School: Issaquah High School
City: Issaquah, WA
Grades taught: 9-10

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

Project Description
Ken Mackie's lab is involved in the study of the interaction of cannabinoids, the active ingredients of marijuana, and the particular receptors they agonize. The principle receptor under investigation, CB1, occurs naturally in the plasma membrane of nerve cells. However, since nerve cells do not replicate and cannot be used for long-term study, the CB1 proteins are transfected into alternate cell lines such as AtT20 (mouse pituitary) or HEK (human embryonic kidney).

AtT20 cells behave like neuronal cells in that once they are transfected with the instructions for CB1 receptors, they not only manufacture them but also translocate them to the cell membrane. HEK cells, on the other hand, do not. Once expressed within the HEK cells, the CB1 proteins linger in the cytosol or aggregate within various organelles. My project was to trick the HEK cells into sending the CB1 molecules to the outer cell membrane where they could be studied in their typical location. This was accomplished by using a preprolactin signal sequence (pplss), a fragment of DNA that codes for a well-known translocation process. Using restriction enzymes, I was able to insert the instructions for the pplss into a plasmid construct used as a vector. The vector also contained instructions for fluorescence which had been incorporated into the carboxyl terminus of the CB1 by a previous StarNet researcher (Bill Monahan). This enabled us to view the results using confocal microscopy.