Selecting the Right Species: Practical Information on Organism Models

The best known phylum of yeast is Saccharomyces cerevisiae which has 16 chromosomes carrying about 6,000 genes. Yeast is a eukaryotic microorganism which has been used as a model of neurological disorders for a long time [1]. It is not easy to model neuronal disorders with yeast, but it can be used as a cellular model. A microtubule defect in a mutant of the DIS3 gene (ortholog of the human exosome complex exonuclease RRP44 gene) has been reported [2]. Apoptotic mechanisms in yeast and in other species such as the zebra fish were compared [3–5]. The real relevance of yeast for medical genetics is for gene expression profiling and functional genetics. By using mRNA profiling, differential expression induced by an identified mutation can be observed. The approach encompasses interactions between genes and/or proteins and cascade effects. It is then possible to use mRNA profiling in yeast as a framework for the mRNA profiling in humans or to select molecules targeting an unwanted overexpression. The use of mRNA profiling has found associations with several rare diseases of the nervous system where the gene in question has an ortholog in yeast: in amyotrophic lateral sclerosis; in Friedrich’s ataxia; in Niemann–Pick disease type C; in ceroid lipofuscinosis, neuronal 3; and in hereditary spastic paraplegia. Research is limited by the small percentage of orthologous genes between the two species (less than 20 %), as mRNA profiling can only be done when the yeast gene has a human ortholog but the difficulty could be overcome [6]. If the required ortholog is not found, a transgene can be inserted into the yeast; the mRNA profiling is then compared to a yeast lacking the gene. This has been done for Huntington’s chorea [7], for Parkinson’s disease, for some forms of Alzheimer’s disease (amyloid β (A4) precursor protein and tubule-associated unit—TAU), for amyotrophic lateral sclerosis, and for frontotemporal sclerosis. We are, however, far from modeling synaptic impairment.

3 Caenorhabditis elegans

Riddle et al. [8] reported the history of this small earthworm. “The potential value of Rhabditis species for genetic research was pointed out very early [9]. C. elegans was initially described and named Rhabditis elegans [10]; it was subsequently placed in the subgenus Caenorhabditis by [11] and then raised to generic status [9]. The name is a blend of Greek and Latin (Caeno, recent; rhabditis, rod; elegans, nice).” The reader may consult Riddle DL, C. elegans II. 2nd edition [8], available also from http://www.ncbi.nlm.nih.gov/books/NBK20127/.

3.1 C. elegans and Its Contribution to Neurobiology

Sidney Brenner later contributed to the popularity of Caenorhabditis elegans by selecting the small soil nematode as a model for deciphering gene–nervous system–behavior correlations. C. elegans has several properties likely to attract neurogeneticists. Hermaphroditism (XX, 99.5 % of the population) makes it easy to rapidly develop inbred lines by a self-fertilizing process, but hermaphrodite individuals can also be crossed with males (XO, only 5 % of the population). The prolific progeny, sometimes reaching 300, provides a good sample of meiotic recombination. C. elegans was the first living organism to have its genome fully sequenced. The six chromosomes encompass 19,735 genes. The number of alternative splice forms (2,685) results in 22,420 proteins [12]. Alternative splicing events occur very rarely compared to fates reported for humans. 90 % of the multi-exon genes are subjected to splicing events in the human genome [13].

The WormBase (http://www.wormbase.org) resource now has 15 species including 7 from the Caenorhabditis family plus related species such as Brugia malayi, Haemonchus contortus, Strongyloides ratti, Meloidogyne incognita, and M. hapla.

C. elegans has an invariant number of cells, as is the case for all nematodes, but specific cellular events characterize hermaphrodites and males (Fig. 1). The adult male has 1,031 nuclei, approximately the number of cells. The adult hermaphrodite has only 959 somatic nuclei, less than the young male that has 1,090. The reason for the difference is that 131 nuclei are lost in early adulthood through apoptosis. The apoptotic phase occurs after cell development. C. elegans cell lineage is another striking property of the model; it is established for all cells in the fertilized egg and appears to be invariant across individuals. White et al. [14] were able to describe the complete neural patterns of connectivity and establish the neural mechanisms—behavior correlations with chemotaxis, thermotaxis, mating behavior, and exploration. Some relatively straightforward methods for investigating the gene functions are available. Homologous recombination is not effective in C. elegans, but other methods can be used for the species. Specific genes can be disrupted by RNAi by soaking or injecting the nematodes with a solution of double-stranded RNA or by feeding them genetically transformed bacteria that express the double-stranded RNA.

Fig. 1

Caenorhabditis elegans. (a) Male C. elegans. Head on the right and male-characteristic round tail on the left; (b) hermaphrodite C. elegans. Eggs, L2, L4, and gravid worm (Photographies Arnaud Blanchard)

C. elegans is a model for a wide range of disorders with a neurological impact: aging and Alzheimer’s disease (particularly in connection with beta-amyloid peptide), alpha-synuclein or dopamine dysfunctions, Huntington’s chorea, oculo-pharyngeal muscular dystrophy, spinal muscular dystrophy, Duchenne muscular dystrophy, Parkinson’s disease, and endoplasmic reticulum disorders.

3.2 Maintaining C. elegans

Looking beyond the rationale of using a simple animal model to decipher complex molecular pathways, this organism has multiple features that make it easy to culture in a laboratory: the small size (1 mm in length), short life span (3 days) (see Fig. 1), and even autogamy are factors behind the success of C. elegans.

The material required to start is basic: the worms are typically grown on 5 cm agar plates seeded with bacteria as a food source and cultured in an incubator at a constant temperature (usually 20 °C). A stereomicroscope is needed to observe them: 50× magnification is sufficient for comfortable conditions to maintain and work with the strains. And a flame device (or equivalent) is needed to avoid any contamination during the different manipulations. Here we present some basic techniques and protocols for getting off to a good start with this animal model. Chapter 6 illustrates the possibilities offered by invertebrates to model synaptic disorders.

3.2.1 C. elegans Strains Providers

The Caenorhabditis Genetics Center (CGC) at the University of Minnesota collects, maintains, and distributes C. elegans strains. Any laboratory creating a new strain can send it to the CGC, thus enriching the collection and sharing the strain with the worm community. Conversely, researchers can easily order a strain by making a request with (1) a lab code given by the CGC and (2) a financial contribution ($25 as an annual fee plus $7 per strain). It takes approximately 1 week for delivery of the worms on an agar plate with the strain information data sheet (name of the strain, genotype, culture conditions, mutagen used, creator, etc.). More information is available on the website: https://www.cbs.umn.edu/cgc.

The National Bioresource Project is a more recent project initiated in 2002 by the Japanese government and operating at the Tokyo Women’s Medical University School of Medicine. The aim of the project is to generate deletion mutants for all genome regions by random mutagenesis. The strains are available for academic research on request (http://www.shigen.nig.ac.jp/c.elegans/).

3.2.2 Culturing the Worms on Solid Media

The worms are usually cultured on a Nematode Growth Medium (NGM) agar plate: this is a potassium-buffered medium containing a source of calcium, magnesium, and cholesterol (as a lipid source for the worm’s molt). The plates are seeded with OP50 an E. coli strain to feed the worms. This is a Uracil Auxotroph strain used to limit the growth of the bacterial lawn on the plate.

Typically, the worms are cultured in an incubator at a constant temperature. The temperature has a great impact on the growth rate of C. elegans which is twice as fast at 25 °C than it is at 15 °C. The standard temperature used is 20 °C.

Worms ordered from the CGC (https://www.cbs.umn.edu/cgc) are sent on an NGM plate. To start a new culture, a slice is cut from the CGC plate using a sterile blade and transferred to a new OP50-seeded NGM plate, placing the upper side of the CGC plate slice on the OP50 lawn to make it easier for the worms to spread to the new plate. The plates are placed upside down and incubated at the desired temperature.

This transfer technique is very suitable for easy maintenance of homozygous strains requiring no specific breeding of worms. In certain cases, however, the experimental protocol requires certain individual worms to be picked from the plate, using a worm picker, to seed a new plate. A worm picker can be made by placing a platinum wire (5 cm long) in a Pasteur pipette with 3 cm of the wire extending beyond from the end of the pipette. The zone with the wire inside is heated then cooled quickly to seal the platinum to the glass. The loose end of the wire (2 mm) is shaped using pliers to form an angle of 45°. Before picking, the end must be sterilized by holding it to a flame until it turns red hot. Next put it in contact with the agar of the new plate to melt it (the picker does not need to be extremely hot), barely touching the edge of the OP50 lawn; with this bacterial coating, the worms will stick to the platinum. On the parent plate, find gravid animals and young adults (a total of 5 or 6 is a good number) and gently touch them with the picker; they will adhere to the bacterial glue. Several worms can be picked at the same time. To release them onto the new plate, stroke the agar with the picker as gently as possible to avoid crushing the worms.

Nematode Growth Media

The following chemicals are required:

3 g NaCl
17 g agar
2.5 g peptone
975 ml mQ H₂O
Autoclave. Let the temperature go down to 55 °C then add in sterile conditions
1 ml CaCl₂ 1 M (autoclaved)
1 ml MgSO₄ 1 M (autoclaved)
1 ml cholesterol 5 mg/ml in ethanol
25 ml KH₂PO₄ 1 M, pH = 6 (autoclaved)

Pour 1 L of NGM on approximately one hundred 5 cm plates (10 ml per plate). A peristaltic pump can be used to have the same volume per plate. Volume has no impact on the growth of C. elegans but makes it easier to observe multiple plates as there is no need to adjust the focal distance of the stereomicroscope.

OP50 Culture

Using a sterile Q-tip, streak an LB plate with bacteria from glycerol stock. Incubate overnight at 37 °C.
The next morning, pick a colony and amplify the bacteria throughout the day in 2 ml LB at 37 °C, shaking gently.
In the evening, seed a 200 ml LB flask with 200 μl of the all-day culture.
Leave the bacteria to develop overnight at 37 °C, shaking.

The culture can be stored for approximately 3 months at 4 °C.

Seeding NGM Plates with OP50

Before seeding, check that the plates are dry. Drop 500 μl of OP50 culture in the middle of a 5 cm plate. The bacterial lawn should not spread over the edge of the plate so as to stop the worms climbing over the sides where they will desiccate on the plastic and die. The plates are left overnight at 37 °C and can then be kept for 1 month at 4 °C.

3.2.3 Culturing the Worms in a Liquid Medium

C. elegans can also be cultured in a liquid medium (S medium inoculated with HB101). This is useful when handling a large quantity of worms but is not the best way to maintain a strain. Even though there is food in the medium, overcrowding usually leads to Dauer formation characterized by stasis and a challenge to survival. The standard recommendation is for the animals to be grown in liquid for no more than one generation.

For a 200 ml culture, add an E. coli pellet from a 1 L LB culture. The start-up quantity of worms can vary and will depend on the final numbers required; as an initial reference, harvest two large plates of worms. Add 3 ml of sterile S medium per plate, swirl the plate to detach the worms, and collect them with a glass pipette (worms stick to plastic). Wash them twice with sterile S medium and inoculate the OP50-S medium with the pellet.

Incubate at 20 °C with shaking to oxygenate the culture.

Preparation of the Liquid Medium

5.9 g NaCl
50 ml potassium phosphate 1 M, pH = 6
H₂O to 1 L
Autoclave
Add 1 ml cholesterol (5 mg/ml in ethanol)

Trace metal solutions are also required in the following quantities:

1.86 g disodium EDTA.
0.69 g FeSO₄ (7H₂O).
0.2 g MnCl₂ (4H₂O).
0.29 g ZnSO₄ (7H₂O).
0.025 g CuSO₄ (5H₂O).
H₂O to 1 L.
Autoclave. Store in the dark.

The final S medium is made as follows:

1 L S base medium
10 ml potassium citrate 1 M, pH = 6 (autoclaved)
10 ml trace metals solution
3 ml CaCl₂ 1 M (autoclaved)
3 ml MgSO₄ (autoclaved)

Collecting the Worms from a Liquid Culture

Spin the culture at 1,000 × g for 3 min.
Resuspend the pellet in 100 mM NaCl (1/2 volume of the culture) chilled on ice.
Spin for 3 min at 500 × g (4 °C). Resuspend the pellet with prechilled 100 mM NaCl (1/20 volume of the culture).
Add an equal volume of prechilled 60 % sucrose (30 % final) and spin quickly for 5 min at 1,200 × g. This keeps the upper layer (containing the worms) on the top of the solution.
Quickly add 4 volumes of cold 100 mM NaCl to dilute the sucrose and spin for 3 min at 1,000 × g.
Wash the worm pellet twice with S medium.

Decontamination

Bacteria or fungi sometimes contaminate the plates. The best way to decontaminate them is to collect the worms in M9 buffer (1 ml per 5 cm plate), to wash the pellet twice with M9 and resuspend the pellet in 100 μl of decontamination solution. Incubate at room temperature for 3 min with gentle agitation. This will kill the remaining contaminants and worms, but the worm eggs will survive the treatment. The 100 μl solution is then divided and put on five plates.

This treatment can also be used to synchronize large populations of worms. At 20 °C, eggs hatch within 9 h which will therefore be the maximum age difference between worms on any one plate.

The decontamination solution contains:

4 ml household bleach
6 ml NaOH 5 N
12 ml M9

The M9 buffer is made as follows:

6 g Na₂HPO₄
3 g KH₂PO₄
5 g NaCl
0.25 g MgSO₄ (7H₂O)
H₂O to 1 L

The solution is autoclaved.

3.2.4 Storing the Worms

Short Term

The ability of C. elegans to enter the Dauer stage is a characteristic often used in laboratories to effortlessly maintain a strain for 4–5 months. One warning: work must be conducted under sterile conditions to stop any contaminants from developing over this period; pick a worm onto a new plate, parafilm the plate, and place it at 15 °C. The main risk is that the plate might desiccate and therefore kill the worms.

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Tags: Organism Models of Autism Spectrum Disorders

Jun 12, 2017 | Posted by admin in NEUROLOGY | Comments Off

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Selecting the Right Species: Practical Information on Organism Models

2 Yeast (Saccharomyces cerevisiae)

3 Caenorhabditis elegans