible T7 polymerase and a disruption of the RNAse III gene (a dsRNAse), the latter marked with Tetracycline resistance (Timmons et al., 2001). There is some overlap between the two libraries; together they can target about 94% of C. elegans genes. Individual clones and whole libraries are available from Geneservice. The Vidal library is also available from Open Biosystems.
As there are some errors in any library, you should sequence any clone you are going to work with to ensure you know which gene is being targeted (single genes or positives from a screen). It is also a good idea to look for potential cross interference (where RNAi of one gene inhibits the intended target gene as well as a closely related gene) using BlastN to compare the sequence of the RNAi template with C. elegans genes. So far, there is no hard and fast rule for predicting cross interference. A reasonable guide is that if there is 80% nucleotide identity over 200bp, cross-interference is likely. Wormbase Gene Summary pages give information on which gene(s) an RNAi clone is likely to target.
The Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK
Synopsis: Prepare dsRNA corresponding to gene of interest by in vitro transcription. Inject dsRNA into young adult hermaphrodites, wait for RNAi to take effect, then score progeny of injected mother. This is a modification of the protocol in Zipperlen et al. (2001).
Use PCR to prepare a gene specific fragment containing bacterial polymerase promoter sequences (T7 or T3) at each end. Fragments of 500 bp – 2kb can be used, though dsRNA yields decrease over 1 kb. If possible, use the same bacterial promoter sequence at both ends, then only one transcription reaction is necessary for dsRNA preparation. There are several ways to obtain a template:
Use T3 and T7 primers to PCR amplify a gene specific fragment cloned into a Bluescript or similar vector that has these primer sequences flanking the insert site.
Design gene specific primers and add T7 promoter sequences to the 5' ends. Use primers to PCR amplify desired fragment directly from cDNA or genomic DNA.
Use T7 primers to amplify the insert from a clone in a double T7 feeding vector (e.g., L4440); 5'-CGTAATACGACTCACTATAG-3'.
Below is a general method for making a template from an RNAi feeding clone:
With a yellow tip, pick a small amount of a bacterial clone into 100 μl of water.
Use 1 μl of bacterial solution as a template for PCR using a standard Taq enzyme in a 25 μl reaction, with 1 μM T7 oligo, 0.2 mM dNTPs and the following cycling conditions: 95°C 50s, 52°C 30s, 72°C 90s for 25 cycles. For a good transcription reaction in the next step, the PCR reaction should yield ~200ng/ul of product.
For high efficiency high yield transcription reactions, we recommend using an in vitro transcription kit (e.g., Promega RiboMAX or equivalent). Use 1 μl of unpurified template from the PCR reaction above in a 5 μl in vitro transcription reaction, incubating 4.5hrs at 37°C. If different polymerase promoters are at each end, carry out each transcription reaction separately.
Dilute the transcription reaction 4X with 20 μl sterile DEPC water or 10mM Tris 8.0, 0.1mM EDTA and run 2 μl on a gel for quantification. The concentration should be 0.2 – 1.0 μg/ul. If two separate reactions were performed, pool them together, heat to 72°C for 10 minutes and allow to cool to RT for annealing.
Inject the dsRNA solution without purification (see note 1) into the intestine, body cavity or gonad of young adult hermaphrotides (see note 2).
Put injected worms onto plates. Move to new plates every 24 hours and score progeny produced at different times after injection (see note 3).
Note 1:
Many investigators purify their dsRNA prior to injection, either by ethanol precipitation or using an RNA purification kit. However, we obtain equivalent RNAi injection results with purified and unpurified dsRNA. For RNAi by soaking, purification is necessary to prevent death during the soaking process.
Note 2:
In general, equivalent results are seen irrespective of the site of injection, but some RNAi phenotypes might be stronger after a gonad injection. For example, progeny might inherit more dsRNA with a gonad injection, which might be helpful if the gene is active in mid-embryogenesis. This can be tested empirically for your gene. If the needle clogs during injection, try diluting the dsRNA a further 2X or more. Strong RNAi effects are seen by injection at quite low dsRNA concentrations (e.g., 50 ng/ul). However, mixing dsRNAs together can significantly reduce the RNAi effect for a given gene (see Table 1 in Gonczy et al., 2000). Therefore, it is important to use care in experiments where two or more genes are to be inhibited simultaneously by RNAi (e.g., use antibodies to confirm that the knockdowns have been successful).
Note 3:
Typically, 24 hours post-injection is a good starting point for a good RNAi effect. For many genes, the strength and penetrance of RNAi phenotypes are increased in progeny laid more than 24 hours post-injection, especially for genes with a strong maternal contribution. It is a good idea to do a time course to find the optimum time of scoring post-injection, looking for a time when the phenotype is strongest and most penetrant. For some genes, shorter times post-injection will give a stronger effect, particularly for genes with a zygotic but not a maternal function. If antibodies are available, it can be helpful to stain progeny at different times post-injection to see when the protein is maximally reduced. In order to maintain progeny production at later time points, mate the injected hermaphrodites with N2 males after injection. Using an RNAi supersensitive strain can increase the strength and penetrance of phenotypes. rrf-3 and eri-1 both display sterility at 25°C and smaller broods than N2 at lower temperatures (Simmer et al., 2002; Kennedy et al., 2004), but this can be overcome by mating with N2 males after injection, as they are cross fertile at all temperatures (J. Ahringer, unpublished).
Laboratory of Developmental Genomics, RIKEN Center for Developmental Biology, Kobe, 650-0047, Japan
DNA template for dsRNA synthesis can be prepared by PCR with primers designed to add T7 promoter sites at both ends. Below are examples of the primer sets for yk cDNA clones (available from Yuji Kohara).
yk001-yk699: These clones are in lambdaZAP II vector, which can be converted to pBluescript plasmid by in vivo excision. A phage suspension or plasmids can be used as the PCR templates.
| T7: 5' - GTAATACGACTCACTATAGGGC-3' |
| Cmo422: 5'-GCGTAATACGACTCACTATAGGGAACAAAAGCTGGAGCT-3' |
(T7 sequences are underlined.)
yk700 and up: These clones are in pME18S-FL3 vector.
| T7ME774FW: 5'-TAATACGACTCACTATAGGGCTTCTGCTCTAAAAGCTGCG-3' |
| T7-ME1250RV: 5'-TAATACGACTCACTATAGGGTGTGGGAGGTTTTTTCTCTA-3' |
(T7 sequences are underlined.)
Perform PCR reaction at a 30μl scale; reactions will typically produce ~0.4 μg/μl of DNA fragment. The PCR reaction can be used for in vitro transcription without purification.
Use 7μl of the PCR reaction mixture (above) for in vitro transcription with T7 RNA polymerase (e.g., Thermo T7 polymerase: TOYOBO (#TRL-201)) in a 100μl scale reaction. Both sense and antisense RNAs are transcribed from the PCR product in this single reaction. An annealing step is unnecessary. Typical yields are ~0.4 μg/μl of RNA (40μg total).
Digest the DNA template with DNase I.
Extract the reaction with phenol/chloroform. Repeat 2–4 times.
Alternatively, “Wizard Plus SV Miniprep DNA Purification System” (Promega #A-1330) can be used for dsRNA purification. Add 700 μl 0.01 M Tris/HCl pH 7.5 to 100 μl transcription reaction mixture before putting onto the column. Follow the supplier's instruction for washing and elution.
EtOH precipitate the extracted reaction. Rinse with 70% EtOH.
Resuspend pellet in 40μl H2O. A concentration of 0.5–5 μg/μl dsRNA is suitable for RNAi by soaking. dsRNA can be stored at −20°C.
Check the concentration and integrity of dsRNA by agarose gel electrophoresis.
By allowing investigators to select the developmental stage for dsRNA delivery, the soaking method can be used to conduct stage-specific RNAi experiments.
L4-soaking: L4 worms are soaked in dsRNA, and their progeny (as well as soaked worms) are examined for phenotypes. In many cases, the first embryo laid is already fully affected by RNAi.
L1-soaking: To perform post-embryonic development specific knockdowns, L1 larvae are soaked in a dsRNA solution. During soaking (which is done in the absence of food), the development of the L1 larvae is arrested but the RNAi response continues, making it possible to suppress gene function from the onset of post-embryonic development.
Day 1: Soaking
In a 200μl PCR tube, put 2μl 5X soaking buffer and 8μl dsRNA solution (prepared as above).
| 5X soaking buffer |
| 1.25 X M9 (Mg2+ free) |
| 15 mM spermidine (SIGMA S2626) |
| 0.25 % gelatine |
| (Store at −20°C) |
Pick L4 worms to a fresh NGM plate without bacteria, and let them crawl for several minutes to remove bacteria completely.
Put 6 - 10 L4 worms (=P0 worms) in the dsRNA solution, and incubate at 20°C for 24 hours.
Day 2: Recovery
Transfer the soaked worms on a 6cm NGM plate with OP50 (max. 4 worms/plate). This is Plate 1.
Incubate at 25°C for 24 hours. (Other temperature can also be used.)
Day 3
Transfer the P0 worms to a new NGM plate with OP50. This is Plate 2.
Check the condition of the P0 worms (they may also show phenotypes).
Incubate Plate 1 and 2 at 25°C for 24 hours.
Day 4
Transfer the P0 worms on Plate 2 to a new plate (Plate 3).
Check the condition (and phenotypes) of the P0 worms.
Count the F1 embryos and larvae on Plate 1.
Day 5
Count the F1 embryos and larvae on Plate 2.
Examine the phenotypes of F1 worms on Plate 1.
Day 6
Examine the phenotypes of F1 worms on Plate 2.
|
Day 1: Egg preparation
| Collect gravid adult worms in a 15 ml tube. Collect the embryos by hypochlorite/NaOH treatment. Wash eggs with M9 several times. Let the eggs hatch at 20°C in 1ml M9, or on a NGM plate without bacteria. |
Day 2: Soaking
Collect the L1 larvae at a concentration of ~100 larvae/ ~l M9.
In a 200 ~l PCR tube, put 2 ~l 5X soaking buffer and 8 ~l dsRNA solution.
Add 1 ~l L1 larvae suspension to the tube.
Incubate at 20°C for 24–48 hours.
Day 3 or 4: Recovery
| Recover L1 larvae on a NGM plate with OP50. Incubate at 25°C. |
Day 4–6: Phenotype observation
| Check the growth and phenotype of the soaked worms. |
|
More than one gene can be targeted simultaneously by the soaking method in wild-type animals. Because the efficiency of double RNAi might be lower than that of single RNAi for each gene, protein or mRNA levels for each gene should be assessed whenever possible.
Some RNAi supersensitive strains (e.g., rrf-3) exhibit developmental phenotypes, such as temperature-sensitive sterility, at a low penetrance. Because these background phenotypes tend to be enhanced by the soaking process, be cautious about interpreting results when these strains are used.
If the soaked worms die, become sick, or sterile, check the following points:
Worms may be physically damaged when they are picked and transferred into the tube. Pick them gently!
dsRNA may not be clean enough. Use highly purified RNA. Do not use DEPC-treated water for soaking method.
The P0 generation can also be affected by RNAi—P0 sterility or lethality may be THE phenotype. Always perform control experiments in parallel (soaking buffer only, dsRNA that cause no phenotype, and dsRNA that cause a known phenotype).
The Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK
Synopsis: Grow RNAi bacteria and seed plates. Feed worms with RNAi bacteria and score for phenotypes. The following is based on the protocol in Kamath et al. (2001).
Pour plates: make standard NGM agar and add carbenicillin to 25 μg/ml and IPTG to 1mM just prior to before pouring. Pour plates 4–7 days before seeding, to allow them to dry. If plates are too wet, the bacteria won't dry after seeding and RNAi phenotypes will be weaker. Feeding can conducted using any format plates (e.g., single plates, 6-well, 12-well).
Spot individual desired bacterial strain(s) from glycerol stock to an LB plate containing 50 μg/ml ampicillin (or 25 μg/ml carbenicillin) and 10 μg/ml tetracycline. Use a 96-pin replicator to spot on a rectangular flat plate if growing in 96-well format. Grow overnight at 37°C.
Grow cultures in LB medium containing 50 μg/ml ampicillin. If using 96-well format, add 800 μl medium to each well of a 96-well deep well plate. To inoculate the cultures, use individual yellow tips or tips in a multichannel pipettor to scrape bacteria from a row or column and eject tips into the correct row or column of medium. When finished innoculating, remove the tips and cover the plates with plastic microtitre lids. Grow cultures with shaking at 300rpm for 6–8 hours.
Seed NGM agar feeding plates the bacterial culture. Use two 30 μl drops if using 12-well plates, and three 50 μl drops if using 6-well or individual plates. Let dry and induce overnight at room temperature.
Grow desired worm strain on standard NGM plates seeded with OP50 bacteria. Carry out standard bleaching/washing protocol to obtain embryos, and leave to hatch into L1s overnight in M9 buffer. These starved L1s will be synchronized at the beginning of the L1 stage. If feeding will be done with larvae older than L1, then put hatched L1s onto standard NGM plates containing OP50 and grow to the desired stage.
Wash worms off plates using M9 buffer, then wash 3X to remove bacteria. It is critical to remove OP50 as residual non-RNAi bacteria will interfere with the feeding results. Resuspend final worm pellet in M9 buffer containing 0.1% Tween-20 to prevent them from sticking to plastic. Adjust the volume of buffer so that the number of worms you want to aliquot per plate is in 10–15 μl.
This part of the protocol will differ slightly depending on your assay. After feeding, either the aliquoted worms or their progeny can be scored. For some assays, scoring is easier if progeny are synchronized. In this case, fed gravid mothers are allowed to lay eggs on a new plate, then removed, and the progeny subsequently scored. This step is time consuming and not always necessary.
In this protocol, a semi-synchronized population of progeny laid in a 24-hour window are scored.
Aliquot 10–15 μl of L3/L4 worms per plate or well (10–20 worms).
Leave 72 hours at 15°C (or 36–40 hours at 22°C) for RNAi to take effect, then replica plate single adults onto other plates or wells seeded with the same bacteria.
After 24 hours, remove the adults from the replica and score the progeny for phenotypes at appropriate time points.
In this protocol, all the progeny laid by fed mothers are scored. It has the benefit of being quick as there is no replica plating involved. Progeny laid early and late in the feeding protocol are in a single well, producing a range of RNAi knockdowns, from weak to strong. This can be helpful if screening for post-embryonic phenotypes, where a strong knockdown might cause embryonic lethality. Low percentage embryonic lethality is difficult to score using this method.
Aliquot 10–15 μl of L3/L4 worms per plate or well. As the adults and all the progeny will remain in this initial plate or well, it is important not to have too many worms for the food available. The optimal number should be determined empirically. For 6-well plates, using 10 worms per well should allow scoring of adult progeny in most cases.
Score when the progeny reach the desired age.
L1s can be used instead of L4s in either of the above protocols. An advantage of using L1s is that some phenotypes can be scored in the fed worms instead of the progeny, allowing an easily scored synchronized population to be used. However for some genes, inherited maternal product will be sufficient for gene activity, preventing induction of a phenotype in the fed worms. Also, as many genes are required at multiple times in development, different phenotypes may be seen when using L1s compared to L4s. For example, RNAi of some genes induces sterility of the fed L1s whereas L4 feeding induces embryonic lethality of the progeny. This will preclude scoring of progeny for these genes if L1s are fed. In contrast, using L1s is beneficial if the assay is for any form of lethality (e.g., sterility, larval lethality, or embryonic lethality).
Notes: Aliquoting bleached embryos directly onto feeding plates instead of hatching them into starved L1s first is not recommended because it is more difficult to aliquot the same number of animals/well due to embryos sticking together. Also, variable hatching rates between bleached preparations will cause inter-experiment variation.
The protocol is identical to feeding a single bacterial strain except that the two bacterial cultures are mixed prior to seeding. Double feeding works much better and more reproducibly in an RNAi supersensitive strain (e.g., rrf-3; Simmer et al., 2002; eri-1; Kennedy et al., 2004 or eri-1; lin-15B; Wang et al., 2005; S. Woods, D. Rivers, and J. Ahringer, unpublished; A. Fraser, pers. comm.). In a test of 17 control double feedings, 4 were successful in eri-1, 7 in rrf-3, and 14 in eri-1; lin-15B (S. Woods, D. Rivers, and J. Ahringer, unpublished). Double feeding is less reliable than single feeding, so in some cases, only one gene may be significantly inhibited, or both genes may be only slightly knocked down. Controls should be carried out to test for knockdown of each gene.
I thank members of the lab for comments. This is a modification of the protocol developed by Ravi Kamath (Kamath et al., 2001), with modifications from Gino Poulin, David Rivers, and Shane Woods.
The Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK
RNAi feeding in liquid is a good method to use when the assay is growth or lack of growth. High embryonic lethality, sterility, larval lethality, and severe slow growth phenotypes are easy to score in liquid culture. Scoring a population for a large change in expression of a GFP reporter can also be done in liquid culture. Screening for post-embryonic phenotypes or low percentage lethality (e.g., under 30%) is better done using RNAi by feeding on plates.
Synopsis: Grow bacteria in 96 well format, spin, resuspend in feeding buffer, and aliquot into 96 well plates. Synchronize worms by bleaching and grow to desired stage. Wash and aliquot worms into 96 well plate containing RNAi bacteria using a 96-well plate dispenser. Incubate for desired length of time and score.
Use a 96-pin replicator to spot bacteria from a glycerol stock to a flat LB plate containing 50 μg/ml ampicillin (or 25 μg/ml carbenicillin) and 10 μg/ml tetracycline. Grow overnight at 37 degrees.
Add 500μl of LB medium containing 50 μg/ml ampicillin into each well of a 96-well deep well plate. To inoculate the cultures, use tips in a multichannel pipettor to scrape bacteria from a row or column and eject tips into the correct row or column of medium. When finished innoculating, remove the tips and cover with a plastic microtitre plate, taped on. Grow cultures with shaking overnight (300 rpm).
Add IPTG to 1mM final concentration. Shake at 37°C for 1 hour.
Spin the 96 well plate cultures in centrifuge at 4000 g for 20 minutes.
Discard the supernatant.
Resuspend bacteria in 160 μl S-Basal containing standard S-Basal additives plus 100 μg/ml ampicillin and 1mM IPTG.
Aliquot 30μl into each well of shallow flat bottomed 96 well plates with lids.
Notes: Some investigators leave the bacteria in LB amp then add IPTG to the worms in M9 buffer before aliquoting, instead of spinning the bacteria down and resuspending in S-Basal. In our hands, this has variable success. There is a higher incidence of contamination and bacteria continue to grow, making scoring more difficult, as the wells do not clear. We find that removing the LB and resuspending the bacteria in S-Basal produces more reproducible and easier to score results.
Bleach adult worms using standard procedure and leave eggs to hatch into L1s in M9 buffer overnight.
If feeding a stage later than L1, aliquot the worms onto plates with OP50 bacteria and grow to the desired stage.
Wash synchronized worms off plates using M9 buffer, spin 600g for 2 minutes. Repeat wash 2X.
Resuspend worms in S-Basal (containing standard S-Basal additives plus 1mM IPTG, 100ug/ml Amp + and 0.01% Tween -20) at a concentration of 10 worms/20 μl and transfer to a tube compatible with your dispenser.
Use 96-well plate dispenser to aliquot 20 μl worm solution into each well of 96-well plates containing the RNAi bacteria from above. The number of worms/well will probably range from 5–15.
Put plates in a humid chamber at the desired temperature with no shaking. Score 4–7 days later for phenotypes. Depending on your assay, this can be done directly in the microtitre plates (e.g., lethality) or worms can be transferred to agar plates or other assay containers.
Note 1: The number of worms and the amount of bacteria suggested above are a good starting point. These may need to be adjusted to suit your screen.
Note 2: Feeding of different larval stages may give different results. For example, many genes that induce an embryonic lethal RNAi phenotype from an L4 feeding induce sterility with L1 feeding. Which stage or stages to use will depend on the assay and your specific phenotype and should be determined empirically using controls.
Note 3: If you want to use a larger volume of media per well (e.g., 150 μl), the plates will need to be shaken for aeration or worms may die due to lack of oxygen.
The choice of strain will be somewhat limited by your assay, but it is a good idea to try several different backgrounds. In particular, we recommend testing whether your phenotype is induced more strongly in an RNAi supersensitive strain e.g., rrf-3 (Simmer et al., 2002), eri-1 (Kennedy et al., 2004) or eri-1; lin-15B (Wang et al., 2005).
It is possible to carry out a liquid culture screen feeding two bacterial strains at once. For example, one common bacterial strain can be mixed with each individual strain to look for interacting genes. The bacteria should be grown separately, mixed in equal amounts, then spun down and resuspended in S-basal feeding buffer as above, or else prepared completely separately before mixing. In most cases, the use of an RNAi supersensitive strain is necessary for the success of feeding two RNAi bacterial strains together (S. Woods, D. Rivers, and J. Ahringer, unpublished; A Fraser, pers. comm.).
Department of Genetics, Yale University, New Haven, Connecticut 06520 USA
Department of Genetics, Yale University, New Haven, Connecticut 06520 USA
Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, Connecticut 06520 USA
This gene knockout strategy uses a random mutagen, trimethylpsoralen (psoralen) to mutagenize a very large number of worms. The worms are divided into many small subcultures and allowed to have progeny. A portion of each subculture is stored alive in the freezer, and genomic DNA is made from the rest of the culture. This DNA is thus made from the siblings of the frozen worms and carries the same mutations as the frozen worms carry. At a very low frequency (~1/200,000 mutagenized genomes) the mutagenesis will produce a small deletion (100-1000 bp) in any gene of interest. If PCR primers flanking an area of the gene of interest are used to amplify from the genomic DNA samples, deletions between the primers can be detected since they will bring the primer sites closer together and thus will generate a PCR amplicon smaller in size than that amplified from wild-type genomic DNA. When the PCR reaction is carried out under appropriate conditions, smaller deletion amplicons are amplified much more efficiently than the larger wild-type amplicon. Thus DNA representing several thousand mutagenized genomes can be amplified in a single reaction and a deletion amplicon generated from just one of those genomes will still be detected on an ethidium bromide-stained agarose gel. Once a DNA pool containing a deletion is identified, one can work back to identify the subculture of worms in which the deletion occurred. The frozen worms from that subculture are thawed, and individual live animals carrying the deletion mutation can be identified. It is important to note that deletion mutations are detected and recovered in heterozygous form, so recessive mutations that cause lethality or sterility are not selected against and can easily be obtained.
A standard mutant library consists of psoralen-UV mutagenized worms representing about 920,000 haploid genomes. Twenty F1 progeny of mutagenized worms are cultured and allowed to have F2 progeny in each well of 240 96-well microtiter plates. Half of each culture is frozen (generating a 240 microtiter plate frozen library of live animals), 1/4 of each culture is used to generate genomic DNA preps from pools of 96 wells: these pooled DNA preps are used as PCR templates for first level screening. The remaining 1/4 of each culture is lysed in the original culture well and frozen for use as PCR templates in second level screening (generating a second set of 240 plates that must be stored in a freezer). 240 microtiter plates fit on one shelf of a standard -80 degree freezer, so two freezer shelves are required to store the library.
This procedure requires an initial investment of about two weeks of part-time work to pilot the methods followed by ~three weeks of full-time work (if two individuals work together) to construct a frozen mutant library. The library can be stored indefinitely and can be screened at least 400 times. Once the library is constructed one can isolate a live mutant in a gene of interest in only 2–3 weeks of work.
We suggest that no more than two people should do the actual work of library construction. Spreading the work among a larger number of people may sound appealing, but the amount of work involved is not so much that more than two people are needed, and a larger group can be detrimental to quality control. We also suggest that at least one of the individuals constructing a deletion library should have several years of bench experience. These protocols are written based on the assumption that the reader has some familiarity with C. elegans and standard worm culture methods. If you have no experience with C. elegans we do not recommend you attempt this method without help.
The time, manpower, space, and financial resources required to use this procedure are modest enough that this technology is suitable for even a small laboratory that needs to generate several knockout mutations in several genes. However, we do not recommend it for labs that only need to knockout a single or just a few genes. For such laboratories, requests can be made to two C. elegans gene knockout consortiums. Information about such knockout requests can be found online at: http://celeganskoconsortium.omrf.org/ or http://shigen.lab.nig.ac.jp/c.elegans/ index.jsp.
A key feature of these protocols is that they include quantitative quality control assessments at each stage of library construction. Thus the success of each part of the procedure can be verified along the way, and any unsuccessful batches can be discarded immediately if a failure occurs. The production of a successful deletion library can be essentially guaranteed as long as the individuals constructing the library are committed to meeting all the quality control criteria. Every failure we are aware of resulted from individuals failing to take the quality control targets seriously and forging ahead without meeting them.
A standard 920,000 mutagenized genome library is adequate to identify at least one deletion allele in most genes. The size of the library can easily be increased to improve the success rate and average number of alleles obtained per gene. At Yale we have two mutant libraries representing a total of about two million genomes. Since completing the second library, we have succeeded in isolating at least one deletion mutation for every gene we have attempted to knock out. Two publications that contain examples of using this technology to knock out whole families of genes are Hess et al. (2004) and Chase et al. (2004).
These protocols are based on methods originally developed and described previously by three different groups: 1) Bob Barstead's lab at the Oklahoma Medical Research Center (Edgley et al., 2002); 2) the NemaPharm Group at Axys Pharmaceuticals (Liu et al., 1999); and 3) Ron Plasterk's lab at the Netherlands Cancer Institute (Jansen et al., 1997; Jansen et al., 1999). The library construction methods described here are most similar to those originated at NemaPharm, and include the microtiter culture/frozen worm strategy pioneered there. Rajesh Ranganathan and Peter Reddien in the Horvitz lab developed the special container used to freeze worms in microtiter plates. The library screening protocols are based on the “poison primer” method described in Edgley et al. (2002). Most aspects of the procedure have been varied to improve efficiency and success rate. Thus, these methods differ from those of others in a number of details.
The protocols below describe in detail each aspect of library construction and screening, but they are so lengthy that it can be difficult when initially reading them to get the overview needed to start organizing the necessary efforts. Therefore, described first is a brief outline and flowchart of the actions required while constructing the library. The purpose and logic of each of the various activities described will become clearer after having read the detailed protocols. Therefore, first become familiar with the strategy by reading through the whole document and then return to the overview and flowchart (Figure 1).
For mutagenesis, order psoralen and find or buy an appropriate UV illuminator and a UV dose meter.
For microtiter culture of worms, buy 96-well culture plates and Tupperware-style boxes.
For freezing of worms in 96-well format, order flexible plastic 96-well plates and film to seal the tops of the plates. Obtain four or more New England Bioloabs styrofoam coolers and construct the foam rubber pieces that will go inside to make the specialized freezing boxes.
For storing the library, clear two shelves of a -80° C freezer. The library is stored in Tupperware-style boxes that hold 24 plates each, and ten such boxes must fit on each freezer shelf. Find freezer boxes that will hold 24 plates and which are the right dimensions to fit 10 per shelf in your freezer.
Obtain two 12-channel pipettors, one for small volumes (5-50μl) and one for larger volumes (up to 200 μl).
For library construction, an oven is needed for 55°C and 95°C incubations (a hybridization oven can be used for 55°C, but a high temperature oven is required for 95°C).
For screening the library, at least one 96-well format PCR machine is needed.
There are other minor supplies required that are described in the detailed protocol.
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Figure 1. Deletion library flowchart. Each step in the construction and screening of the library is shown underlined on the left. The protocols are briefly summarized in the order in which they are carried out. Use this flowchart as a reference when following the more detailed library construction and screening protocols.
Optimize the psoralen-UV mutagenesis of C. elegans. Monitor the success of the mutagenesis by quantitating the percent lethality in the F1 progeny of the mutagenized animals.
Optimize the growth of 96-well liquid cultures of the F1 progeny of mutagenized animals. Monitor the success by 1) counting the number of F1 animals per well to make sure the target of about 20 animals per well is attained; 2) counting the number of F2 progeny produced per well to make sure the target of >1500 per well is obtained; 3) making sure the cultures starve with F2 animals that are predominantly arrested as L1 and L2 larvae; and 4) making sure the cultures are not contaminated. The appropriate amount of bacteria supplied to the worms as food must be empirically determined in order to optimize the culturing conditions.
Optimize the freezing of live animals in 96-well format. Using the practice 96-well cultures as tests, construct and test the special freezing containers. Monitor the success by counting the number of viable animals recovered after thawing test aliquots. A successful freeze/thaw should recover >200 live animals per frozen culture.
The library is produced in batches of 24 96-well plates. A total of 10 batches are required to produce the entire 240 plate library.
Each 24-plate batch requires a mutagenesis, five days of culturing the animals in microtiter dishes, and then one day for processing the grown cultures. Processing involves 1) producing a set of 24 microtiter plates of frozen worms, stored at -80°C; 2) producing a corresponding set of 24 “single well lysates” that can be used as PCR templates, also stored at −80°C; and 3) producing 24 “pooled DNA” preps, one for each plate, that are also used as PCR templates. The day a batch is harvested, do items 1 and 2 from this list. When all the growths are done, complete the DNA preps for all the batches at the same time.
It is recommended to start by just setting up one batch and processing it from beginning to end to make sure all the technical steps work before beginning full-scale production. Proceed to full-scale production by setting up five batches, one per day for five days, and then spending five days harvesting those batches, one per day. Repeat this procedure once more to produce enough batches for a complete library. At the end, complete the processing of the pooled DNA preps for all the batches. This schedule distributes the work over a three-week period in a fairly even fashion.
Grow N2 worms on three 15 cm NGM plates with thick bacterial lawns. Use a healthy bacterial strain like HB101 rather than the more sickly standard strain OP50 to get a thick lawn. For instructions on standard C. elegans culture and media there are several reference books, such as Wood et al., 1988. The library is constructed in batches of 24 microtiter plates, and the three NGM plates will provide enough animals for several batches. Let the plates grow until there are a large number of gravid adults but the plates have not yet starved.
Add 10 mls sterile S medium to each of the three plates and use a sterile Pasteur pipette to transfer the worm suspension to three sterile 15 ml centrifuge tubes. (The recipe for S medium is given below in section 7.2.2). Spin the tubes in a clinical centrifuge (30 sec at ~1000 rpm) and remove the supernatant.
Add 5 mls alkaline bleach solution to each tube. Incubate at room temperature for 3 minutes with occasional gentle agitation. This kills all adults and larvae, while leaving the eggs alive. The eggs must be washed from the bleach solution in less than 5 minutes to prevent them from dying.
| Alkaline bleach solution |
| 0.25 M NaOH |
| 20% (by volume) commercial Chlorox (final concentration = 1% hypochlorite) |
Spin as before and remove the supernatant. Add 10 mls S medium (within 5 minutes after adding the bleach solution), mix, spin, and remove the supernatant. Repeat this wash twice more. Fill the tubes one last time with S medium and pour the contents of all the tubes into a sterile 250 ml Erlenmeyer flask containing ~50 mls S medium.
Place the flask on a 20°C shaker overnight (or longer). All the eggs will hatch and the animals will arrest as starved L1 larvae. This step produces almost perfect synchrony of the population. The worms can be left on the shaker for up to two weeks. During library construction several 24-plate batches can be started from this same flask of L1's. The following steps describe how to restart growth of L1's for a 24-plate batch.
Late in the afternoon, pour some of the L1 solution into sterile 15 ml centrifuge tubes, spin the animals down, remove most of the supernatant, and combine all the animals into one tube.
Quantitate the concentration of L1's. Use serial dilutions to reduce the L1 concentration to something easier to count: be sure that you shake up the suspensions of animals immediately prior to any pipetting, as the animals settle rapidly. To count the animals in a suspension, use a P20 pipetteman to remove 10 μl of suspension and then place it onto an unseeded NGM plate in a long, thin line. Count the worms under a dissecting microscope. The long thin line makes it easy to count the worms without skipping any or counting any twice.
Pipette a volume containing 10,000 worms onto each of six 15 cm NGM plates with thick bacterial lawns. There should be enough food on the plates to allow the animals to grow to the L4 stage without starving. Set the plates at 20°C.
Psoralen-UV mutagenesis is less used and somewhat more difficult than EMS mutagenesis, which is the standard method for most C. elegans genetics. However, psoralen-UV mutagenesis is thought to produce a higher frequency of the class of small deletions detected by the deletion screening methods described below.
Animals should be mutagenized at the late L4 stage. Use a dissecting microscope to verify the staging: almost all the animals should have a white crescent visible where the vulva is undergoing morphogenesis; a few will be just past this stage. This should be at about 43–48 hours after they were put on plates with food. The animals should be almost perfectly synchronous.
Keep all solutions of psoralen and worms treated with psoralen in the dark; ordinary room light has ~3-4 μW/cm2 of 360 nm UV light.
Wash the worms off the NGM plates with M9 medium, using sterile glass pipettes, and transfer the worms to a sterile 15 ml plastic centrifuge tube. Spin the worms down (1000 rpm in a clinical centrifuge for 30 sec.) and remove the supernatant or add M9 until the total volume of the worm suspension is 7 mls.
| M9 Medium |
| 5.8 g Na2HPO4 |
| 3.0 g KH2PO4 |
| 0.5 g NaCl |
| 1.0 g NH4Cl |
Bring to 1 liter w/ dH20. Dispense into 100 ml bottles and autoclave.
Place 7 mls M9 in a separate 50 ml tube that is covered with aluminum foil to block out light. While working in a hood and wearing gloves, add 210 μl of 2 mg/ml psoralen in DMSO (this requires heating and stirring to get into solution). Follow proper procedures for disposing of psoralen waste. Our experience suggests that psoralen solutions go bad over time. Therefore, make a fresh solution of psoralen, store frozen aliquots at −80°C, and use these aliquots for no more than a few weeks before discarding them.
Transfer the 7 mls of worms into the tube of psoralen and rock on a rocker at 20°C for 15 minutes. In the meantime, turn on the UV light of the UV illuminator to warm up for >10 minutes. Note that an appropriate UV illuminator and dose meter, along with specifics on other equipment and supplies, are described at the end of this document.
Pour the worms into a 15 cm sterile plastic petri dish. Use a UV dose meter to monitor the 360 nm UV dose (through a petri dish bottom) on the UV box. For example, if the machine gives an intensity of 3.05–3.2 mW/cm2, expose the worms for ~7–8 seconds. Quickly place the petri dish at the center of the machine, time the 7–8 seconds, and remove the dish. Afterward transfer the worms with a Pasteur pipette back to a 15 ml tube and spin down in a clinical centrifuge. Remove the supernatant to a psoralen liquid waste container. Wash the worms twice in ~5 mls of M9. Remove all of the last supernatant except for ~200 μl. Using a sterile glass pipette, transfer the worms to the edge of the bacterial lawn on 15 cm NGM plates. Eight 15 cm plates should suffice to give enough food to allow the worms to grow to gravid adults. Place plates at 25°C in the dark.
This is a very heavy mutagenesis and results in significant lethality of the F1 progeny of the mutagenized animals. Raising the UV dose even slightly leads to a rapid increase in the F1 lethality, so the UV dose must be monitored and controlled carefully. The best way to monitor the mutagenesis is to measure the F1 lethality and adjust the UV dose until you reach the desired range of 10–15% lethality, as described below.
A more convenient way to carry out the psoralen mutagenesis is as follows. After the 15 minute incubation in psoralen solution, spin down the worms and remove the supernatant. Transfer the animals in a small amount of liquid to the surface of unseeded NGM plates. Place the plates in a “Stratalinker” (Stratagene) equipped with long-wave (360 nm) UV bulbs (note that the standard bulbs in these machines are for short-wave UV). The Stratalinker can then be used to deliver a measured dose of UV radiation. While this method has been very successful for psoralen mutagenesis for other purposes, it has not been used to generate a deletion library and thus this method of mutagenesis cannot be guaranteed to work for library production.
When the mutagenized P0's are gravid adults, about 24–30 hours after the end of mutagenesis, they are ready for processing. The plates should be recently starved, there should be lots of F1 L1's on the plate, and the P0 adults should be bloated with unlaid eggs.
To assess the success of mutagenesis, remove ~20 worms to a freshly-seeded plate at 25°C for a few hours, remove the adults, and then replace the plates at 25°C overnight. The next day count the number of hatched larvae and dead eggs among the F1. The mutagenesis worked well if there is ~10–15% lethality in the F1's. This is the first quantitative quality control criterion to be met for each batch. Keep a set of records noting the numbers measured at each quality control point for each batch during the library construction.
After setting up the quality control check, treat the remainder of the mutagenized worms with alkaline bleach as described earlier. Plate the surviving eggs on two unseeded 6 cm NGM plates. The hatched eggs will arrest as starved and perfectly synchronized L1's that will then be ready to transfer to liquid culture.
An alternative method of getting gravid adults bloated with eggs prior to the second bleaching may be used to greatly increase the yield of F2 animals. At 24–30 hours after the end of the mutagenesis, while the 15 cm plates are not close to starvation, wash off the worms with 5 mls of M9 per plate and transfer to a 15 ml centrifuge tube. Spin the worms down, remove the supernatant, and wash twice with S medium, leaving 14 mls final volume. Pour the worms and S medium into an empty, sterile 15 ml plastic petri dish (not filled with agar) and let sit on the lab bench for 4–5 hours. Shake the dish occasionally to keep the worms aerated, and to avoid them sticking to the bottom of the dish. Inspect the culture periodically. When the worms appear bloated with eggs (they do not lay eggs well in M9 medium) transfer the liquid to a 15 ml centrifuge tube, spin, remove the supernatant, and bleach the eggs as stated in section 7.2.1.1 and above. Transfer the pellet of eggs to two unseeded plates. Add 10 mls of S medium to the remaining empty petri dish, which will still have eggs stuck to its surface. Incubate the three petri dishes overnight in the 25°C incubator. This technique will produce reliable yields of about 80–100 thousand L1 animals.
Commentary: The synchrony induced by starving the F1 animals is critical to the success of the procedure. It guarantees that an F1 animal carrying a deletion mutation of interest will be the same age as the ~19 animals it is cultured with (see section 7.2.2). If the animal of interest is younger than the others in the well it will have few F2 progeny and the deletion will therefore be difficult to detect and recover.
Use sterile technique when culturing the mutagenized worms. The growth medium contains an antibiotic/antimycotic to reduce contamination. Visually inspect some of the plates for contamination when the cultures are harvested. Contaminated plates must be discarded.
The three critical parameters for growth in microtiter wells are 1) amount of bacteria added, 2) adequate aeration, and 3) growth temperature. If too much bacteria is added the worms will die; if too little bacteria is added the worms will produce few progeny before the culture starves. With the proper amount of food, 1000–2000 progeny will be obtained from the 20 worms added to each microtiter well, and these progeny will starve as L1, L2, and L3 larvae 4–5 days after the culture is started. Inadequate aeration causes the cultures to grow slowly or not at all. Cultures grow slowly at temperatures below 20°C or above 25°C.
| S-medium (4 liters) |
| 23.2 g NaCl |
| 200 ml 1M potassium phosphate, pH 6.0 (136.1 g KH2PO4 per 1000 ml; start with 800 ml and adjust to pH 6.0 with solid KOH (approx. 15g) before bringing up to volume.) |
| 3.8 L dH20 |
| 4 ml 5 mg/ml cholesterol in 95% EtOH. Warm to 37°C to dissolve. |
| Aliquot 400 ml into each of 10 bottles; autoclave. |
Just before use, supplement each sterile 400 ml aliquot of S Medium with six solutions (I-VI). These must be added after the S Medium has been autoclaved. Each supplement should be sterile and added to the S medium using sterile technique.
1.2 ml 1M MgSO4
1.2 ml 1M CaCl2
4 ml 100X trace metals solution
| 0.346 g FeSO4.7H2O |
| 0.930 g Na2EDTA |
| 0.098 g MnCL2.4H2O |
| 0.144 g ZnSO4.7H2O |
| 0.012 g CuSO4.5H2O |
| bring up to 500 ml final volume with dH20 |
| Autoclave. Store trace metals solution in the dark. |
4 ml 1M potassium citrate, pH 6.0 (21.01 g citric acid monohydrate per 100 ml; start with 80 ml and adjust to pH 6.0 with solid KOH (approx. 17 g) before bringing up to volume. Autoclave.)
4 ml Gibco 100X penicillin/streptomycin
4 ml 100X nystatin suspension 10,000 units/ml.
| L broth (2 liters) |
| 20 g tryptone (Difco) |
| 20 g NaCl |
| 10 g yeast extract (Difco) |
| dH20 to 2 liters |
| Autoclave. |
