Now that the sequence of the C. elegans genome has been completed, the next important task is to determine the function of each of the predicted genes. Once a scientist choses a gene to focus on, an important question one might want to ask is whether it is essential for embryonic development. One can take a genetic approach to addressing this question and construct a true gene knockout. However, making and isolating a true gene deletion mutant can take quite a long time in C. elegans. Fortunately, RNAi can be applied to give a good indication of the likely loss-of-function phenotype of a mutation in that gene.
If you have a cloned gene of interest and you want to know if it is necessary for embryonic development, you can perform an RNAi experiment to interfere with the expression of that gene in a developing worm embryo. Of course you will first need to subclone that gene into a dual promoter expression plasmid such as that described on page 6. But what if you don’t even know what gene you are testing? Could you test random genes from C. elegans for a role in embryogenesis? Yes! There exist several libraries that consist of random C. elegans cDNAs that have been cloned into a dual promoter vector. Screening such a library using a functional assay such as RNAi would reveal clones that contain a C. elegans gene that is essential for embryonic development. Clones which test positive for the RNAi assay are then identified by DNA sequencing. This is a very good approach and has been done in C. elegans. Once a C. elegans gene has been identified, the next logical step is to search the DNA databases for orthologs (functional homologs) in other organisms. For example, if a C. elegans gene has been shown by RNAi to be necessary for embryonic development and the gene is highly conserved between worms and humans it is reasonable to infer that the human gene counterpart is required for embryonic development in humans. (We can learn a lot about ourselves by studying simpler animals such as worms, which has many similar genes to humans and is more amenable to study!)
What if you want to more directly identify human genes that you believe to be necessary for embryonic development? Rather than screen a C. elegans cDNA library and make inferences about the role of its human gene counterparts, a more direct approach would be to screen a human cDNA library using RNAi in worms. If the human cDNA is sufficiently similar in sequence to a worm gene needed for embryonic development it is again reasonable to infer that the human gene is also needed for embryonic development of humans. Therefore, to identify human genes likely to be needed for embryonic development you will be screening a human cDNA library and testing for arrest of embryonic development in worms by using the RNAi assay described on pages 9-11. This experiment has never been done before!
Can you think of any potential problems with the experimental approach described above?
 Wild type animals lay egss at the 40-cell stage. These hatch and develop outside the parent.  Mutants that cannot lay eggs (Egl1) have progeny that hatch inside the parent, creating a "bag of worms".  Mutants that are double defective so that they cannot lay eggs and the eggs connot develop (inhibted in early embriogenesis) look like bags of eggs. |
For this reason we will take advantage of mutants that are egg-laying defective (Egl phenotype you will work with an Egl mutant known as lin-2). In Egl mutants, eggs are fertilized normally, but the parent lacks the structures needed for egg laying. As a consequence, eggs that are fertilized hatch inside the parent. These ultimately consume the parent from the inside called the bag-of-worms phenotype (Bagging).
There is one way to block this terrible fate by preventing embryos from developing! In the case that a nematode is both Egl and defective in embryonic development (Emb phenotype) then the parent will be sterile but will persist as an adult with many unhatched eggs inside (fig. 1).
You can use this stragegy to test your library cDNA for a requirement in embryonic development. Here is the idea (fig. 2).:
Induction of expression of the cloned gene in bacteria.
Remember that the TripleEx vector has bacteriophage T7 and T3 primer sites flanking your inserted DNA. These can be activated by growing the bacteria in the presence of an inducer--IPTG. Once both strands are expressed, they can anneal, because they are complementary. We will perform the induction by adding IPTG to plates on which we grow the bacteria.
Strategy to test embrionic genes that are essential for development.
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Controls. Of course, any good experiment will be conducted with positive and negative controls. For a negative control, we will feed animals plates seeded with vector only. We would expect that all animals on this plate would show the Bag phenotype. For a positive control, we will feed C. elegans a bacterial strain making double stranded pos-1 RNA. pos-1 is a gene known to be required for embryonic development. We expect that all animals will harbor arrested embryos and will not be bags--they will just look full of eggs rather than full of worms.
