Using Caenorhabditis elegans to understand if particular genes have a role in Glioblastoma Multiforme formation:
The goal of this project was to continue to identify and investigate genes that implicate Glioblastoma Multiforme (GBM) formation using Caenorhabditis elegans (C.elegans). To understand whether these genes have a role in GBM, we used C.elegans as a model organism to understand the morphological and cellular significance of the suspected genes. C.elegans is a nematode that possesses a nervous system. Its nervous system comprises of 302 neurons and 56 glial cells. (Oikonomou and Shaham, 2011) These cells have an influence on sensory and motor functions. We implicated in this study that if we knockout a suspected GBM gene from C.elegans, and observed changes in glial cell development using behavioral assay and advanced microscopy, then will be able to confirm the suspected genes association with GBM. Using several sequencing platforms, we were able to find 10 genes from the Backes et al study (2015) that had orthologous genes with C.elegans. We had to research the background of the 10 genes to pinpoint which ortholog had any association with glial cells or the nervous or neuromuscular system in C.elegans. One gene, Lev-9, a levamisole resistance gene, was one of the few genes from the bunch that had a connection to the nervous system in the nematode. Utilizing the services of a company that specializes in knocking out selected genes in organisms, we were able to order two strains of Lev-9: RB1717 and ZZ16. The two strains with similar genotypes allowed us to investigate the effects of the Lev-9 gene knockout.
To understand if cell division is affected in the knockout (KO) C.elegans, we carried out several assays in order to detect any problems with reproduction and embryonic development in the organisms. In the beginning of these assays, two young adult (L4 stage) C.elegans were picked onto a fresh NGM agar plate with OP50 bacterial lawns. This step was required three times to separately plate the two Lev-9 strains and N2 strain (wild-type) C.elegans, which would serve as our control. Using a standard protocol on culturing C.elegans, we allowed the young adults to grow at 20 C for 72 hours. This test was repeated several times and the progeny count of on each plate was recorded. The progeny consisted of eggs and any offspring inhabited the agar plate ranging between L1-L3 stage C.elegans. The test yielded inconsistent and unquantifiable results, which suggested that we develop another test to study the reproductive system in the KO strains of C.elegans. The next test consisted of picking an equal amount of L4 C.elegans per strain and the control onto new agar plates. The plates were incubated at 20 C for 24 hours, then the adult mothers would be picked off or terminated using heat over a flame. The plates are then incubated for another 24 hours in 20 C, and thereafter the plates are counted for the amount of eggs laid and the amount of L1-L3 stage C.elegans inhabiting the agar plates. This allowed us to understand the time it took for a ZZ16 and RB1717 strains of Lev-9 C.elegans to hatch and start developing.
From the data we have collected, we can start discussing how the progeny count is affecting the nematode and whether this data is relevant to glial cells. We predicted that the organisms would have a deteriorated nervous system and that the neural tube would also be affected by the KO of the Lev-9 gene. The organisms did exhibit abnormal behavior and uncoordinated movement when observed under a 10X microscope. Body curling and slowed movements were two of the most obvious observations, when compared to a normal organism (control). This raised questions whether glial cells have been directly affected. To determine whether glia are directly affected, we would need to perform a series of behavioral assays, such as “nose-touch” assay, to confirm their associations. In order to perform this sub-experiment, we will require a strain of C.elegans that would be immune to nose-touch assays, such as Grl-1. There is another prestige university lab that specialized with glia cells in C.elegans and we anticipate on trying to collaborate this their resources to see if the glial in the Lev-9 C.elegans is being affected.
Keeping in mind that there is no research adequate enough to understand what extent GBM is hereditary, and the details of it being linked to cell division, this study has the potential to help provide significant insight for the gap of knowledge in genetic inheritance and variability in GBM. GBM has significant molecular characterization for its category as a tumor, and not all therapies like chemotherapy or radiotherapy have not been found to be completely effective. This study may contribute to biomedical and bioengineering professionals that are working on solutions for cancer development prevention and those who are finding simpler ways for disease prognosis. Additionally, this study may add information to new studies concerning C.elegans and cell reproductive health. It is also greatly anticipated that the contributions from this research can stimulate more direction for the development of both better drugs and therapies for life-threatening mutations in cell division. It is anticipated that the present and future studies may help reshape the current predisposition testing for cancer. For a bigger picture, it is hoped that the small, but significant information from this research and other studies can redesign treatment options, and revolutionize education on how cancer and may other diseases work. This project has definitely enriched and enhanced my education in the fundamental knowledge of the reproductive biology and cellular biology. I hope that my research experience can be a motivation to others interested in the scientific field and that determination and commitment has the potential to create great things.
Backes C, Harz C, Fischer U, et al. New insights into the genetics of glioblastoma multiforme by familial exome sequencing. Oncotarget. 2015;6(8):5918-5931.
Oikonomou G, Shaham S. The glia of Caenorhabditis elegans. Glia. 2011 Sep; 59(9):1253-63. Doi:10.1002/glia.21084.