BaNG - Blaxter Nematode and Neglected Genomics
  Caenorhabditis elegans
     Introduction to the biology of a model nematode
       Mark Blaxter at the Institute of Evolutionary Biology, University of Edinburgh
 

vab-5 mutant L1
a vab-1 mutant L1 larva

Caenorhabditis elegans research techniques

Here you will find a short guide to some of the research techniques used in the analysis of C. elegans . This list is not exhaustive - it just points to important ways of looking at nematodes.

Making Crosses

Though C. elegans is a self-fertilising hermaphroditeit is possible to set up genetic crosses because

  • functional males are found, though rarely
  • male sperm outcompetes hermaphrodite sperm

In wild type C. elegans males are found at about 0.05% of the population. They develop after accidental non-disjunction of the X chromosomes in gametogenesis (males are XO). The rate of nondisjunction can be increased by exposing a population of hermaphrodites to heat shock (30 deg. C for >6 hrs), and "male lines" can be propagated indefinitely by picking individual males and hermaphrodites. In addition, several loci have been described which when mutated result in high levels of males in a population. These Him strains (such as those carrying the him-8 mutation) can be used to generate males carrying desired mutations for crossing by mating with mutant hermaphrodites.

When a hermaphrodite is mated, the male sperm outcompete her own supply, and the progeny are thus almost entirely cross-fertilized.

Freezing nematodes

In order to preserve strains, a method has been developed whereby nematodes can be cryopreserved and then recovered at later dates. First stage larvae are frozen at -80 deg. C in the presence of glycerol and other cryoprotectants. On defrosting (years or decades later) live nematodes can be recovered.

Watching embryos develop

One of the major experimental benefits of C. elegans as a system for developmental biology research is that it is transparent. This is especially true of the 500 micron egg. The process of embryogenesis can be followed in single-cell detail using Nomarski or differential interference contrast microscopy. A microscope can be hooked up to a video camera and recording system and the whole process of embryogenesis (which takes about 6 hrs) recorded remotely for later analysis. This setup is called the 4D camera. The original cell lineage observations on C. elegans were performed (heroically) without this instrument, by dint of many hours (days, weeks) of repeated observation.

Laser Ablation

By using the optics of a high power microscope as a focussing device, it is possible to direct a laser light pulse to a very defined area of an embryo. This technique, laser ablation, can be used to kill cells (by "frying" their nuclei). The development of the treated embryo can then be followed, allowing both the fate of the ablated cell and the ability of those remaining to complete development to be addressed.

Mosaic analysis

Extrachromosomal elements are unstable, and can be lost (in a stochastic manner) at each cell division. An extrachromosomal element tagged with a visible marker can thus be followed and any cells from which it is lost can be identified. Using the knowledge of the lineage of the normal embryo, it is possible to deduce in which cell division the element was mispartitioned, and thus map its presence/absence in the development of the embryo under observation. Any genetic element carried on the same extrachromosomal element will also be lost, and its effects can thus be deduced. The commonest marker used is ncl-1 a mutation which causes the loss of the prominent nucleolus in cells from which it is lost. It has no deleterious phenotypic effects. Thus an animal, chromosomally ncl-1(-), is made ncl-1(+) by providing a wild type copy on an extrachromosomal element. As this element is lost, the mutation is "uncovered" and can be scored.

The Tc1 Transposon

Trasposons are mobile genetic elements which have been isolated from prokaryotic and eukaryotic genomes. Tc1 is a simple transposon which encodes a single protein (a transposase) in a gene flanked by inverted repeat sequences. It is 1.6 kb long. The mechanism of transposition has been examined in detail, and it appears that the transposase is the only protein needed for precise excision of the element and its reinsertion into a new site. Tc1 inserts into TA dinucleotides by a mechanism which results in duplication of the TA. Not all TA dinucleotides are equal however, and individual genes display hotspots for insertion. TA dinucleotides are commonest in introns and in intergenic regions of DNA and thus many (most) Tc1 insertions will be phenotypically silent. In addition, cryptic splice sites at either end of the Tc1 allow Tc1 to be spliced out of pre-mRNAs and thus to have no or little effect on gene expression even if it is inserted into a coding region.

Tc1 elements distributed ranmdomly over the genome provide a useful set of anonymous DNA markers for genetic mapping.

Tc1 and cloning genes 1: RFLP analysis

Tc1 has proved to be of great utility in the analysis of C. elegans genetics and biology. Alleles of genes-of-interest can be isolated in strains in which Tc1 is actively moving (transposing to new sites in the genome; also known as mutator, Mut or high-hopper strains). Thes e new mutations may be due to new Tc1 insertions, and this insertion "tags" the gene with a molecular marker. By looking at all the Tc1 elements in the parental and Tc1 mutated strain it is possible to identify which Tc1 element is responsible for the new mutation. The general method is to look for a restriction fragment length polymorphism assoiciated with the new mutation and detected by Tc1. This Tc1 can be isolated along with flanking DNA: the flanking DNA will derive from the gene of interest.

Tc1 and cloning genes 2: Sib selection PCR

In a high hopper strain, when Tc1 transposition is induced, the population of nematodes will include individuals where the Tc1 has inserted randomly all over the genome. By making a bank or library of these lines, it is possible to search through them for the rare ones that have a Tc1 next to a gene of interest. A large number (3000) of populations are split in two, and half are frozen away. The other half is used for DNA preparation for looking for insertional events. This search is performed by using the polymerase chain reaction to detect the fusion fragment between the gene of interest and the transposon. The mutant nematode line can then be isolated from the population by going back to the cryopreserved nematode stocks and looking for the siblings of those which gave the positive PCR reaction.

Tc1 and directed mutagenesis

When Tc1 leaves a chromosomal site, it leaves a double stranded break in the chromosome. This break is repaired by cellular machinery which is usually efficient, but sometimes deletes flanking sequences in error. This phenomenon allows the Tc1 insertion to be used as a substratte for the isolation of deletion mutants where a significant portion of flanking DNA has been deleted. If the Tc1 ids in a gene of interest this will result in the deletion of coding sequences and thus in the formation of a null or loss of function mutation in the gene. This forward or directed mutagenesis protocol is a powerful method of isolating mutants.

Transgenic Nematodes

The early germ line of C. elegans is syncytial: individual nuclei reside in pockets of cytoplasm but are connected to a central rachis. This allows uptake by multiple nuclei of any substance injected into this syncytial gonad. When DNA is injected into the gonad of a young hermaphrodite, nucleui take it up, and repair machinery catenates it into long extrachromosomal arrays (>100 kb) which can be relatively stably maintained in the resultant offspring. The transgenic nematodes are selected by coinjecting a visible marker gene, usually a dominant mutation in the cuticular collagen gene rol-6(su1006). This gene causes the nematodes to develop a helically twisted cuticle and to roll longditudinally when they move forward.

Transgenesis can be used for several sorts of analysis:

  • proof that a DNA fragment contains the wild type copy of a mutated gene, by rescuing the mutant with the transgene
  • generation of "antisense knockout" strains. If a gene's open reading frame is hooked up "backwards" to its own promoter, antisense RNA will be made, and this will suppress expression (translation) of the wild type mRNA
  • analysis of promoter elements by hooking the 5' region of a gene up to enymatic (lacZ or betagalactosidase) or fluorescent (jellyfish green fluorescent protein) reporters/markers
  • driving the overexpression of a given gene to look at gain-of-function mutations
  • introducing in vitro mutagenised or foreign genes to examine structure-function relationships
  • constructing complexly mutated strains

Transgenic extrachromosomal arrays can be stably integrated into the chromosomes by UV irradiation of transgenic lines, or by coinjection of single stranded DNA. Homologous recombination is very, very rare.

lacZ and GFP as markers of gene expression

By hooking the presumed promoter region of a gene up to a reporter system, an idea ofd the developmental regulation and tissue specific expression of a gene can be derived. C. elegans promoter elements tend to be relatively short (often less than 1 kb). Both transcriptional (where the start codon of the gene is not included in the construct) and translational (where the open reading frame of the gene is fused to the reporter open reading frame) fusions can be made.

lacZ is a stable enzyme, and background activity in C. elegans is negligible. The chromogenic stain is stable, and the enzyme is still active after mild fixation. Often, a lacZ with a nuclear localisation signal (derived from SV40) is used so that the nuclei of cells expressing the constuct can be identified.

GFP is a naturally fluorescent protein which can be used to mark the cells in which a promoter is active. It has the benefit that the animals can be observed live.

It is also possible to add short tags of amino acids to the native genes, and then detect expression patterns by using an antibody which is specific to the epitope tag.

Promoter trapping. The random fusion of genomic DNA fragments to a promoterless reporter gene will result in the stochastic generation of promoter fusions. These can tbe assayed in bulk and constructs generating interesting patterns (for example those staining a subset of early blastomeres) isolated for further study.

The Physical Map

The physical map of the C. elegans genome is made up of two elements: cosmid clones and yeast artificial chromosome clones.

Cosmids The 17,000 cosmids (insert size 35-40 kb) were ordered with respect to each other by fingerprinting each one by its pattern of digestion with restriction enzymes. Clones with overlapping fingerprints were assumed to overlap in the genome, and thus coniguous sets of cloned DNA (contigs) were built up. Not all the DNA of C. elegans is stable in E. coli and thus the contigs dis not simply join up to make six chromosomes.

YACs Yeast has a different tolerance for DNA sequences than E. coli and thus a second library (3000 clones) of large insert (>200 kb) yeast artificial chromosome (YAC) clones was constructed. A subset of cosmid clones was hybridiased to the YACs and thus the cosmid contigs were stitched together into chromosome-sized overlappign clone sets. The YAC library has been ordered with respect to chromosomal position and gridded on filters to allow others to access the resource.

Sequencing The cosmid contigs form the substrate for the sequencing of the C. elegans genome. this international effort will be complete in late 1998. Gaps between cosmid contiga are filled by sequencing from bridging YACs.

Stitching the genetic and physical maps together. As more genetic loci were cloned, and the clones placed on the physical map, the two maps were stitched together. It is now possible for a researcher to go from a genetic position to a candidate, sequenced gene in a matter of weeks rather than years of work.

The genome database WormBase

In order to present the genetic, physical, sequence and biological information of C. elegans in a single unified format, a database called WormBase has been developed. I presents the information known about C. elegans in the context of all biological information, and is available at http://www.wormbase.org/ .

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These pages were written by Mark Blaxter and colleagues.
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