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 o....

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 liqui....

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 that .......

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

List of materials used in this article

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