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

Summary

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.

Abstract

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.

Introduction

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 screens^1,2. 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 system^3-6. 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 source^7,8. 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.

Protocol

1. Worm Strains

1. Obtain C. elegans wildtype Bristol N2 strains from the Caenorhabditis Genetics Center (CGC) (http://www.cbs.umn.edu/cgc) and maintain them on NGM plates seeded with E. coli strain OP50⁷. Note: Transgenic worm strains IQ6011 (Phrg-1::GFP) utilized were generated as previously described¹¹. IQ6011 can be requested from the corresponding author.

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.

1. Obtain ultra pasteurized fat-free milk from a grocery store. Use the refrigerated product and not the room temperature product. Prior to use in mCeHR medium, streak the milk on Brain Heart Infusion (BHI) agar plates and incubate for 3 days at 30 °C and 37 °C to check for sterility. Transfer the milk to 50 ml sterile tubes and store at -80 °C
2. Prepare each of the following components and combine in the order and amounts given to prepare 1 L of mCeHR (Table 1A): 10 ml of 2 mM choline diacid citrate, 10 ml of vitamin and growth factor mix (see recipe in Table 1B), 10 ml of 2.4 mM myo-inositol, 10 ml of 2 mM hemin chloride, 250 ml of deionized water. Apply suction and filter through a 0.22 μm filter unit.
3. Add 20 ml of nucleic acid mix (recipe in Table 1C), 100 ml of mineral mix (recipe in Table 1D), 20 ml of 170 mg/ml lactalbumin hydrolysate, 20 ml of essential amino acids, 10 ml of non-essential amino acids, 20 ml of 450 mM KH₂PO₄, 50 ml of 1.45 M D-glucose, 10 ml of 1 M HEPES, sodium salt, 250 ml deionized water.
4. Apply suction to filter sterilize the components. Remove the filter and add 1 ml of 5 mg/ml cholesterol. Ensure that the pH of the media is approximately 6-6.5. Note: This solution can be stored at -80 °C for up to one year.
5. Add 20% (v/v) ultra pasteurized organic skim milk to the mCeHR medium before use. Use scrupulously sterile technique (for example in a laminar flow hood) when opening and aliquoting the milk. Store the media at 4 °C.

3. Prepare C. elegans for Culture in Axenic mCeHR Liquid Medium

1. Grow worms on ten 60 mm NGM plates until there are many gravid worms and a minimal amount of OP50 E. coli on the plates.
2. Remove the worms from each plate by rinsing with 5 ml of M9 buffer (recipe in Table 1F), and combine in a 50 ml conical tube.
3. Allow the worms to settle by leaving the tube to stand for 5 to 10 min then carefully remove the supernatant containing the E. coli.
4. Repeat steps 3.2 and 3.3 2x to remove as much of the residual bacteria as possible before continuing the procedure.
5. Resuspend the worms in 0.1 N NaCl in a volume that is a multiple of three, for example 1.5 ml, 3 ml or 6 ml in which individual worms can be seen in the tube.
6. Bleach the worms by adding 5 N NaOH and 5% sodium hypochlorite (bleach) solution in a 1:2:6 ratio to the worm suspension. For example, 1 ml 5 N NaOH: 2 ml 5% sodium hypochlorite: 6 ml of the worm suspension. Mix the NaOH and bleach before adding to the worm suspension.
7. Vortex the solution vigorously until the gravid worms are dissolved and only eggs are visible within the suspension (about 5 to 10 min). Monitor the process using a phase contrast microscope with a 10X objective.
8. After the worms have completely dissolved, immediately pellet the eggs at 800 x g for 45 sec at 4 °C. Remove the supernatant and rinse the pellet twice with 10 ml of sterile water by centrifuging the pellet at 800 x g for 45 sec at 4 °C.
9. After bleaching, transfer the egg pellet to a 25 cm² tissue culture flask containing 10 ml of mCeHR media supplemented with 100 μg/ml tetracycline. Carry out this procedure using sterile techniques. If desired, add additional antibiotics, including 250 μg/ml nalidixic acid, to prevent bacterial growth in the axenic liquid cultures.
10. Incubate the liquid cultures at 20 °C on a shaking incubator at approximately 70 rpm.
11. Check the worms daily to note the stage of development and the rate of growth.
    Note: Worms will initially grow slowly and require 7 to 10 days to become gravid. However, as the worms acclimatize to the liquid medium the generation time will decrease to approximately 4 days for wildtype (N2) worms.
12. After the worms have developed to the gravid stage in liquid media, transfer the worm culture to a conical tube and pellet the worms at 800 x g for 5 min at 4 °C using a swinging bucket rotor.
13. Remove the supernatant and gently resuspend the worm pellet in an equal volume of 0.1 M NaCl. For example, use 10 ml of 0.1 M NaCl for worms cultured in 10 ml of mCeHR. Allow the worms to settle for 5 min on ice.
14. Carry out the bleaching procedure as described above in steps 3.5-3.8, and allow the worms to grow for a second generation in the axenic liquid media. If necessary, add the antibiotic again to inhibit bacterial growth.
15. Again allow the worms to become gravid then pellet and resuspend the worms in 0.1 M NaCl as described in steps 3.12 and 3.13, then bleach again as described in steps 3.5-3.8, to produce a synchronized population. Grow the L1 larval worms in liquid media without antibiotics from now on.

4. Synchronizing Worms from Liquid Culture

1. Pellet and resuspend the worms in 0.1 M NaCl as described above in steps 3.12 and 3.13. Bleach as described above in steps 3.5-3.8.
2. Resuspend the egg pellet in 10 ml of M9 buffer and allow the larvae to hatch O/N at 20 °C on a rotating platform shaker. Note: The eggs will hatch into L1 larvae, but will not develop further, synchronizing the worms at the L1 stage.
3. To maintain and subculture worms, pellet L1 larvae at 800 x g for 5 min, then resuspend the larvae in 10 ml of mCeHR and transfer to a 25 cm² flask. Allow the worms to grow at 20 °C on a rotating platform. Maintain a maximum density of 3,000 worms/ml/cm² to ensure adequate nutrition for the worms.
   Note: At this stage, antibiotics are not required to maintain axenic conditions when the procedures are carried out using sterile techniques in a laminar flow hood. Worms should be checked daily to monitor growth.

5. Freezing and Thawing Worms for Axenic Medium Cultures

1. Freeze axenic asynchronous worm cultures or synchronized L1 larvae in liquid nitrogen. Pellet worms at 800 x g for 5 min then resuspend in S buffer (6.45 mM KHPO₄, 43.55 mM KHPO₄, 146.25 mM NaCl) using 0.5 ml for each vial. Add an equal volume of S buffer with 30% v/v glycerol to each vial and transfer the worms to -80 °C before long term storage in liquid N₂. Freeze approximately 1 x 10⁶ worms per ml in each vial.
2. Thaw worms by incubating the vials at 37 °C for approximately 2 min until almost all of the ice is melted. In a laminar flow hood, transfer the worms to a sterile flask containing mCeHR and place them at 20 °C.

6. Determine the Effect of Hemin Concentration on Growth and Reproduction in mCeHR

1. Use axenic mCeHR media to assay nutrient dependent growth of C. elegans. To determine the effect of hemin concentration on worm growth and development, use synchronized axenic N2 worms or other strains of interest. Synchronize L1 larvae as described above in section 4.
2. On the following day, pellet and count synchronized L1 larvae and dilute to 1 worm per μl of axenic media. Make 10 mM hemin chloride in 0.3 M NH₄OH and adjust to pH 8 using concentrated HCl.
3. Add 800 μl of mCeHR media to a 24-well plate. Add 100 worms to each well in 100 μl of media. Add hemin chloride to each well in concentrations ranging from 0 μM, 1.5 μM to 1,000 μM in triplicate. Ensure that all wells contain the same concentration of 0.3 M NH₄OH. Gently resuspended the worms before pipetting to ensure that 100 L1 larvae are added to each well.
4. Examine the worms daily and note the growth in each concentration in each well. Count the number of worms after 9 days of incubation and plot the number of worms/ml corresponding to each heme concentration (Figure 1).

7. Effect of Hemin Concentration on Heme Sensor Worms

1. Incubate synchronized L1 heme sensor worms (Phrg-1::gfp) in mCeHR containing 4 μM, 8 μM, 10 μM and 20 μM hemin.
2. Image the worms using fluorescence confocal microscopy, 48 hr post incubation (Figure 2).

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.

1. Worm Preparation
   1. Transfer approximately 1 x 10⁶ asynchronous unc-119(ed3) worms in 90 ml of mCeHR media in a 175 cm² flask one week prior to bombardment. Allow the worms to grow at 20 °C on a shaking platform at ~70 rpm (Figure 3-1).
      Note: One week later, the worm density should be significantly greater with many adult gravid worms. Approximately 3 x 10⁷ worms will be required for microparticle bombardment.
   2. Chill JM109 E. coli seeded 10 cm NGM plates on ice.
   3. Transfer the axenic worms to two 50 ml conical tubes and centrifuge the worms at 800 x g for 2 min. Aspirate the supernatant and resuspend the worms in each tube in 50 ml of M9 buffer (Figure 3-2).
   4. Allow the adult worms to pellet by gravity for 10 to 15 min on ice.
      Note: The 2 ml pellet should now contain >90% adult worms. This 2 ml pellet should have approximately 5 x 10⁶ worms and is sufficient for one microparticle bombardment.
   5. Coat the entire surface of the chilled JM109 seeded NGM plate with the 2 ml pellet of worms. Allow the liquid to be completely absorbed then leave the plate for 30 min on ice before proceeding (Figure 3-3).
2. Preparation of gold particles
   1. Weigh 30 mg of gold particles into a siliconized microcentrifuge tube. Add 1 ml 70% ethanol and vortex the tube for 5 min.
   2. Allow the tube to sit for 15 min, then centrifuge at 6,000 x g for 10 sec to pellet the gold particles. Remove the supernatant using a pipette tip by carefully touching the tip to the side of the tube opposite to the gold particles.
   3. Wash the gold particles with 1 ml of sterile water, vortex for 1 min and briefly centrifuge the tube at 6,000 x g for 10 sec. Repeat the wash step twice, then resuspend the gold particles in 0.5 ml of sterile 50% glycerol.
      Note: The final concentration of the gold particles will be 60 mg/ml and it can be stored for 1 to 2 months at 4 °C.
3. Prepare the DNA-gold particle mix
   1. Combine 10 μg of the desired plasmid DNA with 10 μg of the unc-119 rescue plasmid in a siliconized 1.5 ml microcentrifuge tube. Add 50 μl of DI water and 100 μl of well resuspended gold particle solution then vortex for 1 min.
   2. Add 150 μl of 2.5 M CaCl₂ and vortex the solution again for 1 min. To this solution, add 60 μl of 0.1 M spermidine and vortex for 3 to 5 min.
   3. Pulse centrifuge the solution at 6,000 x g for 10 sec, remove the supernatant and add 300 μl of 70% ethanol. Vortex well for 1 min, pulse centrifuge at 6,000 x g, remove the supernatant and resuspend in 170 μl of 100% ethanol. Vigorously vortex the solution for 5 to 10 min then pulse centrifuge and remove the supernatant.
4. Bombardment (Figure 3-3)
   Note: This protocol is carried out with the particle delivery system specified in the Table of Materials/Equipment. Follow the instructions for the particle delivery instrument available.
   1. Place a sheet of tissue paper into a 15 cm plate. Using tweezers, dip seven macrocarriers individually into 100% ethanol and lay them on the tissue to dry.
   2. Thoroughly clean the biolistic chamber, macrocarrier holder, and target plate with 70% ethanol. Clean and autoclave the stopping screens before each bombardment procedure.
   3. Load the macrocarrriers into the holder using tweezers and press into the holder using the seating tool.
   4. Evenly spread 20 μl of the DNA coated gold particles in the center of each macrocarrier and then allow the solution to dry completely.
   5. Clean the two parts of the pressure divider with ethanol and assemble with ethanol cleaned rupture disks. Screw the assembled pressure divider and rupture disks into the bombardment chamber.
   6. Assemble an autoclaved stopping screen with the macrocarrier holder and place the apparatus on to the metal shelf required and lock in place.
   7. Insert the assembled macrocarrier apparatus into the biolistic chamber in the allotted slot below the pressure divider and align with the pressure divider.
   8. Tape the open NGM plate with the worms onto the target plate shelf and close the door of the chamber.
   9. To bombard worms, adjust the pressure to that needed to break the rupture disks. Turn on the vacuum to 28 inches of Hg pressure, then press the fire button until the disks rupture.
   10. Release the vacuum and remove the NGM plate from the apparatus. Wash the worms from the plate and redistribute in twenty 10 cm NGM plates seeded with JM109 E. coli (Figure 3-4).
   11. Allow the worms to grow for 10 to 14 days at 25 °C and starve the plates. Worms rescued from the unc-119 defect will have normal movement and can be identified at this time. Pick at least 10 “wildtype” worms from separate plates for further analysis as these represent independent transgenic lines (Figure 3-5).

Results

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 ...

Discussion

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 m...

Materials

Name	Company	Catalog Number	Comments
MgCl2•6H2O	Sigma	M-2393
Sodium citrate	Sigma	S-4641
Potassium citrate monohydrate	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' - and 3' -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, sodium 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