Investigations Into PRG-2 and its Involvement in Developing Gallus Gallus Retinal Neurons

Jeffrey Parham

OAI: oai:digitalcommons.winthrop.edu:graduatetheses-1144

I am interested in the development of the nervous system, especially since during development nerves grow and extend, but in adults, they do not regenerate if damaged. We are specifically interested in the molecules that guide nerves to the correct target during their development. Lysophosphatidic acid (LPA) is a bioactive molecule that has been shown to play a role in neural development. LPA, through repeated studies, has been shown to stop neurons from growing by causing a physical change in a neuron's growth cone (a structure used for navigation and growth). Recently, a novel set of genes, called PRGs, have been demonstrated to interact with LPA and LPA receptors. However, interaction between PRGs and LPA is not well understood. Thus, I investigated the role of PRG and LPA in neuronal development, focused on the visual system in chicken embryos. During this study I determined expression of PRG genes in the developing chicken eye using RT-PCR. Then I designed target sequences for mutation of chicken PRG gene using CRISPR and cloned targeting guides. I designed six guide RNAs (gRNAs) to target one specific PRG-2 gene determined by the expression studies. I then evaluated these gRNA constructs to determine if they efficiently mutate PRG-2 in cells. This project then required delivery of my gRNA DNA constructs to embryonic chicken eyes via electroporation to induce mutations in the retinal ganglion cells (RGCs) of the eye that compose the optic nerve which I optimized. I then overexpressed PRG-2/GFP fusion protein in chicken fibroblast cells to confirm my construct would be a candidate for overexpression in chicken retina.

PRG-2 PPCR plasticity related gene Biochemistry, Biophysics, and Structural Biology Biology Cell and Developmental Biology Cell Biology Molecular Biology