The beginning of this semester we continued our work on the C. elegans’ gene T20B12.7 and it was a little shaky due to the snowstorm and school closure. The first RNAi experiment we performed was unsuccessful because we could not get into the lab to record the results. We then performed another RNAi experiment, which show similar results as the previous semester. The RNAi T20B12.7 worms had only a small difference from the L440 worms in unhatched embryos. Another RNAi experiment was performed with rff-3 worms in the hope a more significant difference would be seen. The rff-3 worms are more sensitive to the RNAi treatment. However, the results from the RNAi experiments with rff-3 worms still had an insignificant difference. These results could have been for two different reasons. The first reason was that the RNAi was effecting the worms in another way besides embryo lethality. This was possible, but previous studies showed that this gene influences embryo development so not as likely as the second reason. The second reason for the insignificant difference could have been that something in our RNAi experiment was contaminated or defective. We decided since we were getting deterred by the RNAi experiments for T20B12.7, that we would hold on further experiments for this gene and complete experimentation on another gene, F55A3.3, that had had successful RNAi results.
F55A3.3 is a gene in C. elegans that is involved in embryo development, molting cycle, nucleus organization and reproduction. F55A3.3 is a human ortholog, spt16, that facilitates chromatin remodeling in the FACT complex. Previously performed RNAi experiments in our lab to knockdown F55A3.3 resulted in a sterile phenotype. We hypothesize that the loss of F55A3.3 may be necessary for normal embryo development. Our current experiments will analyze this hypothesis and help further understand the role this gene has in C. elegans. The next step was to use fluorescent microscopy to identify cause of sterility. We have performed two RNAi experiments and used microscopy to image the worms after they were treated. The first experiment we saw several abnormalities in the RNAi treated worms compared to the L44o worms. The abnormalities consisted of changed in the germline shape and irregular dividing in the embryos. The second RNAi experiment was not as successful. The imaging could not be performed properly. We are now in the process of troubleshooting the imaging process to see if the microscope is not working properly or if the worms that are tagged with GFP are contaminated.
It is now the end of the fall semester and my research group has performed several RNAi experiments on four genes. About half have yielded positive results. My gene (T20B12.7) did not yield any positive results. When the experiment was performed, the data for my gene came out similar to the L440 gene results. The L440 and npp-19 genes both came out positive. The L440 had normal reproduction as expected, which was shown by a lot of worms on the plate with little to no eggs on the plate. The npp-19 had interrupted reproduction, shown by increased amounts of unhatched eggs and little to no hatched worms. Since no difference in reproduction was seen in my gene we evaluated our experimental design. We decided that the lack of results could be for a few reasons. The bacteria could have needed more time to grow. The worms could have needed a different temperature to grow. Starting up next semester we will change both factors and see if we can yield positive results for my gene. If the RNAi results remain the same for my gene, then we will start to question when my gene is active during reproduction and development. There are other experiments that could be performed to test reproduction stages.
We also considered a project called the Million Mutation Project. C. elegans were exposed to a mutagen resulting in several thousand mutations. We ordered the strains of worms that had our genes mutated. However, the strains did not come until this last week so we will begin our experiments with these worms at the beginning of the spring semester. Since these worms have mutations beyond just our genes of interest it will allow us to see possible connections with other genes and effects to our gene of interest. We will also begin experiments with microscopy, which will give us a closer look at the reproduction process of the worms and the eggs.
Hello everyone. My name is Mikayla Bonnett and I am now working with Dr. Matthew Marcello and his research group to study the reproduction and gene function in Caenorhabditis elegans. The title of my project is The Function of T20B12.7 in Caenorhabditis elegans. The purpose of this project is to develop a better understanding of the C. elegans genome and possibly be able to apply what is learned to the human genome. The goal of this project is to learn the function of the T20B12.7 gene. The objectives in the lab are to perform RNA interference and deletion experiments on the C. elegans’ gene T20B12.7.
C. elegans are a nematode with a genome about 30 times smaller than the human genome. Even though it is smaller it still encodes for more than 22,000 proteins, which is slightly lower than the human genome. They make very good model organisms for laboratory work because they are small in size, transparent, and easy to grow, so they are very easily managed and taken care of in the lab. Additionally, they have a rapid life cycle and well annotated genome which is helpful to receive results in a timely manner.
The main method used to answer the research question is RNA interference (RNAi). C. elegans respond very well to RNAi experiments. RNA interference is a tool used in experiments to prevent gene expression/function. This interruption can lead to the identification of the function of a gene. The RNAi effect is achieved when double stranded DNA (dsDNA) in the cytoplasm is cut up by a protein called Dicer. The dsDNA into single stranded DNA (siDNA) pieces and then binds to another protein called Argonaute, which forms RISC, RNA-induced silencing complex. The target messenger RNA (mRNA), which is a perfect base compliment to target site, goes through this complex and is cleaved and degraded. The mRNA of a gene is the RNA that is translated into a protein and a protein is what performs gene function. If the mRNA is degraded before it can be translated then the protein of that gene will never be formed, so the gene function cannot be carried out.
T20B12.7 is a C. elegans gene that is involved in embryo development, gamete generation, nematode larval development and receptor-mediated endocytosis. The gene is a ortholog of human ciapin1, which is the cytokine induced apoptosis inhibitor 1. It has several phenotypes such as slow growth, early larval lethal, sterile progeny, and embryonic lethal. The protein associated with this gene is an Anamorsin homolog. When RNAi is applied to this gene, the effect will be observed and compared with wildtype (normal) worms. The phenotype that is observed is what occurs when the worms lacks the protein from the interrupted gene. From there the function can be determined.
So far during this semester I have selected my gene of interest by using the C.elegans’ database Wormbase, which has a record of all the information known about C. elegans genomes. I also used Ortholist, which helps identify orthologs between humans and C. elegans. And to learn about the proteins associated with each gene I search through the databases UniProt and SMART Genomes. Additionally, I have started to examine wildtype worms under the microscope and practice picking and transferring the worms to new plates. I have also started to learn how to perform exploratory data analysis so I will be able to analyze the data that I collect form my experiments.