Fluorescence Microscopy in Caenorhabditis elegans

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Fluorescence Microscopy in Caenorhabditis elegans
NOTES and PROTOCOLS
Marta Kostrouchová and Zdenek Kostrouch
Caenorhabditis elegans – A powerful model organism of molecular biology
Why to work on model organisms:
Each organism - each individual is unique. In this line, we have to be aware that
each patient is unique and medicine that will be shaped for individually adopted
therapeutic approaches will continue to gain prevalence in the future.
On the other hand, mechanisms that constitute the basis of individual metabolic,
developmental and disease-linked states of individual organisms are to some
extent shared.
The closer the individual organisms are related, the more similar are the
regulatory mechanisms that form the basis of the metabolism, development,
reaction to stress and disease causing factors.
These relations are valid not only for organisms of the same biological species
but also for organisms in the whole domain of living organisms or cellular life
(Cytota).
Contemporary medicine is a biological discipline and in reverse, medicine is an
important part of contemporary biology. The mechanisms that constitute
existence of all species are connected by their evolutionary history, and reflect
the similarities and differences between species.
In order to understand the structural and regulatory mechanisms, we have to be
aware of the context on which these mechanisms perform their biological roles.
This means that biology of any system needs to be done in a complex way as a
complex biology of the given species. Nevertheless, for many reasons, technical
as well as ethical, some approaches are unthinkable on humans, vertebrates or a
particular species.
This is another reason, why biological research has to be done on certain
species, which are called model organisms.
Biology of Caenorhabditis elegans was elaborated to a very detailed and further
dynamically developing complex biology of this specific organism and
contributed to large number of critical discoveries, including developmental
regulatory pathways, apoptosis, regulation by RNA interference and many other.
The experimental tools and open informatics of this research system were
enlarged for other experimental systems including human biology and medicine.
Since many tools are shared between the C. elegans system and human biology
and medicine, it may be inspirational and instructive for medical students to
have a closer look on methods of the C. elegans research system and their link to
medicine.
Techniques and literature are freely accessible on WWW:
1/ Wormbase http://www.wormbase.org/
2/ individual webpages
e.g. http://hymanlab.mpi-cbg.de/hyman_lab/methods/c_elegans/techniques_fluo.shtml
Lesson I: C. elegans biology. Handling worms in the laboratory.
Points that will be discussed:
Caenorhabditis elegans – an excellent model organism
1. It is an 1 mm long nematode, lives in soil and or water
2. Why we study model organisms
3. Several examples of model organisms
4. What we study in model organisms
5. C. elegans was introduced by Sydney Brenner in 1975 as a simple multicellular
organism for genetic and behavioral studies and for study of the nervous system
6. It belongs to the phylum of nematodes, has a simple body, its body is transparent
7. They have fixed number of somatic cells, cell divisions are conserved,
hermaphrodites 959, males 1031
8. It is easy to grow it in the laboratory, Petri dish, OP50, lives longer at lower
temperature and with less food
9. It has a short life span, develops during 3 days from embryos to adults, reproduces for
3-4 days and lives 2 more weeks
10. It has many progeny – 300 (good genetic model), two sexes - hermaphrodites and
males, hermaphrodites reproduce by self fertilization and by crossing with males
11. It is a very good experimental model – the first multi-cellular organism with
sequenced genome (1998), 100Mbp and 22 000 genes x human 3000 Mbp and 22-25
000 genes
12. C. e. genes: -an average of five introns
- exons comprise 27% of the genome.
- 42% of predicted protein products match those of organisms in other phyla
- the non-coding RNAs include: dispersed transfer RNA genes, tRNA- derived
pseudogenes, spliceosomal RNA genes, and ribosomal RNA genes.
the chromosomes have a GC content of 36% and have no localized
centromeres.
13. Genetics: - forward – classical (from mutation to the gene)
- reverse (from a gene to its loss of function phenotype)
(by mutagenesis, RNA interference)
14. Preparation of transgenic organisms is easy
- transcriptional fusion genes with gene, which codes for green fluorescent
protein
- translational fusion gene
15. RNA interference (A. Fire and G. Mello) – posttranscriptional effect
16. Communication – WormBase (http://www.wormbase.org/)
Fig. 1 A scheme of an adult hermaphrodite:
Fig. 2 A schema of gonads in hermaphrodite:
The reproduction system:
Fig. 3. An example of regulatory cascade discovered in C. elegans - Apoptosis:
Note. Figures 1 – 3 are reproduced from the open source of Wormbase
(http://www.wormbase.org/).
Practical demonstration:
Complex informatics of C. elegans – The C. elegans server and The Wormbase
(http://www.wormbase.org/).
Lesson II: Studying gene expression in living intact organisms
Transgenic organisms - their preparation and detection
1. GFP
Genes are tagged with the gene for green fluorescent protein from Aequorea Victoria (medusa
- jelly fish).
It produces a fluorescent product, when expressed in prokaryotic (E. coli) or eukaryotic (C.
elegans) cells.
Because exogenous substances and cofactors are not required for this fluorescence, GFP
expression can be used to monitor gene expression and protein localization in living
organisms.
In Aequorea a light is produced when calcium binds to the photo protein aequorin. It produces
a blue light. Green light is a result of a second protein that derives its excitation energy from
aequorin, the GFP.
Purified GFP:
- has 238 amino acids
- absorbs blue light maximally at 395nm
- emits green light at 509nm
- the fluorescence is very stable, with no photo bleaching
The expression of the protein in C. elegans starts when natural expression starts,
- it lasts 10 minutes when illuminated with 450 to 490 nm light
- it does not interfere with cell growth and function
2. Reporter gene fusions
A. Transcriptional reporters consist of
- a promoter fragment, a region upstream of a gene of interest driving GFP (in frame
fusion);
- The length of the promoter is for genes in C. elegans usually a few kb immediately
upstream of the start codon (1.5 – 2)
- ATG from the first methionine should be included
B. Translational reporters consist of
- an entire genomic locus of a gene: 5’ upstream region , all gDNA and 3’UTR
- GFP can be inserted at any point in the open reading frame, not to disrupt protein
function or topology
Sometimes the regulatory sequences can be found within introns, if they are very large,
or at long distances from the gene of interest
3. Multicolor GFP expression systems
The existence of GFP variants with non-overlapping emission spectra:
Cyan-FP
Yellow-FP
Allows the possibility for multi-color labeling of C. elegans cells.
4. Artifacts
- Natural auto-fluorescence of gut granules and nucleoli of hypodermal cells.
- Non specific fluorescence in posterior gut cells.
5. Co - injection markers:
Rol-6 (su1006): is a dominant allele allowing easy selection of transgenic lines
6. Is this real expression what we see?
The expression pattern has to be conformed by preparation of several transgenic lines, by
other methods as by in situ hybridization, by antibody staining etc.
Fig. 4 Caenorhabditis elegans at laboratory conditions. A – Mixed stages of C. elegans on an
agar plate with bacteria E. coli. B – A hermaphrodite with embryos developing inside the
uterus. Shead cuticle of some other worm is visible on the right. C – A two cell stage embryo.
D – An embryo in a comma stage. E – An embryo in two fold stage. F – A larva in L4 stage.
G – A L3 larva expressing nhr-153::gfp in pharynx and in intestinal cells.
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