C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.
In this protocol, we present the required materials, and the procedure for making modified C. elegans Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.
The soil nematode, Caenorhabditis elegans, is a powerful model organism used in numerous studies from genetics to toxicology. As a result of its 1 mm size, rapid generation time of four days, ease of cultivation, and large progeny numbers, these nematodes have been utilized in a number of genetic and pharmacological screens1,2. Researchers take advantage of this worm to identify molecules and pathways conserved in vertebrate systems. These pathways include cell death signals, pathways of aging and metabolism, and the nervous system3-6. Additionally, the transparency of C. elegans allows for the generation of transgenic lines using fluorescent protein reporters, which can be directly visualized to analyze gene expression patterns and protein localization.
In many studies this nematode is cultured on a solid agar-based surface using nematode growth medium (NGM) plates or in liquid cultures seeded with Escherichia coli as a food source7,8. These bacterial food sources can confound biochemical and toxicology studies with interference from bacterial by-products affecting the interpretation of results. In order to avoid these compounding effects, C. elegans can be cultured in an axenic liquid media that is devoid of bacteria as a food source. Using this media, we successfully cultured millions of highly synchronized worms for many standard C. elegans protocols including microarray analysis of differentially regulated genes in C. elegans exposed to different heme concentrations, and production of transgenic worms using gene bombardment. This media is chemically defined and modified from an original recipe formulated by Dr. Eric Clegg9. Using this mCeHR media, we have successfully identified genes involved in heme homeostasis, referred to as heme responsive genes (hrgs)10, which would have not been possible in regular growth conditions which utilize NGM agar plates seeded with E. coli.
In this protocol we describe the procedure for introducing and maintaining C. elegans grown on E. coli to the axenic mCeHR and utilize this method to obtain a large number of worms for producing transgenic C. elegans lines using microparticle bombardment. We also present studies that show the utility of using axenic media for determining the nutritional requirement of C. elegans using heme as an example. These studies show that using mCeHR media allows for rapid growth of a large number of C. elegans for many downstream applications utilized by worm researchers.
1. Worm Strains
2. Preparation of Modified C. elegans Habitation and Reproduction Medium (mCeHR)
Prepare mCeHR liquid media as described below12. This axenic liquid media allows the worms to grow without any additional food sources. Carry out all manipulations of axenic liquid media and axenic worms using strictly sterile conditions such as a laminar flow hood.
3. Prepare C. elegans for Culture in Axenic mCeHR Liquid Medium
4. Synchronizing Worms from Liquid Culture
5. Freezing and Thawing Worms for Axenic Medium Cultures
6. Determine the Effect of Hemin Concentration on Growth and Reproduction in mCeHR
7. Effect of Hemin Concentration on Heme Sensor Worms
8. Utilizing mCeHR to Generate Transgenic C. elegans Using Microparticle Bombardment
Note: The procedure for generating and carrying out microparticle bombardment using unc-119(ed3) worms grown in mCeHR is outlined in Figure 313.
Culturing C. elegans in axenic liquid medium aids in the determination of nutrients that are required by worms, without interference from secondary metabolites produced by E. coli. Wildtype N2 worms acclimatize to mCeHR media within three generations and show growth comparable to worms grown on NGM bacterial plates. Indeed, these worms become gravid within 4 days as compared with 3.5 days for worms grown on OP50 bacteria.
One advantage of using mCeHR was seen in studies that examined the exact nutrient requirement of these worms14. In Figure 1 worms were grown in mCeHR supplemented with increasing amounts of heme, up to 1 mM. Observations of these worms showed a distinct delay in growth at heme concentrations below 4 μM, with worms developing to the L4 larval stage but unable to progress to the gravid stage after nine days in mCeHR. At concentrations of 10 μM and 20 μM heme the worms develop to the gravid stage in 4 days and produce large number of progeny. A maximum number of progeny was seen when worms were grown in mCeHR containing 20 μM heme. Worms continued to develop and produce progeny at 100 μM and 500 μM heme. However, the number of larval progeny significantly declined in comparison to worms grown at the optimal heme concentration of 20 μM heme. Heme concentrations at or above 800 μM resulted in stunted, sickly worms at the L3 larval stage, which indicated that these heme concentrations were toxic to the worms.
In addition to determining the optimal heme concentration and the effect of heme deprivation and heme toxicity on C. elegans growth, heme reporter strains could be utilized to indirectly assess the heme status of the worm within a smaller concentration range. The IQ6011 worm is a transgenic worm that expresses a heme responsive transcriptional reporter, Phrg-1::GFP, that inversely expresses GFP in response to environmental heme concentrations. When this worm is exposed to low environmental heme in mCeHR, GFP is highly expressed. This response is reversed under heme replete conditions, as seen in Figure 2. GFP expression is repressed at 20 μM heme and increases as the heme concentration is decreased. The incremental changes in heme concentration can be accurately correlated with gene response and expression in mCeHR media.
In addition to being able to carefully control nutrient concentrations provided to the worm, mCeHR axenic media allows for the efficient growth of a large number of synchronized worms. This feature can be exploited for microparticle bombardment (Figure 3). Using this procedure at least one integrated line have been developed from every microparticle bombardment carried out (Table 2).
Figure 1. C. elegans heme growth curve. Worms were grown in a range of heme concentrations from 0 μM to 1,000 μM in mCeHR for 9 days. The number of worms in each concentration was counted and plotted. At 1.5 μM heme concentrations worms were L4 and unable to further develop. At 800 μM heme the worms were stunted at L2-L3 stages and showed effects of heme toxicity.
Figure 2. Response of heme sensor (Phrg-1::GFP) strain to different heme concentrations in mCeHR. Synchronized transgenic C. elegans expressing hrg-1::GFP were grown in mCeHR media supplemented with 4, 8, 10, or 20 µM heme for 48 hr. Images were taken with a Zeiss LSM 710 confocal microscope. Scale bar is 100 µm. Please click here to view a larger version of this figure.
Figure 3. Schematic of microparticle bombardment in C. elegans using unc-119 worms grown in mCeHR. (1) Approximately 3 x 107 unc-119(ed3) worms are grown in 90 ml mCeHR media. (2) Gravids were allowed to settle on ice for 10-15 min in 50 ml conical tubes. (3) A 2 ml pellet of approximately 5 x 106 gravid worms was spread evenly onto an unseeded NGM plate. The worms were bombarded with 12 μg of plasmid of interest and 6 μg of unc-119 rescue plasmid complexed to gold particles. (4) The bombarded worms were split onto twenty 10 cm plates seeded with the E. coli strain JM109. (5) After 2 weeks incubation at 25 °C, plates with wild type worms were selected for analysis of transgene expression strength and segregation rates.
Table 1A | |
CeHR, 1 L | |
Using sterile technique and a 1 L (0.22 μm) vacuum filter unit, filter the following volumes of stock solutions and water in the order described. | |
Choline diacid citrate | 10 ml |
Vitamin and growth factor mix | 10 ml |
myo-Inositol | 10 ml |
Hemin chloride | 10 ml |
Deionized water | 250 ml |
Nucleic acid mix | 20 ml |
Mineral Mix | 100 ml |
Lactalbumin hydrolysate | 20 ml |
Essential Amino Acid Mix | 20 ml |
Non-essential Amino Acid Mix | 10 ml |
KH2PO4 | 20 ml |
D-Glucose | 50 ml |
HEPES, sodium salt | 10 ml |
Deionized water | 250 ml |
Volume will be 800 ml at this point Remove filter unit from vacuum then add: |
|
Cholesterol | 1 ml |
Ultra-pasteurized skim milk | 200 ml |
Table 1B | |
Vitamin and growth factor mix, 100 ml | |
Solution 1: To 60 ml of water add: | |
N-acetyl-α-D-glucosamine | 0.15 g |
DL-alanine | 0.15 g |
Nicotinamide | 0.075 g |
D-pantethine | 0.0375 g |
DL-pantothenic acid, hemi calcium salt | 0.075 g |
Folic acid | 0.075 g |
Pyridoxamine 2HCl | 0.0375 g |
Pyridoxine HCl | 0.075 g |
Flavin mononucleotide, sodium salt | 0.075 g |
Thiamine hydrochloride | 0.075 g |
Solution 2: Prepare the following chemicals in 5 ml 1 N KOH: | |
p-aminobenzoic acid | 0.075 g |
D-biotin | 0.0375 g |
Cyanocobalamin (B12) | 0.0375 g |
Folinic acid, calcium salt | 0.0375 g |
Nicotinic acid | 0.075 g |
Pyridoxal 5-phosphate | 0.0375 g |
Solution 3: 0.0375 g (±) α-L-lipoic acid, oxidized form in 1 ml ethanol: | |
Combine solutions 1, 2, and 3 and bring the final volume to 100 ml. Store in dark at 4 °C or freeze aliquots at -20 °C. Make small volumes of stocks for this mix so it is used quickly. |
|
Table 1C | |
Nucleic acid mix, 100 ml | |
To 60 ml of water add: | |
Adenosine 5' -monophosphate, sodium salt | 1.74 g |
Cytidine 5' -phosphate | 1.84 g |
Guanosine 2' – and 3' -monophosphate | 1.82 g |
OR | |
Guanosine 5' -phosphate | 2.04 g |
Uridine 5' -phosphate, disodium salt | 1.84 g |
Thymine (add last) | 0.63 g |
Bring solution to 100 ml and store in the dark at 4 °C or freeze aliquots at -20 °C. Make small volumes of stocks for this mix so it is used quickly. |
|
Table 1D | |
Mineral Mix, 1 L | |
MgCl2•6H2O | 4.1 g |
Sodium citrate | 2.9 g |
Potassium citrate monohydrate | 4.9 g |
CuCl2•2H2O | 0.07 g |
MnCl2•4H2O | 0.2 g |
ZnCl2 | 0.1 g |
Fe(NH4)2(SO4)2•6H2O | 0.6 g |
CaCl2•2H2O (always add last) | 0.2 g |
Make small volumes of stocks for this mix so it is used quickly. | |
Table 1E | |
Other Components | |
KH2PO4 | 450 mM |
Choline di-acid citrate | 2 mM |
myo-Inositol | 2.4 mM |
D-Glucose | 1.45 M |
Hemin chloride | 2 mM in 0.1 N NaOH pH 8.0 |
HEPES, sodium salt | 1 M stock solution |
Cholesterol | 5 mg/ml in ethanol |
Lactalbumin enzymatic hydrolysate | 170 mg/ml |
Table 1F | |
M9 Buffer, 1 L | |
KH2PO4 | 3 g |
Na2HPO4 | 6 g |
NaCl | 5 g |
H2O | 1 L |
Autoclave 30 min | |
1 M MgSO4 (sterile) | 1 ml |
Table 1. Recipes for components of mCeHR and mCeHR.
Bombardment | Lines with wild type rescue | Lines with rescue / transgene | Stable lines |
1 | 2 | 2 | 1 |
2 | 8 | 5 | 0 |
3 | 4 | 4 | 2 |
4 | 5 | 2 | 1 |
5 | 5 | 3 | 1 |
Average | 4.8 | 3.2 | 1 |
Table 2. Average number of transgenic C. elegans generated using microparticle bombardment.
In this protocol we present a modified axenic liquid media mCeHR that allows for rapid C. elegans generation with production of a large number of worms. This media shows several advantages as the worms are grown without contaminating E. coli or bacterial byproducts and can be exploited in nutritional and toxicological studies. The use of E. coli or other bacteria in such studies has several drawbacks. For example, the growth of the bacteria can change under various conditions and the bacteria may metabolize molecules that are being assayed, confounding the interpretation of results. Therefore, the development of a defined medium to perform these studies is highly advantageous.
Although C. elegans have been grown in liquid media in previous studies15, worms grown in the C. elegans maintenance medium (CeMM) show a distinct delay in generation times16, unlike what we observe in mCeHR medium. Our main goal was to exploit C. elegans to study nutrient homeostasis with specific emphasis on heme and metal metabolism. With this in mind, mCeHR and modifications generated by our group accomplish this goal and has directly led to the identification of a number of genes required for maintaining heme homeostasis in the worm and ultimately in vertebrate model systems17,18. Reformulated mCeHR-1 media can also be exploited for other nematode species including Panagrellus redivivus, Oscheius myriophila, and C. remanei. In addition low metal formulations mCeHR-2 and mCeHR-3 can be exploited in studies examining heavy metal toxicity and requirements12.
Prior to acclimatization, N2 worms exhibit a longer generation time of approximately 7 to 10 days in mCeHR media. As the worms are subcultured, this generation time decreases to 4 days, similar to that of worms grown on OP50 bacteria. However, the generation time may be longer for certain mutant worms, plausibly because of defects in feeding, movement, or specific nutritional requirements.
When using axenic liquid media it is critical that fastidious sterile techniques are utilized. Usually, the use of antibiotics is minimized and eventually eliminated as the worms acclimatize to the medium and the residual bacteria are eliminated. Antibiotics are initially required to ensure that the cultures are axenic when established as it prevents the growth of residual bacteria that can overwhelm the worm cultures. After two successive rounds of bleaching, antibiotics are omitted from the cultures as continual usage of antibiotics only masks poor aseptic techniques that are essential for growing worms axenically. Using these techniques in a laminar flow cabinet, the authors have been able to grow the worms axenically without contamination. Additionally, proper storage of the media and components is essential for worm maintenance and growth. The worms should be checked for the rate of growth and subcultured to prevent crowding, which can lead to dauer formation as essential nutrients are depleted. This can be prevented by ensuring that the density of worms does not exceed 3,000 worms/ml/cm2. Researchers that are new to growing C. elegans axenically can achieve this by checking the worms daily and counting the worms weekly.
C. elegans researchers take advantage of its transparent properties by generating transgenic worms expressing fluorescently-tagged markers that allow for visualization of gene expression and protein localization. Utilizing biolistic bombardment allowed for generation of low copy integrants that avoided the issues of extrachromosomal arrays and elevated gene expression noted with transgenics generated using injection19. Previously, one drawback of generating transgenics using microparticle bombardment of C. elegans was the requirement for egg plate preparation to generate the large number of unc-119 (ed3) worms necessary for the procedure20. Each transformation required 20 egg plates for the number of worms needed. Using axenic mCeHR liquid culture allows more efficient growth and subsequently more worms for bombardments. Additionally, bombardments can be used in conjunction with drug selection to avoid using unc-119 (ed3) worms21.
The authors have nothing to disclose.
This work was supported by the National Institutes of HealthGrants DK85035 and DK074797 (I.H).
MgCl2.6H2O | Sigma | M-2393 | |
Sodium citrate | Sigma | S-4641 | |
Potassium citrate.H2O | Sigma | P-1722 | |
CuCl2.2H2O | Fisher | C455-500 | |
MnCl2.4H2O | Fisher | M87-100 | |
ZnCl2 | Sigma | Z-0152 | |
Fe(NH4)2(SO4)2.6H2O | Sigma | F-1018 | |
CaCl2.2H2O | Fisher | C70-500 | |
Adenosine 5 -monophosphate, sodium salt | Sigma | A-1752 | |
Cytidine 5 -phosphate | Sigma | C-1006 | |
Guanosine 2 – and3 -monophosphate | Sigma | G-8002 | |
Uridine 5 -phosphate, disodium salt | Sigma | U-6375 | |
Thymine | Sigma | T0376 | |
N-Acetylglucosamine | Sigma | A3286 | |
DL-Alanine | Fisher | S25648 | |
p-Aminobenzoic Acid | Sigma | A-9878 | |
Biotin | Sigma | B-4639 | |
Cyanocobalamine (B-12) | Sigma | V-2876 | |
Folinate (Ca) | Sigma | F-7878 | |
Niacin | Sigma | N-0761 | |
Niacinamide | Sigma | N-3376 | |
Pantetheine | Sigma | P-2125 | |
Pantothenate (Ca) | Sigma | P-6292 | |
Pteroylglutamic Acid (Folic Acid) | ACRCS | 21663-0100 | |
Pyridoxal 5'-phosphate | Sigma | P-3657 | |
Pyridoxamine.2HCl | Sigma | P-9158 | |
Pyridoxine.HCl | Sigma | P-6280 | |
Riboflavin 5-PO4(Na) | Sigma | R-7774 | |
Thiamine.HCl | Sigma | T-1270 | |
DL-6,8-Thioctic Acid | Sigma | T-1395 | |
KH2PO4 | Sigma | P-5379 | |
Choline di-acid citrate | Sigma | C-2004 | |
myo-Inositol | Sigma | I-5125 | |
D-Glucose | Sigma | G-7520 | |
Lactalbumin enzymatic hydrolysate | Sigma | L-9010 | |
Brain Heart Infusion | BD | 211065 | |
Hemin chloride | Frontier Scientific | H651-9 | |
HEPES, Na salt | Sigma | H-3784 | |
Cholesterol | J.T. Baker | F676-05 | |
MEM Non-Essential Amino Acids | Invitrogen | 11140-076 | |
MEM Amino Acids Solution | Invitrogen | 11130-051 | |
Nalidixic acid sodium salt | Sigma | N4382 | |
Tetracycline Hydrochloride | MP Biomedicals | 2194542 | |
Biolistic Delivery System | BioRad | 165-2257 | |
Gold particles (Au Powder) | Ferro Electronic Material Systems | 6420 2504, JZP01010KM | |
or | |||
Gold Particles 1.0 μm | BioRad | 165-2263 |