School: Mercer Island High School
City: Mercer Island
Grades taught: 10 (Biology)

Principle Investigator: Ken Mackie, Ph.D.
Department: Anesthesiology, School of Medicine
Institution: University of Washington

Project Description
Ken Mackie’s Lab works with cannabinoid receptors and the development of tolerance.  Cannabinoid receptors are found on the membrane of neurons and produce down stream signaling when bound by an agonist, such as THC found in marijuana.  I worked with the CB1 receptor and the protein b-Arrestin.  b-Arrestin is involved with desensitizing, stopping the down stream signaling, and internalization, pulling the receptor into the cell, of CB1 after is has been stimulated by an agonist.

In particular, my project involved taking a plasmid containing the b-Arrestin gene and inserting a red fluorescent protein gene (mRFP).  This plasmid would then be transfected into HEK293 (human embryonic kidney) cells for expression. A similar plasmid already existed but it contained a green florescent gene.  By inserted mRFP, It would be possible to see the b-Arrestin protein in conjunction with the CB1 receptor which contains a green fluorescence.  Once the HEK293 cells were transfected with both the b-Arrestin vector and CB1 vector, confocal microscopy was used to image them.  They were also imaged after agonist treatment to visualize translocation of b-Arrestin from the cytosol to the membrane, glowing red, in contrast with CB1, glowing green.