Our genome is the “cookbook” of our organism. It contains genes that directly or indirectly influence how we shape up. They either directly enable generation of a protein that serves a function in our body, or they have an indirect impact through influencing other genes or processes that help in shaping proteins. The proteins that make up our bodies each have a particular structure and function vital to our survival. However, not everything is known about the function of each gene – what’s more, many genes have not been recognised just yet. In this article I’m going to tell you how to investigate genomic function as I did at Uppsala University with some wriggly friends, who (sadly for them) became short and chunky after our brief encounter…
Most initial work done in discovering how genomes work was by use of forward genetics – moving from a particular phenotype (an organism’s observable features) to a particular function; from there scientists could track down the gene responsible for this function. Some genes were not discovered this way due to them either being expressed under certain conditions or only at certain point in an organism’s life; or because they had the same function as some other gene that was identified. If a gene has the same function as other genes, a mutant that lacks this gene will have the same phenotype as normal individuals, so in that case this approach is not very helpful! That is when a reverse genetics method came in; this involves moving from looking at a gene to exploring its function, before then examining the phenotype for which it is responsible. So, imagine you have a map of where all the genes are in a certain organism and you want to find out whether a change in one of the genes will have an impact on the morphology. How would you go about finding a specific function of a gene? This is exactly what we are about to tell you in this article. Let’s start with a question – what organism should I use for my DIY genetic manipulation?!
The best organisms for this kind of work are model organisms (organisms that have been extensively studied, so positions of their genes in the genome have been identified). Unlike models in the fashion world, these individuals are actually representative of the genetic makeup of other organisms in their species. One of these models is a humble worm, Caenorhabditis elegans – an organism which has become very useful in studies of gene function, because its genome has been sequenced (C.elegans sequencing consortium, 1998) – parts of the genome that code for proteins have also been identified (see Wellcome Trust Sanger Institute Caenorhabditis genome sequencing and analysis for more detail). That’s one useful worm!
So now imagine you have an organism and you have to find out what a particular gene does. What on earth do you do next? One of the methods involves you switching off the gene’s function. In other words, you silence the gene. The method used for this particular purpose is commonly RNAi (RNA interference). This is a process that involves silencing the expression of a gene, accomplished by destroying mRNA that ‘translates’ information from DNA regarding how to create a particular protein. If you chop up a gene’s mRNA, no protein is produced. This may result in an observable change to your model organism. The exact method consists of introducing a double-stranded RNA molecule into the model; this destroys the RNA serving as a template for protein production in the target organism (produced from the DNA of the organism). Despite the variability and incompleteness of knockdown (not all the protein is degraded, or the effect can be rescued by other proteins produced) and the potential non-specificity of reagents (binding to other target RNAs) it is generally easy to use and has long-term effects.
In C.elegans this process is super easy! Well, not quite – don’t try it at home just yet, but read on for one I did earlier. The double-stranded RNA is expressed by a plasmid in a bacterium ingested by C.elegans. Yummy. The silencing RNA is taken up in the worm intestine and incorporated into the genome, causing gene silencing in the organism. Surprisingly, the worm’s progeny also exhibit gene silencing for several generations! Recently for my Master’s degree I carried out my own experiments with other researchers which involved RNAi and C.elegans. In our experiment we fed 120 worms with a bacterium expressing double-stranded RNA targeting dpy13 and 120 fed with bacteria not containing any silencing mechanisms, as our controls (or comparisons). Our aim was to discover whether the gene has any influence on C.elegans morphology.
According to our literature review the gene dpy13, also called the dumpy gene, causes the worms to become short and chunky. Dpy13, according to von Mende et al (1988), has a unique function in cuticle growth and belongs to one of the three collagen gene families. After three days of letting our worms reach adulthood we did indeed observe a considerable change in the phenotype when comparing the negative control and the RNAi treated worms. Not sure if the worms were happy about their sudden change of morphology (I know I wouldn’t be) – but they were much shorter and chunkier and moved at a slower pace. This change was observed in all of the 120 worms that were used for the modification treatment.
In conclusion, we discovered targeting a single gene can have major effects on the phenotype of the organism. What we are not entirely sure through this experiment is if it is a direct impact of the gene or if the dpy13 gene targeted other genes which in turn produced the effect. This requires more study. Anyone interested in working on the C.elegans genome? Give me a call!
N. (Swedelicious Master’s Student).