Tuesday, 3 April 2012

Glowing Genes 

The green fluorescent protein - revolutionising cancer imaging and surgery.

GFP – what is it?
Aequorea Victoria
The green fluorescent protein (GFP) is, as its name suggests, no ordinary protein. First isolated during the 1970’s from the jellyfish Aequorea Victoria, the protein glows when irradiated with UV light, and since its discovery the possible applications of this protein have continued to grow in scope and complexity. In 1987 American molecular biologist Douglas Prasher first suggested utilising the protein’s unique properties by using GFP as a tracer molecule (Zimmer, 2011), and today contemporary research capitalising on this function is quite common, especially when it comes to understanding the molecular biology of cancer and assisting in both its diagnosis and treatment.

How does it work? 
The gene that codes for GFP was cloned in 1994 and can now be found in laboratories all over the world, where it is used in countless plants and animals.
Using biomolecular techniques, the gene for GFP can be inserted into another gene before the stop codon, so that the encoded protein will be synthesised with the GFP attached. This protein will glow when illuminated by ultra-violet light and can now be traced within an organism. As GFP is a relatively small protein, it attaches easily to other proteins without hindering their original function, and can follow them into neurons without obstructing the fused proteins’ passage. Unlike other bioluminescent molecules, which require modifications before they fluoresce, GFP is intrinsically fluorescent (Zimmer, 2011).

Applications in cancer imaging and surgery

Cos-1 cells (cell line from African green monkey kidney) 
expressing the different colour fluorescent proteins.
Nuclei are counterstained in blue.
Researchers have devised techniques to distribute GFP amongst cancer cells, meaning that they can now image tumours and metastatic cancers down to the single-cell level, even in soft tissue, organs and bone, which can frequently prove challenging when using other imaging methods such as MRI (magnetic resonance imaging) (Hoffman, 2002 & Yorkshire Cancer Research, 2010). The use of GFP has a number of advantages over other methods of imaging and visualisation: it glows much more strongly than luciferases, which are a class of bioluminescent enzymes used for similar imaging processes; unlike luciferases, fluorescent proteins come in a variety of colours, which means that multiple proteins and events can be imaged at the same time (Teicher, 2010); and furthermore many other fluorescent molecules are toxic to cells, whereas GFP causes little or no damage (Zimmer, 2011).

One of the more advanced applications of GFP is as a method of labelling tumours for surgical navigation, which requires very precise identification and differentiation between malignant and benign tissue (Hoffman, 2002 & Teicher, 2010). Scientists have recently developed technology to insert and activate the GFP in the malignant tissue within an organism using an adenovirus that expresses the protein. This technique was trialled in two mouse-models manifesting widespread occurrence of cancer, which in humans would usually present as very challenging cases when using current imaging methods. However in both models only the metastatic tissue fluoresced, meaning that the GFP was specifically localised only in areas of malignancy. These promising results suggest that adenoviral-GFP labelling of tumours could provide greater precision in cancer treatment through fluorescence-guided surgical navigation (Teicher, 2010).

Click here for more information on cancer imaging using GFP.

Other uses
Neuron expressing the molecule Arch, 
here fused to green fluorescent protein.

However, the imaging of cancers and tumours is only one of the many possible applications of GFP. Other uses currently include utilising GFP to analyse brain circuitry, measure neural membrane potential and track the activity of certain receptors on cell membranes (Baker et. al, 2008). These applications continue to grow in scope and every month, over 100 papers are published reporting yet another novel method of using GFP (Zimmer, 2010).

Overall the green fluorescent protein gene is highly versatile: its simple but elegant application may eventually prove indispensable to all fields utilising molecular techniques and it also has the potential to revolutionise current methods used in these fields.

Further reading:
For more information on the green fluorescent protein's structure and applications, follow these links: 

Baker et al 2008, ‘Genetically encoded fluorescent sensors of membrane potential’, Brain Cell Biology, vol. 36, no. 4, pp 53-67.
Boyden, E 2008, ‘Optogenetics’, Nature Neuroscience, vol. 8, pp 163 – 168.
Hoffman, RM 2002, ‘In vivo imaging of metastatic cancer with fluorescent proteins’, Cell Death and Differentiation, vol. 9, no. 8, pp786-789.
Kiefer, F 2010, Mammalian cell signalling laboratory, Max Planck Insitute for Molecular Biomedicine, viewed 1st April 2011, <http://www.mpi-muenster.mpg.de/en/research/teams/groups/rgkiefer/index.html>
Teicher, BA 2010, Tumour models in cancer research, 2nd edn, Springer, New York.
Yorkshire Cancer Research, 2010, Luminous cells from jellyfish could diagnose cancers deep within human bodyScience Daily, viewed 18 March 2010, <http://www.sciencedaily.com/releases/2010/11/101103083906.htm>
Zimmer, M 2010, Green fluorescent protein: a molecular microscope, viewed 1st April 2012, <http://www.photobiology.info/Zimmer.html>
Zimmer, M 2011, The history of green fluorescent protein, viewed 17 March 2010,

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