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Green Fluorescent Protein:

Green Fluorescent Protein:. In vivo real-time imaging of nuclear- cytoplasmic dynamics. Jason Gregorin. Background. Discovered in 1960s by Osamu Shimomura, Martin Chalfie , and Roger Tsien . Nobel Prize awarded in 2008 Gene for GFP successfully cloned in the early 90s

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Green Fluorescent Protein:

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  1. Green Fluorescent Protein: In vivo real-time imaging of nuclear-cytoplasmic dynamics Jason Gregorin

  2. Background • Discovered in 1960s by Osamu Shimomura, Martin Chalfie, and Roger Tsien. • Nobel Prize awarded in 2008 • Gene for GFP successfully cloned in the early 90s • Comes from the jellyfish Aequoreavictoria

  3. Basic Structure • 238 amino acids • 11 β strandsforming a β barrel • 1 central alpha helix • Fluorophore is p-hydroxybenzylideneimidozolidinone • Fluorophore forms as post-translational modification from internal cyclisation and oxidation • Residues involved: Ser65-Tyr66-Gly67

  4. Left: AequoreavictoriaRight: GFP structure

  5. Forming of Fluorophore

  6. Red Fluorescent Protein • Similar in function to GFP • Isolated from the Discosoma coral • Emits a longer wavelength producing a red fluorescent color • Used to contrast with GFP

  7. In vivo real-time technique • Nuclear-cytoplasmic dynamics • Requires the use of fluorescent labeling of the nucleus and cytoplasm • GFP is linked with the histone protein H2B • This labels the nucleus green • RFP is expressed normally in cytoplasm • Various microscopy methods used • In vitro/In vivo • Skin fold chambers, exteriorization of organs, subcutaneous windows, non-invasive whole body imaging

  8. Purpose • Further understanding of nuclear and cytoplasmic ratios, shape changes, cell cycle in living cells • Further knowledge of cancer mechanisms on the inter and intra cell level GFP/RFP labelled mouse mammary cancer tissue

  9. Mitosis,in vitro, 5 minute intervals

  10. Apoptosis Induced with staurosporine A= no treatment B-G 2 hour intervals

  11. Cancer Circulation • GFP/RFP cancer cells introduced to mice • Deformation of cancer cells leads to arrest in capillaries • Allowed for determination of cancer “flow rate”

  12. Genetic Exchange in Cancer Cells This demonstrates genetic exchange in cancer cells (human pancreas cells used). Possibility for better understanding the mechanisms for creating highly metastatic cells

  13. Discovery of Harmful Side Effects Pre-treatment with cyclophosphamide. A) Pre-treated mouse B) Normal, non-treated mouse Though cyclophosphamide is typically used as an effective cancer drug, use at improper times may have side effects allowing easier spread

  14. The Future of Testing for the Cure • Left- Mouse mammary tumor • Right- 12 hours after treating with doxorubicin

  15. References • HOFFMAN, R. (2008). In vivo real-time imaging of nuclear-cytoplasmic dynamics of dormancy, proliferation and death of cancer cells. APMIS, 116(7/8), 716-729. doi:10.1111/j.1600-0463.2008.01036.x. • Haldar, S., & Chattopadhyay, A. (2009). Green fluorescent protein: a molecular lantern that illuminates the cellular interior. Journal of Biosciences, 34(2), 169-172. Retrieved from Academic Search Complete database.

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