Since my last post, things have been somewhat slow in the research lab. We are aiming to conduct experiments on male worms. To obtain male worms of the correct mutant strain. First, we have to conduct RNAi on wild-type worms. Once these worms have successfully grown into their L4 stage we pick them off and place them on a separate plate which we then place in an incubator which is ~5 degrees Celsius higher than their optimal growth temperature. This is called heat shocking. The reason we do this is that under increased temperatures, the worms experience reproductive stress which is supposed to give a higher yield of male worms, which at standard temperatures occur only 0.1% of the time by spontaneous non-disjunction. After the worms are in the intense heat for about 6 hours we then transfer them back to the standard temperature and await their reproductive cycles completion. After 2 days we then check the plates every few hours for 2-3 days in hopes of finding male worms. So far we have been unsuccessful and so we will continue to repeat these steps until we find males so that we can proceed with more experiments.
Since my first blog post, I have learned a lot of techniques commonly used in a genetics research laboratory. I have made plates on which the worms will grow, spotted them with bacteria for RNAi, and cultivated different mutant strains of worms and observed various phenotypes. To set up this experiment, the first thing I did was grow my bacterial cultures, which contained siRNA, that would be used to silence a targeted gene. The bacteria were then incubated for 16 hours. After 16 hours a 100 microliter spot of each bacterium was placed on a RNAi plate and incubated at room temperature for 24 hours.
The next morning I set up my experiment, placing 5 L4 N2 C.elegans hermaphrodites on each plate and incubating them at 22 degrees Celsius overnight. A week later I observe the phenotypes produced and repeat the experiment. Along with collecting the data from these experiments, I have also been learning how to perform statistical analysis using a program called R.
I will be continuing a project started by a previous undergraduate researcher which consists of investigating the role of certain genes in the fertilization of Caenorhabditis elegans necessary for sperm capacitation that can be compared to a similar complex in humans. The gene we will be focusing on, M05D6.2, is an ortholog for human t-complex protein 11 (TCP11) which is a testis specific gene product. TCP11 is hypothesized to be essential for proper sperm activation in mammals. Men with mutations in the M05D6.2 ortholog have fertility issues. By understanding the function of this gene, we could help these patients find better treatment or determine if they need to seek assisted reproductive technologies to have children.
Previously used techniques in the lab include RNAi interface which has revealed that males exposed to the treatment show a decrease in fertility while hermaphrodites exposed to the same treatment have no reduction in fertility. Using my background as a technician in the Biology department, a research assistant in a pathology lab at the University of Pennsylvania , and working with my supervisor I plan to present my work at the annual Eastern Colleges Science Conference in Spring 2018 and publish my results in a peer-reviewed journal when it is complete. My goals include being able to participate in undergraduate research until graduation from Pace where I then will apply to attend graduate school with a PhD track. Research will fit into these goals because it is my ultimate career goal when I finish schooling.