Download Green Fluorescent Protein:

In vivo real-time imaging of nuclear-cytoplasmic dynamics
Jason Gregorin
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Discovered in 1960s by Osamu Shimomura,
Martin Chalfie, and Roger Tsien.
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Nobel Prize awarded in 2008
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Gene for GFP successfully cloned in the early 90s
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Comes from the jellyfish Aequorea victoria
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238 amino acids
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11 β strands forming a β barrel
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1 central alpha helix
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Fluorophore is p-hydroxybenzylideneimidozolidinone
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Fluorophore forms as post-translational modification
from internal cyclisation and oxidation
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Residues involved: Ser65-Tyr66-Gly67
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Similar in function to GFP
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Isolated from the Discosoma coral
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Emits a longer wavelength producing a red
fluorescent color
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Used to contrast with GFP
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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
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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
Mitosis,in vitro, 5 minute intervals
Apoptosis
Induced with staurosporine
A= no treatment
B-G 2 hour intervals
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GFP/RFP cancer
cells introduced to
mice
Deformation of
cancer cells leads to
arrest in capillaries
Allowed for
determination of
cancer “flow rate”
This demonstrates genetic exchange in
cancer cells (human pancreas cells used).
Possibility for better understanding the
mechanisms for creating highly metastatic
cells
Pre-treatment with
cyclophosphamide. A) Pretreated 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
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Left- Mouse mammary tumor
Right- 12 hours after treating with
doxorubicin
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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.16000463.2008.01036.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.