An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

Summary

Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.

Abstract

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.

Protocol

Part 1: Preparing nematode growth media (NGM) Petri plates

Experimental nematodes such as C. elegans and P. pacificus are typically cultured in the laboratory on 6 cm Petri plates containing Nematode Growth Medium (NGM) ¹. Worms are kept at 20°C, but can be incubated at a range of temperatures (between 4°C - 25°C), depending on the nematode species and experimental design. To prepare plates with NGM, standard sterile techniques have to be used to prevent fungal and bacterial contamination. Following is the protocol to prepare NGM plates:

1. Weigh 15 g agar, 2.4 g NaCl, 2 g Tryptone, and 2.72....

Discussion

Mutagenesis is a widely employed technique for genetic studies in various experimental model organisms. Mutagenesis experiments are often used to identify the function of a gene². The mutagenesis procedures described here can be used not only for P. pacificus, but also for C. elegans and other related nematode species. Here we describe two methods, EMS and TMP/UV mutagenesis.

EMS is a chemical that induces point mutations or small deletions throughout the genome