Green Fluorescent Protein:
In vivo real-time imaging of nuclear-cytoplasmic dynamics
Jason Gregorin
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 Aequorea victoria
Background
238 amino acids
11 β strands forming 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
Basic Structure
Left: Aequorea victoriaRight: GFP structure
Forming of Fluorophore
Similar in function to GFP
Isolated from the Discosoma coral
Emits a longer wavelength producing a red
fluorescent color
Used to contrast with GFP
Red Fluorescent Protein
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
In vivo real-time technique
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
Purpose
GFP/RFP labelled mouse mammary cancer tissue
Mitosis,in vitro, 5 minute intervals
ApoptosisInduced with staurosporine
A= no treatment
B-G 2 hour intervals
GFP/RFP cancer cells introduced to mice
Deformation of cancer cells leads to arrest in capillaries
Allowed for determination of cancer “flow rate”
Cancer Circulation
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
Discovery of Harmful Side Effects
Pre-treatment with cyclophosphamide. A) Pre-treated mouseB) 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
Left- Mouse mammary tumor Right- 12 hours after treating with
doxorubicin
The Future of Testing for the Cure
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.
References
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