Date of Graduation

Fall 2009

Degree

Master of Science in Cell and Molecular Biology

Department

Biomedical Sciences

Committee Chair

Richard Garrad

Keywords

P2Y2 receptor, GFP, DNA sequencing, transfection, cystic fibrosis

Subject Categories

Medical Molecular Biology

Abstract

The G protein-coupled P2Y2 receptors have found application in an array of human pathologies. Cystic fibrosis is a lethal, autosomal recessive disease of Caucasians for which treatment is largely symptomatic. Mutations cause the Cystic Fibrosis Transmembrane Regulator (CFTR) to be defective, leading to decreased secretion of chloride ions and water with thickening of the mucous membrane in the respiratory tract. The P2Y2 receptor agonists activate a calcium ion dependent chloride channel leading to increased chloride and water efflux through a CFTR independent mechanism. Unfortunately, this response is transient due to rapid desensitization leading to sequestration of the receptor. To study the sequestration of the P2Y2 receptor, it was tagged at its N-terminus with the Green Fluorescent Protein (GFP) using a GFP expression vector. Expression in 1321N1 astrocytoma cells was achieved using lipofectamine mediated transfection. The presence and orientation of the receptor sequence in the construct was verified by DNA sequencing, though no evidence suggestive of GFP could be found. The cells were analyzed for functional expression of the receptor and GFP in a single cell imager and fluorescence microscope respectively. Transfected cells showed an increase in intracellular Ca2+ concentration on stimulation with UTP and exhibited higher fluroscence compared with the controls. Put together, these results suggest the successful expression of the chimeric GFP-P2Y2 receptor in 1321N1 human astrocytoma cells.

Copyright

© Raja Virendhra Bharath Thotakura

Campus Only

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