C.elegans Flashcards
How many cells, genomes and genes does C.elegans have?
959 Cells (302 neurons; ~7000 synapse)
97 Mb Genome
6 chromosome
~19000 Genes ( ~ 5000 essential genes)
Describe the anatomy of the C.elegans intestine.
The intestine is made from only 20 cells, which form 9 intestinal rings of which 1 ring is made of 4 cells, and the other 8 rings are made from 2. The nutrients travel in each of the cells.
Describe three differences between the dauer larva and the L3 larva in C.elegans.
The dauer larva is a response to the environment. The worm will enter this stage if the population density is high if there is no food etc (otherwise it will continue from L2 into L3)
The dauer larva can stay in this stage for up to a year while a C.elegans entering L3 stage will live for 14 days. The year as a dauer doesn’t count.
A dauer larva has closed mouth and anus. Which a L3 larva doesn’t
Please list six features that make C.elegans such a great model organism.
C-elegans can be grown cheaply and in large numbers on plates.
Can be frozen
Produce over 1000 eggs everyday
They have a short life cycle, which is useful for studying their development.
C.elegans is a very small organism so is convenient to keep in the lab.
The anatomy and development of C.elegans can be examined easily under a microscope.
What is “pseudocoelomic fluid” in C.elegans, and what function does it serve?
The pseudocoelom is a fluid-filled body cavity lying inside the external body wall of the nematode that bathes the internal organs, including the alimentary system and the reproductive system
With respect to C.elegans development what are “cytoplasmic determinants”
Cytoplasmic determinants are different segregated to specific cells during early embryogenesis, specifying their identities.
Technique: C.elegans Transgenic
You first inject both plasmids into the ovary of a hermaphrodite, where the plasmids will form extrachromosomal arrays. About 50 ng/l of each plasmid. You let the worm self-fertilize. The transgenic offspring will be mosaic, i.e. cells within the organism will have different genotypes. You then select for offspring that are rollers, and check which ones are fluorescent with a microscopy. Since we provide hundreds of plasmids simultaneously we are making sure that at least some of the offspring got both plasmids.
Microinjection of two plasmids (e.g a test plasmid and a phenotype marker plasmid) inte the gonad syncytium of C.elegans can lead to the generation of transgenic animals.
a) Will the eventual transgenic transgenic animals carry both plasmid? why?
b) Why are transgenic animals said to be “mosaic”?
a) It might, but cannot know for sure unless it has a marker. Injected plasmids form extra chromosomes, but there is no way to control how many plasmids that do so. If we inject many plasmids it is highly likely that both will be present. But we cannot know that for sure unless there is a specific marker for that plasmid.
b) During cell division, the chromosomes might not separate properly and the added DNA ends up in only one cell. This means that the transgenic animal will be made up of both transgenic cells and wildtype cells. This can easily be seen if the injected DNA has a marker like GFP.
Please mention two methods by which RNAi can be administered to C.elegans.
E.coli can be made to express the RNA and then the worms can be grown on the E.coli. The worm will eat the bacteria , crush it in the grinder and then the RNA will be released in the intestine and spread to the rest of the cells (which is done easily in C.elegans)
Injecting the RNA into the gonad syncytium, will be picked up by oocytes and the effect of the RNAi that we wanted can be seen in the next generation of worms.
Mention three different methods to achieve RNAi against a specific gene of interest in C.elegans.
- In vitro transcription & annealing, then microinjection or bathing of worms in dsRNA
- Feeding of worms with E.coli expressing hairpin RNA
- Expression of hairpin transgenes under heat shock promoters.
Forward genetics: Feno to geno
I would begin with outcrossing the mutations to a wildtype. This will reduce the amount of other mutations. I would do this at least six times and for each time select worms with the phenotype- can understand French poetry. The outcrossing will reveal a hotspot which is a cluster of mutations. it is likely to find our mutation there. The next step is to sequence the genome of the mutants and compare it to a wildtype. I would then rescue each of the wildtype genes for that mutation. When the phenotype of interest (understanding French poetry) disappears, we have found the mutation.
Explain in what form a plasmid microinjected into the gonad syncytium of C.elegans will be retained in future generations of transgenic worms.
It’s used to make transgene worms. When injected, the plasmids will form artificial chromosomes. and these will be successfully integrated into daughter cells. We expect 10-50% transgenics per parent. The offspring will be mosaic, these are screened for, other with the help of a co-injected phenotypic marker plasmid.
Several important differences exists between C.elegans and Drosophila development. For example, the oocytes of each organism are fundamentally different: one is formed with pre-established asymmetries while the other is not. Explain briefly how the primary embryonic axis are established in both organisms.
In C.elegans, the side where the sperm enters the egg will be the posterior pole.
In drosophila, the cytoplasmic determinant determine which side of the embryo that is anterior and posterior respectively. (preestablished asymmetries)
Operons exist in C.elegans. Describe the structure of the primary transcript(s) and of the final transcript(s) when expressing an operon that carries two genes.
Operons usually don’t exist in eukaryotes, but in C.elegans they do. They are expressed under the same promoter. The genes are then transpliced to SL1 or SL2 leader sequences.
