Group 9 Project - Fluorescent Proteins

From CellBiology
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Introduction

The Green Fluorescent Protein (GFP) was first observed in the Aequorea victoria jellyfish in 1962 and since then this protein has become a vital tool for viewing many biological processes that were once invisible. The GFP can be bound on many of the thousands of different proteins in living organisms, in real time and in vivo or in vitro. Many new colours of fluorescent proteins are now available and allow multiple cell structures or processes to be imaged simultaneously. Fluorescent proteins, in their many colours and properties, have revolutionised modern cell biology as we know it, and continue to play a part in cutting-edge research into normal function and disease.

The Theory of Fluorescence

YouTube video explaining fluorescence theory

Fluorescence is the spontaneous emission of radiation (ultraviolet, visible or infrared photons) from an excited atom. The Latin word luminescenz describes all phenomena in which light is released and where a rise in temperature does not occur, whereas incandescence involves heat. The various types of luminescence are classified according to the mode of excitation, fluorescence occurs via the mode of absorption of light to release another wave of light. A prime example is the reaction of luciferase where the oxidation of a substrate by an enzyme releases light. [1] [2]


There are three principles of fluorescence

1. Excitation; energy/ photons are absorbed by the atom of an appropriate wavelength by the fluorophore which becomes excited.
Summary Explanation

2. Excited state; the electron jumps to a higher energy level.

3. Soon, the electron drops back to the ground state, the atom will emit light as the atom's electron reverts to a lower, more stable, energy level, thus fluorescing.

NB; fluorescence is cyclical therefore a fluorophore usually is excited repeatedly.


Fluorescence microscopes emit a specific wavelength to excite the specimen, the microscope has a filter that only allows the specific emitted wavelength which helps make up the image without other emitted light to get through and scatter desired light. [1] [2]



The History of the Green Fluorescent Protein

Major Milestone Timeline [2] [3]

The Aequorea victoria Jellyfish demonstrating bioluminesence

1565 N. Monardes observed the emission of light by an infusion of wood lignum Nephriticum (first reported observation of fluorescence).

1640 Licetus studied the Bolognese stone and defined it as a non-thermal light emission.

1842 E. Becquerel made the first statement that the emitted light is of longer wavelength than the incident light.

1853 G. G. Stokes introduces the term fluorescence.

1858 E. Becquerel created the first phosphoroscope.

1944 Lewis and Kasha Triplet state

1955 Green fluorescent substance in jellyfish first described.

1962 GFP identified as protein, extracted from Aequorea victoria jellyfish

1979 Osamu Shimomura characterized structure of chromophore.

1985 Prasher clones GFP gene from Aequorea victoria.

1993 The Structure of GFP chromophore confirmed, side amino acid residues corrected from Shimomura's 1979 structure.

2000 onwards The development of GFP variants, as described below in "The Development of New Fluorescent Proteins"


GFP and The Nobel Prize


The 2008 Noble Prize in Chemistry "for the discovery and development of the green fluorescent protein" was awarded jointly to:


Youtube video tells how and why
Osamu Shimomura
Osamu Shimomura- When Shimomura first obtained GFP, he did so by squeezing strips of the jelly fish Aequorea through filtercel and then purified it by ammonium sulfate and column chromatography. [4]


Martin Chalfie
Martin Chalfie- conducted experiments by tagging GFP in six individual cells in the transparent roundworm Caenorhabditis elegans, supporting previous ideas that GFP can be used as a molecular tagger or “light bulb”. Chalfie also contributed to the ideas of transfecting cells and with his experiment was able to revolutionize the biosciences.


Roger Y. Tsien
Roger Y. Tsien- -was able to mutate GFP and create a variety of new colours now termed. Tsien was also able to make the DsRed FP compact thus more versatile and finally created mutants that start fluorescing faster and are brighter. [5]







The Extraction and Purified of GFP from the Aequorea victoria Jelly fish[6] [7] [8] [9]

1. Transfection into cells via vector for amplification (See Current Research section for detailed methodology)

2. Cell are lysed by sonication, then centrifuged

3. Products are put in ammonium sulfate, ethanol and chloroform and mixed it, resulting in GFP at upper aqueous phase and others proteins at bottom. Alternatively, the hydrophobic nature of the GFP molecule can be utilised for purification with a size column

4. To confirm the extraction of GFP, the sample is excitated at 395nm and checking if a 510nm wavelength emission results.


Further links to purification methods:[32] , [33]

Green Fluorescent Protein

Structure and Classification

GFP is compromised of 238 amino acids and weighs 26.9kDa, discovered in 1962 by Osamu Shimomura in the jellyfish Aequorea victoria, it glowed green when under ultraviolet light. Its structure was found in 1972 by Shimomura, it is an 11-stranded beta-barrel threaded by an alpha-helix running up the axis of the cylinder, it is predicated that the beta-can structure protects the chromophore and is presumably responsible for GFP’s stability. This protein is now the most commonly used biological marker tool in molecular biology, medicine, and cell biology. [10] [11] [12]

GFP Molecule and light emission
GFP use in cell
GFP use in 3D model

The Protein Data Bank currently lists 22 GFP’s, there are many mutants, this is done by changing amino acids in them, they actually all have similar structures to aequorea GFP. The chromophore is in the centre of the beta can and is a 4-(p-hydroxy-benzylidene) imidazolidin-5-one attached to the peptide backbone through the 1- and 2-positions of the ring.


Tsien [5] has classified GFPs into seven major classes based on their spectral characteristics:


(i) Wild-type GFP. The chromophore is in equilibrium between the phenol and phenolate form. It has two excitation peaks at 395 and 475 nm.

(ii) Phenolate anion. Ser65 has been substituted with Thr, Ala, or Gly. Does not have the 395 nm excitation peak.

(iii) Neutral phenol. Mutation of Thr203 to Ile results in a mutant that only has the 399 nm excitation.

(iv) Phenolate anion with stacked ð-electron system. Mutation of Thr203 to His, Trp, Phe, or Tyr results in yellow fluorescent proteins.

(v) Indole in chromophore. Cyan fluorescent proteins have properties intermediate to those of BFP and EGFP.

(vi) Imidazole in chromophore and phenyl in chromophore. Blue fluorescent proteins have an excitation peak at 383 nm.

(vii) Phenyl in chromophore. It has the shortest excitation wavelength and no apparent uses.


Advantages of GFP [5] [13] Disadvantages of GFP [5] [13]
Applicable to nearly all organisms and in live tissue Gene transfection required which is time consuming
Can be targeted to specific tissues, cells, organelles, or proteins GFP is most efficient temperatures well below 37 °C, thus searches for ones functional in mammalian cells were made.
Unlikely to diffuse well enough to blur spatial gradients GFP only fluoresces in presence of oxygen as the chromophore requires an autocatalytic reaction, thus GFP only works in aerobic environments.
Good optical properties: visible excitation, high photo-stability Some of GFP’s limitations are the slow post-translational chromophore formation.
Cheap and relatively easy to replicate and distribute Difficulty in distinguishing GFP from background fluorescence when the GFP is not densely localized or highly expressed.
It’s chromophore does not require a cofactor
It is highly stable as GFP is resistant to heat, alkaline pH, detergents, photo-bleaching, organic/ non-organic salts, and many proteases.
Shimomura showed GFP was a relatively small protein which allows little hindrance to the protein or its function.




Tagging GFP onto Targets

GFP is so versatile and can be tagged by means of labelling reagents with functional groups on amino groups via covalent binding, Wang and Hazelrigg discovered that GFP can be used as a fluorescent tag for the N- or C-termini of proteins. Since there are so many proteins it can be labelled on nearly all aspects of the cell. [5] [12] [14]



Development of New Fluorescent Proteins

Since the revolutionary development of GFP, extensive research has been conducted with the aim of producing fluorescent proteins (FPs) which cover a broader colour spectrum. These colour mutant FPs are also being genetically engineered to exhibit faster maturation rates, greater photostability, increased brightness, pH insensitivity and reduced oligomerisation and toxicity Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title.Due to the sheer number of FPs that have been produced, this section will focus on the most current and valuable colour mutant FPs and touch on FPs with other specialisations such as photoactivation and photoconversion.

New FP Colours

A sample of the fluorescent protein colour palette


Blue

Enhanced BFP (EBFP) was one of the first spectral variants engineered from Aquorea GFP, but due to its low brightness and poor photostability it is now unappealing for most research Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title [15]. EBFP2 is the most photostable and brightest blue FP Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title.


Cyan

The cyan FPs (CFPs) began with the production of enhanced CFP (ECFP) from Aequorea GFP[16]. mCerulean followed in 2004 offering a brighter and better general-purpose CFP [17]. In 2006, a monomeric teal-coloured variant mTFP1 was obtained from a Clavularia soft coral protein[18]. mTFP1 is brighter, less pH sensitive and more photostable than the traditional CFPs, making it an excellent alternative to its predecessors Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title.

Most recently, site-directed mutagenesis of ECFP has produced super CFP (SCFP) which is twice as bright as ECFP when expressed in bacteria. SCFP shows promise for use as a fusion tag or as a biosensor for the detection of calcium ion fluctuations, pH changes, metabolites or enzyme phosphorylation Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title. Other proteins which fall into this class include mCFPm [19] and CyPet Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title .



Green

Following the discovery of the original Aequorea GFP discussed previously, many other proteins which express in the green region of the spectrum have been isolated from other Aequorea species, copepods, amphioxus and reef corals Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title.

Most of these novel GFPs exhibit no discernable advantage over EGFP Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title and hence will not be discussed. mEmerald, a derivative of EGFP, is currently the best choice for live-cell imaging due to its more efficient folding than EGFP at 37°C Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title [20].

YFP expression (yellow and green) in Arabidopsis root cells


Yellow

Yellow FPs (YFPs) are among the brightest and most versatile probes developed in any of the spectral classes Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title. EYFP was developed in 1999[21] and is still widely used despite its high pKa and sensitivity to halides Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title. EYFP has proved effective in tracking the distribution patterns of single proteins on the membranes of live cells[22]. mCitrine, derived from the addition of a single mutation in EYFP, is less halide sensitive and twice as resistant to photobleaching as its predecessor[23].

mVenus is a popular YFP mutant with a greatly reduced maturation time, however it has low photostability. The fact that mVenus requires only two minutes in vitro or seven minutes in vivo to produce fluorophores makes it ideal for monitoring cellular processes with fast dynamics such as gene expression[24].

Super YFP (SYFP), the product of site-directed mutagenesis of EYFP, may hold similar applications as those discussed previously for SCFP. Yellow fluorescent protein for energy transfer, YPet, is the brightest YFP variant. YPet is also reported to have very good photostability and superior acidic resistance to mVenus and other YFP derivatives Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title.


Orange

Compared to the other areas of the spectrum, few probes have been constructed to emit in the orange and red wavelengths. Probes such as DsRed, TagRFP and tdTomato actually have emission profiles in the orange range of the spectrum and not the red range as suggested by their names Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title. mOrange, a member of the ‘Fruits’ series (see ‘Red’ section for details), once dominated this spectrum in terms of brightness, but has average photostability and is unstable at low pH Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title. In an attempt to overcome these problems mOrange2 was engineered from mOrange and shows significantly improved photostability but is still pH sensitive and shows an almost doubled maturation time.

Kusabira Orange (KO), derived as a tetramer from the mushroom coral Fungia concinna, was later modified to give monomer KO (mKO)[25]. mKO demonstrates extremely good photostability and brightness similar to that of EGFP, making it a good candidate for long-term and wide-fluorescence illumination experiments Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive titleCite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title.

The redistribution of GFP-labelled myosin and mCherry-labelled tubulin in a single Drosphila cell during Anaphase

In 2007, TagRFP was cloned as a dimer from Entacemaea quadricolor sea anemone and appears to be a useful tool for localisation and FRET studies[26] . Random mutagenesis of TagRFP produced the highly photostable, bright and pH resistant TurboRFP.

Many of the problems associated with Discosoma DsRed FP- including slow maturation, an intermediate green state, and tetrameric character- have been the target of many attempted modifications of this protein through both random and site-directed mutagenesis. The production of the monomeric mRFP1 from DsRed was promising, but reduced emission and quick photobleaching still means that it is less useful than monomeric GFPs and YFPsCite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title.

tdTomato, another of the 'Fruits' proteins, is the brightest of all available FPs, emits at closer to the true red range and is very photostable Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title. The major drawback in the use of tdTomato is its comparatively large size which is proposed to interfere with fusion-protein packing Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title.


Red

FPs that emit in the far red area of the spectrum are desirable due to this wavelength of light being less phototoxic and more able to probe deeper into biological tissues Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title. The most promising develops have arisen from the site directed mutagenesis of mRFP1 to give monomeric FPs which emit in the 560-610nm wavelength which are collectively referred to as the ‘Fruit’ proteins Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title . Despite their improved emission colours, many Fruits lack the brightness and photostability needed for most experiments Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title. According to Shaner and team, mStrawberry and mCherry are the best reds, with brightness levels of 75% and 50% of EGFP Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title. mCherry is more photostable than mStrawberry Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title and as well as a better alternative to mRFP1 for long-term imaging experiments Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title.

Developed in 2004, mPlum is one of the first true far-red probes, emitting at 649nm[27]. mPlum has limited brightness (10% of EGFP) but good stability and is recommended for use in multicolour imaging experiments, the imaging of thicker tissues and as a FRET partner for GFPs and YFPs Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive titleCite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title.

Kutushka, a dimeric protein that emits at 635nm, was developed in 2007 and is commercially available from Evrogen as TurboFP635Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title. Despite being less bright than EGFP, Kutushka has the highest brightness level of any of the FPs in the 650-800nm wavelength area.

mKate (Evrogen, TagFP635) has similar spectral characteristics to Kutushka, brightness on par with mCherry and is reported to be very photostable, making it a good candidate for localisation experiments in this area of the spectrumCite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title. mKate has been reported to exhibit complex photobleaching behaviour which is yet to be well characterised and it is suggested that mCherry remains a more reliable choice for single-molecule imaging Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title.


Summary Table of Useful FPs


For more information on the range of currently available colour palette of FPs visit:

1) The Clontech "Interactive Guide" (found as a link on the righthand side of the page)- allows you to explore their range of comercially available FPs by colour, application, spectra and antibody choice [34]

2) The Nikon Microscopy Education page- gives the basics of FP use in live cell imaging [35]

3) The Olympus FluoView Resource Centre- gives information on the colour palette for confocal microscopy [36]

Other Novel FPs

Infra-Red FPs

The development of a protein that emits in the infra-red spectrum is the holy grail of fluorescent protein engineering. Accurate in vivo imaging of animal tissue using FPs requires emission wavelengths of 650 to 900nm to minimise absorbance by water, lipids and haemoglobin and to reduce light scattering. Infra-red fluorescent proteins (IFPs) which can be genetically expressed would be incredibly valuable for whole-body imaging in cancer and gene therapyCite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title.

A paper published in 2009[28]reports the production of a monomeric IFP from a bacteriophytochrome which incorporates biliverdin (an intermediate of heme catabolism) as the chromophore. The IFP was effectively expressed in both mammalian cells and mice, demonstrated the spontaneous incorporation biliverdin and produced infra-red fluorescence.


Photoactivated FPs

These proteins display negligible fluorescence until excited by irradiation at a specific wavelengthCite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title. This allows for the highlighting of molecules within a discrete region of a cell as well as a way to study the lifespan and behaviour of proteins independently of other newly synthesised proteinsCite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title. PA-GFP is one such protein that is activated by light of wavelength 488nm and emits in the green wavelength (38). Other FPs which demonstrate “reversible on/off switching” are Dropna, rsFastLime, rsCherry, rsCherryRev and PAmCherryCite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title.

Use of the photoconvertable Kaede fluorescent protein, before (Red Kaede) and after UV Conversion (Green Kaede)


Photoconvertable FPs

This class of FPs show “light-driven modulation of fluorescence properties”Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title and demonstrate green-to-red conversion when activated at around 400nmCite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title. These proteins, such as Kaede (as shown in the picture to the right), KikGR, EosFP, Dendra2, mKikGR, tdEosFP, mEosFP and mEosFP2, are useful for the tracking of fusion proteins, organelles or the fate of embryonic cells during developmentCite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive titleCite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title.


Destabilised GFP variants

These FPs allow the characterisation of the expression timing or lifetime of a target protein due to their rapid turnover by proteolysis resulting in only younger protein chimeras fluorescing[29].


"Fluorescent Timer" Protein

This protein, similarly to the destabilised GFP variants, allows the measurement of protein turnover and expression timingCite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title. It initially fluoresces in the green area of the spectrum before conversion of the fluorophore after several hours leads to emission in the red[30]. Using the ratio of green to red fluorescence allows the age of the tagged protein to be determined.


Future Engineering Endeavours

Criteria for future engineering endeavours as proposed by Shaner et al.Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title. :

• Improved brightness

• Monomeric structure

• High contrast

• Uncomplicated photoconversion

• Reversible photoactivation

• Red-to-green photoconversion

• Improved expression in the far-red or near-infrared regions

Current research

Current research in cell biology with fluorescent proteins is a vast field of study. We shall focus our investigation on a specific technique that has significant clinical consequences. This technique is the production of individual transgenic cells and whole transgenic organisms using spliced fluorescent genes. The principle behind this technique is based on the model for protein synthesis and thus an explanation of the protein synthesis model is an essential requisite. Protein synthesis occurs in 3 stages:

Stages of protein synthesis

The first stage: (Transcription)

1) Inside the nucleus of a cell, the double stranded DNA helix unwinds in the area of the gene containing the information for synthesizing a specific protein. [31] [32]


2) The enzyme RNA polymerase moves along the unwound DNA strand at the location of the gene using it as a template to create a complementary strand known as a messenger RNA strand (mRNA). This complementary strand is made by linking RNA nucleotides that are complementary to the gene nucleotides on the DNA strand. Note: In any RNA strand, the base thymine is replaced with uracil and the start codon (AUG) and a stop codon (UAA or UAG or UGA) control the length of the strand. [31] [33]


3) The mRNA strand then undergoes modifications in the nucleus to contain only the base sequence that will code for the protein.[32] Most genes contain non-coding regions known as introns and coding regions called exons.[31] [32] Thus its complementary mRNA strand will also contain coding and non-coding regions. The non-coding regions are excised by splicing introns from the mRNA and joining the exons together.[31] [32]



The second stage: (Amino acid activation)

mRNA and tRNA are chemically and structurally different from DNA

This stage occurs in the cell cytoplasm where the aminoacyl-tRNA synthetise enzyme attaches amino acids to transfer RNA molecules (tRNA). Each type of amino acid is attached to its specific tRNA.[33]



The final stage: (Translation)

1) The mRNA strand binds onto a ribosome in the cytoplasm at the end with the start codon (AUG).[31] [33]


2) A tRNA molecule binds to the start codon on the mRNA within the ribosome.[33]


3) A second tRNA binds to the next codon of the mRNA within the ribosome. [33]


4) The first tRNA is released from the ribosome and the ribosome moves along the mRNA strand one codon at a time (42). Two tRNAs are temporarily bound within a ribosome at any one time and their amino acids linked together forming a polypeptide chain. This process ends when the stop codon is reached on the tRNA molecule and the polypeptide is then released. [32] [33]


5) The polypeptide then undergoes further modifications in the cell cytoplasm particularly in the Golgi apparatus before becoming the final protein.[32]



Z Tf2.jpg
Z Tf3.jpg
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Transgenic Organism

A transgenic organism is one whose genome contains genes from another species.[32] The aim of creating a transgenic organism is to obtain a favorable characteristic in the organism’s phenotype. This desired characteristic of phenotype is obtained by altering an organism’s normal genotype to include the gene from another species coding for the desired characteristic. [33] Once a desired gene is incorporated into an animal’s genotype, the process of protein synthesis allows the transgenic organism to express this new characteristic in its phenotype. [32] [33]


In the case of fluorescent research, the gene responsible for fluorescence phenotypic characteristics in one organism can be spliced and inserted into the genotype of another organism for experimentation. The recipient organism is then able to produce the fluorescent proteins (coded for by the inserted gene) in its phenotype. [34]



The production of a fluorescent transgenic organism or cell involves several steps summarized below:


1) Organisms containing fluorescent proteins are identified. The chromosome and the gene coding for the fluorescence are identified in the organism.[32]


2) A cell sample is taken from the organism and the gene coding for fluorescent protein synthesis is isolated from its DNA strand. This involves “cutting” the gene out of its DNA strand using enzymes called restriction endonucleases. The restriction endonucleases cut DNA at specific site so the desired gene can be removed from the DNA strand. The cut ends are known as “sticky ends”.[31] [32]


3) The gene is then incorporated into a recipient organism’s DNA. To do this, the recipient organism’s cell DNA must first be cut with the same restriction endonucleases at a desired location (usually just before the stop codon at the end of a gene coding for a particular protein).[31] The foreign gene is then inserted with a promoter sequence by microinjection or vector transfer into the fertilized egg cell (zygote) and as the embryo develops, the entire organism will become transgenic.[34] Alternatively, the foreign gene can be injected into a somatic cell or sex cell of a post natal recipient organism, this in effect will result in the fluorescence of a particular type of cell and thus tissues rather than the entire organism. [31] [34]


4) The inserted foreign gene will be attracted to and connect at the recipient’s cut DNA in a process called “annealing”.[33] The DNA is now known as recombinant DNA as it contains a combination of the recipient’s normal genes as well as foreign genes. [32]


5) DNA ligases are added to the annealed DNA fragments to help strengthen the bonds of the new recombinant DNA. [32] [33] DNA ligases are sealing enzymes found in all living organisms that help make and repair DNA. [32]


6) As the cell develops and its genetic information read, the recombinant DNA will not only code for the production of the normal protein, but will also code for the production of the fluorescent protein that will be attached to the normal protein. [32] This will be true of the recipient cell and all its descendants. [32]






The creation of transgenic organisms with the capability to produce fluorescent proteins has significant clinical consequences particularly in the understanding of inexorable diseases such as cancer and malaria.


Red fluorescent primary tumour inside a green fluorescent mouse. The normal tissue of the mouse is clearly distinguishable from abnormal tumour
Tumor traffic.jpg

Cancer

Cancer is the leading cause of death worldwide claiming approximately 8 million people in 2009. The WHO estimates this figure to rise to 12 million by 2030. [35] In Australia, cancer claims approximately 42,000 lives every year and an estimated 114,000 new cases are expected in 2010. [36] A cancer or malignant neoplasm is a class of disease in which cells display uncontrolled mitotic divisions, invasion of surrounding tissues and metastasis (spread via the circulatory or lymphatic system). [37] Cancer cells are unspecialized and have no other function other than reproduction. These cells invade tissues and organs diminishing their functioning gradually until failure. [34]


Fluorescent proteins provide a means by which cancer can be studied in vivo. The implications of this are significant as it allows researchers to study the characteristics of this disease as it develops in the body over time. [38] A histologically intact primary tumour from a cancer patient is obtained by surgical means and transfected with a specific fluorescent protein. The transfected tumour cells are then transplanted into immunodeficient (nude) transgenic mammals (usually mice) which themselves have been genetically modified to display a different coloured fluorescent protein.[38] [39] This creates a contrast between the healthy cells of the recipient organism and those of the tumour cells as both display different coloured fluorescence. [40] [41] The fluorescent protein fluoresces for the entire life of a cancer cell and after it divides mitotically, fluoresces in the daughter cells. [40] Using this contrast, researchers are able to visualize, in real time, important aspects of cancer in living animals, including tumour cell angiogenesis, mobility, invasion and metastasis. [39] [41]


This technique of studying cancer has so far yielded some interesting results. GFP expressing tumour cells reveal that angiogenesis occurs early in tumour formation when as little as 60 to 80 tumour cells are present inducing vasodialation and changes to blood vessel morphology. [42] [43] Armed with this knowledge, researchers can experiment with various methods to stop the ability of tumour cells to recruit blood vessels that are vital for their growth and spread thereby stopping the cancer in its tracks. [44] Research by Yamauchi and Yamamoto explored the mechanism by which tumour cells migrate down through narrow capillaries. By expressing red fluorescent proteins in the tumour cell cytoplasm and green fluorescent proteins in the cell nucleus they showed that tumour cells are highly motile and capable of changing their morphology to great extents in order to squeeze down narrow vessels. [45] [46] This is critical information on the metastatic ability of cancer cells and explains to some degree the mechanism by which cancer cells are able to spread to body extremities. [45]



Malaria

Plasmodium parasite transfected with GFP inside a mosquito
In vivo Plasmodium parasite transfected with GFP inside a hepatocyte

Malaria is the world’s most serious infectious disease and is responsible for one million deaths annually worldwide.[47] There are currently between 250 to 500 million people affected by malaria most of them in developing countries.[32] [47] In Africa, malaria is an especially serious problem where a child dies from it every 30 seconds.[47] In Australia, malaria was declared eradicated in 1981 but approximately 700 to 800 cases occur each year from travellers infected elsewhere with northern Australia the primary receptive zone for malaria transmission. [48] Malaria is a parasitic infection caused by four species of the genus Plasmodium: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale. The infection begins when Plasmodium sporozoites enter dermis from the bite of an infected female Anopheles mosquito.[31] [32] [33] [48] The parasites then invade blood vessel and travels by blood to the liver where they enter liver cells (hepatocytes).[31] Inside a hepatocyte, the Plasmodium sporozoites divide asexually forming Plasmodium merozoites.[49] The merozoites are then released into the circulatory system where their invade red blood cells (erythrocytes).[49] [50] Once inside erythrocytes, the merozoites continues to proliferate causing each infected red blood cell to burst releasing toxins into the blood stream. [50] Some erythrocytes do not burst as they contain merozoites that do not proliferate but instead develop into male and female gametocytes. When a female Anopheles mosquito bites an infected host, these gamatocytes enter the mosquito abdomen where they form a zygote and develop into new active Plasmodium sporozoites that can begin a new infection cycle. [50] Growing Plasmodium resistance to anti-malarial medicines such as chloroquine and vector resistance to mosquito pesticides has spread very rapidly undermining malaria control efforts across the globe. [47]


Fluorescent proteins provide a unique method of imaging the entire malaria parasite lifecycle from development to transmigration inside both insect and vertebrate hosts. This is achieved by transfecting the malarial parasites with genes encoding for green-fluorescent protein (GFP) or red-fluorescent protein (RedStar) creating a transgenic parasite.[51] Fluorescent microscopy can then be used to viasulize the parasites behaviour inside the mosquito vector and also in cells of the vertebrate host. Using this technique, Rogeiro et. al found that Sporozoites are formed inside oocysts at the mosquito midgut wall. They are then released into the hemolymph and eventually bind to the mosquito’s salivary glands, which they then invade. When the mosquito bites its host, the sporozoites are released into the hosts’ circulatory system.[52] Tracking the movement of the malarial sporozoites inside the mosquito vector helps researchers understand the behaviour of the parasite and devise methods of preventing transmission to humans by stopping the parasite’s advance at the vector level. [51] [52]


Another research focusing on control of malaria found that transgenic mosquitoes were less susceptible to plasmodium infection and had fewer sporozoites in their salivary glands than control mosquitoes. They also found transgenic mosquito parasite transmission was reduced by more than twofold. The team investigated this result to be a consequence of the expression of the SM1 peptide in the mosquito midgut that severely reduced vector competence by inhibiting Plasmodium development. [53] This suggests that gene modifications can inhibit malarial sporozoite development in the mosquito and thus holds great potential for gene experimentation methods to control malaria. Other research has focused on breeding experiments where fluorescent proteins are used to differentiate male and female mosquitoes. Since female mosquitoes only mate once in their lifecycle and are the transmitters of the malaria parasite, sterile males (marked by fluorescent gonads) are sorted and released into the environment where they mate with the infected females. Since the female will be unable to reproduce, the malaria parasite life cycle is ended. [54]

Mosquitoe larvae with fluorescent gonads. This allows the selective sorting of male mosquitoes for sterilization

Advantages of Fluorescent Proteins

Fluorescent proteins allow for in vivo imaging of tissues such neurons (green) and their associated glial cells (red)


Some advantages in using fluorescent proteins include:

• The imaging of structures, cells, tissues, organs and even living organisms in vivo.

• There is no known adverse disruption to the normal functioning of the cell biochemistry.

• Fluorescent proteins are easily obtained from fluorescent organisms mainly corals, jellyfish and marine invertibrates.

• The isolation and transfection of fluorescent proteins is a higly successful process that produces minimal hazardous wastes unlike other fluorescent techniques like radioactive dyes and fluorescent nano dots.

• FP's are available in a vast array of colours covering different emission spectra for imaging different tissues.


Limitations of Fluorescent Proteins

Some problems associated with using fluorescent proteinsCite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive titleCite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title:

• Oligomerisation

• Aggregation

• Cytotoxicity due to FP

• Environmental sensitivity

• Light-induced cytotoxicity

• Potential biological activity exerted by FPs

• No strict criteria under which the FPs are tested- results are often biased and therefore not trustworthy for researchers

• Commercial availability/price

Links to Current Research

AntiCancer Inc. [37]

Bio medicine [38]

Brighter Ideas Inc [39]

Central caribbean marine institute [40]

Clontech [41]

Evrogen [42]

GeneBrite [43]

Glen Research [44]

MetaMouse [45]

Invitrogen [46]

Promega Corporation [47]

Glossary

Angiogenesis: The formation of new blood vessels

Chromophores: Molecules which are able to absorb light

Codon: A specific sequence of three adjacent nucleotides on a strand of DNA or RNA that specifies the genetic code information for synthesizing a particular amino acid

Erythrocytes: Red blood cells

Exons: A segment of a gene that contains a coding region. The exons provide the instructions for making a protein

Filtercel:An industrial filter used to seperate small particles from big ones.

Fluorochromes or fluorophores: Molecules which are able to absorb and emit light Filtercel

FRET: Fluorescence Resonance Energy Transfer. See the Invitrogen website for a short explanation of this technique: [48]

Gametocyte: A eukaryotic germ cell that divides by mitosis into other gametocytes or by meiosis into gametids during gametogenesis

Genotype: The genetic makeup of an individual

Golgi apparatus: An organelle in the cytoplasm that packages proteins and carbohydrates into vesicles for export from the cell

Hemolymph: A circulating fluid in the bodies of some invertebrates that is the equivalent of blood

Introns: An intron is a DNA region within a gene that is not translated into protein

Ligase: Any of a group of enzymes that catalyze the binding of two molecules

Merozoite: Cell that arises from the asexual division of a parent sporozoan during its life cycle

Metastasis: When cancer cells have broken away from the primary tumor and spread to other organs in the body through the blood stream or the lymphatic system

mRNA: The RNA transcript of a protein-encoding gene

Phenotype: Phenotype is any observable characteristic or trait of an organism

Photoactivation:The absorption of energy from a photon in raising a chromophore from the ground state

Photoconversion: The photochemical conversion of a chromophore, resulting in a change in emission spectrum

Photostability: The stability of a chromophore during excitation

Random mutagenesis: Random incorporation of mutations in DNA

Restriction Endonucleases: An enzyme that cuts double-stranded or single stranded DNA at specific recognition

Ribosome: Ribosomes are the components of cells that make proteins from amino acids

Site-directed mutagenesis: Targeted insertion of a mutation in a DNA sequence, usually by use of of a plasmid vector

Sporozoite: Slender, spindle-shaped organism that is the infective stage of malaria

tRNA: A small RNA molecule that "translates" a codon in mRNA by bringing in its corresponding amino acid during protein synthesis

Zygote: A fertilized egg cell, it is diploid

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2010 Projects

Fluorescent-PCR | RNA Interference | Immunohistochemistry | Cell Culture | Electron Microsopy | Confocal Microscopy | Monoclonal Antibodies | Microarray | Fluorescent Proteins | Somatic Cell Nuclear Transfer