Lewis, Ashlynn; Drake, Kendric
College of Science and Mathematics
Professor: Dr. Rachel Hopp
ABSTRACT:
Collagen, a structural extracellular protein, is the fundamental component of bone, cartilage and connective tissue. It is responsible for cell adhesion, migration, and tissue growth in eukaryotes. In humans there are 42 collagen genes; approximately 1300 mutations in 23 of the 42 genes have been associated with phenotypes ranging from skin blistering to early death. Caenorhabditis elegans is an ideal model to study the effects of mutation in the collagen gene due to the relative ease of inducing and observing the mutagenic phenotype. A potential human collagen ortholog, the C. elegans (bli-1) gene encodes the BLI protein, and is responsible for development of the nematode exoskeleton. RNA interference (RNAi) is used to identify genes important to disease processes by reducing the quantity of the gene transcript, or mRNA. RNAi is a mechanism developed by organisms to defend against viral invaders with double stranded RNA genomes. The introduction of an exogenous single strand complement binds target gene mRNA transcripts to stimulate destruction via host cellular machinery. This produces a phenotype similar to loss of function gene mutation. Escherichia coli HT115 (DE3)/pL4440-bli-1 feeding vectors were used to induce blistering phenotype in wild type N2 C. elegans via a plasmid transformed bacteria. IPTG induced expression of transformed bacteria occurred via the use of T7 plasmid promoters for highest levels of gene expression. Wild type worms were chunked onto HT115/pL4440-bli-1 bacterial lawn and observed for RNAi induced blistering under dissecting and light microscopy at 48 and 120 hours. Applications of this study pertain directly to human collagen gene mutations to offer potential avenues for observing phenotypic effects in higher eukaryotic organisms, aiding in the understanding of mechanisms and treatment of collagen disorders.
