Editing Green fluorescent protein (section)

== Applications ==

=== Reporter assays ===

Green fluorescent protein may be used as a [[reporter gene]].<ref>{{cite journal | vauthors = Jugder BE, Welch J, Braidy N, Marquis CP | title = Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH) fusion to gfp (green fluorescent protein) | journal = PeerJ | volume = 4 | pages = e2269 | date = 2016-07-26 | pmid = 27547572 | pmc = 4974937 | doi = 10.7717/peerj.2269 | doi-access = free }}</ref><ref name="pmid15596111">{{cite journal | vauthors = Arun KH, Kaul CL, Ramarao P | title = Green fluorescent proteins in receptor research: an emerging tool for drug discovery | journal = Journal of Pharmacological and Toxicological Methods | volume = 51 | issue = 1 | pages = 1–23 | year = 2005 | pmid = 15596111 | doi = 10.1016/j.vascn.2004.07.006 }}</ref>

For example, GFP can be used as a reporter for environmental toxicity levels. This protein has been shown to be an effective way to measure the toxicity levels of various chemicals including ethanol, ''p''-formaldehyde, phenol, triclosan, and paraben. GFP is great as a reporter protein because it has no effect on the host when introduced to the host's cellular environment. Due to this ability, no external visualization stain, ATP, or cofactors are needed. With regards to pollutant levels, the fluorescence was measured in order to gauge the effect that the pollutants have on the host cell. The cellular density of the host cell was also measured. Results from the study conducted by Song, Kim, & Seo (2016) showed that there was a decrease in both fluorescence and cellular density as pollutant levels increased. This was indicative of the fact that cellular activity had decreased. More research into this specific application in order to determine the mechanism by which GFP acts as a pollutant marker.<ref>{{cite journal | vauthors = Song YH, Kim CS, Seo JH | title = Noninvasive monitoring of environmental toxicity through green fluorescent protein expressing Escherichia coli. | journal = Korean Journal of Chemical Engineering | date = April 2016 | volume = 33 | issue = 4 | pages = 1331–6 | doi = 10.1007/s11814-015-0253-1 | s2cid = 62828580 }}</ref> Similar results have been observed in zebrafish because zebrafish that were injected with GFP were approximately twenty times more susceptible to recognize cellular stresses than zebrafish that were not injected with GFP.<ref name="pmid23143852">{{cite journal | vauthors = Pan Y, Leifert A, Graf M, Schiefer F, Thoröe-Boveleth S, Broda J, Halloran MC, Hollert H, Laaf D, Simon U, Jahnen-Dechent W | title = High-sensitivity real-time analysis of nanoparticle toxicity in green fluorescent protein-expressing zebrafish | journal = Small | location = Weinheim an Der Bergstrasse, Germany | volume = 9 | issue = 6 | pages = 863–9 | date = March 2013 | pmid = 23143852 | doi = 10.1002/smll.201201173 }}</ref>

==== Advantages ====

The biggest advantage of GFP is that it can be heritable, depending on how it was introduced, allowing for continued study of cells and tissues it is expressed in. Visualizing GFP is noninvasive, requiring only illumination with blue light. GFP alone does not interfere with biological processes, but when fused to proteins of interest, careful design of linkers is required to maintain the function of the protein of interest. Moreover, if used with a monomer it is able to diffuse readily throughout cells.<ref name="pmid19553219">{{cite journal | vauthors = Chalfie M | title = GFP: Lighting up life | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 106 | issue = 25 | pages = 10073–10080 | date = Jun 2009 | pmid = 19553219 | doi = 10.1073/pnas.0904061106 | pmc=2700921| bibcode = 2009PNAS..10610073C | doi-access = free }}</ref>

=== Fluorescence microscopy ===
{{Main|Fluorescence microscope}}
[[Image:GFP Superresolution Christoph Cremer.JPG|thumb|200px|Superresolution with two [[fusion protein]]s (GFP-Snf2H and RFP-H2A), Co-localisation studies (2CLM) in the nucleus of a bone cancer cell. 120.000 localized molecules in a widefield area (470&nbsp;μm<sup>2</sup>).]]
The availability of GFP and its derivatives has thoroughly redefined [[fluorescence microscopy]] and the way it is used in cell biology and other biological disciplines.<ref name=Yutse_2005>{{cite journal | vauthors = Yuste R | title = Fluorescence microscopy today | journal = Nature Methods | volume = 2 | issue = 12 | pages = 902–4 | date = Dec 2005 | pmid = 16299474 | doi = 10.1038/nmeth1205-902 | s2cid = 205418407 }}</ref> While most small fluorescent molecules such as [[Fluorescein|FITC]] (fluorescein isothiocyanate) are strongly [[phototoxic]] when used in live cells, fluorescent proteins such as GFP are usually much less harmful when illuminated in living cells. This has triggered the development of highly automated live-cell fluorescence microscopy systems, which can be used to observe cells over time expressing one or more proteins tagged with fluorescent proteins.

There are many techniques to utilize GFP in a live cell imaging experiment. The most direct way of utilizing GFP is to directly attach it to a protein of interest. For example, GFP can be included in a plasmid expressing other genes to indicate a successful transfection of a gene of interest. Another method is to use a GFP that contains a mutation where the fluorescence will change from green to yellow over time, which is referred to as a fluorescent timer. With the fluorescent timer, researchers can study the state of protein production such as recently activated, continuously activated, or recently deactivated based on the color reported by the fluorescent protein.<ref>{{cite journal | vauthors = Terskikh A, Fradkov A, Ermakova G, Zaraisky A, Tan P, Kajava AV, Zhao X, Lukyanov S, Matz M, Kim S, Weissman I, Siebert P | title = "Fluorescent timer": protein that changes color with time | journal = Science | volume = 290 | issue = 5496 | pages = 1585–8 | date = November 2000 | pmid = 11090358 | doi = 10.1126/science.290.5496.1585 | bibcode = 2000Sci...290.1585T }}</ref> In yet another example, scientists have modified GFP to become active only after exposure to irradiation giving researchers a tool to selectively activate certain portions of a cell and observe where proteins tagged with the GFP move from the starting location.<ref>{{cite journal | vauthors = Patterson GH, Lippincott-Schwartz J | title = A photoactivatable GFP for selective photolabeling of proteins and cells | journal = Science | volume = 297 | issue = 5588 | pages = 1873–7 | date = September 2002 | pmid = 12228718 | doi = 10.1126/science.1074952 | bibcode = 2002Sci...297.1873P | s2cid = 45058411 }}</ref> These are only two examples in a burgeoning field of fluorescent microcopy and a more complete review of biosensors utilizing GFP and other fluorescent proteins can be found here <ref>{{cite journal | vauthors = Lin W, Mehta S, Zhang J | title = Genetically encoded fluorescent biosensors illuminate kinase signaling in cancer | journal = The Journal of Biological Chemistry | volume = 294 | issue = 40 | pages = 14814–14822 | date = October 2019 | pmid = 31434714 | pmc = 6779441 | doi = 10.1074/jbc.REV119.006177 | doi-access = free }}</ref>

For example, GFP had been widely used in labelling the [[Spermatozoon|spermatozoa]] of various organisms for identification purposes as in ''[[Drosophila melanogaster]]'', where expression of GFP can be used as a marker for a particular characteristic. GFP can also be expressed in different structures enabling morphological distinction. In such cases, the gene for the production of GFP is incorporated into the genome of the organism in the region of the DNA that codes for the target proteins and that is controlled by the same [[regulatory sequence]]; that is, the gene's regulatory sequence now controls the production of GFP, in addition to the tagged protein(s). In cells where the gene is expressed, and the tagged proteins are produced, GFP is produced at the same time. Thus, only those cells in which the tagged gene is expressed, or the target proteins are produced, will fluoresce when observed under fluorescence microscopy. Analysis of such time lapse movies has redefined the understanding of many biological processes including protein folding, protein transport, and RNA dynamics, which in the past had been studied using fixed (i.e., dead) material. Obtained data are also used to calibrate mathematical models of intracellular systems and to estimate rates of gene expression.<ref name="pmid20550887">{{cite journal | vauthors = Komorowski M, Finkenstädt B, Rand D | title = Using a single fluorescent reporter gene to infer half-life of extrinsic noise and other parameters of gene expression | journal = Biophysical Journal | volume = 98 | issue = 12 | pages = 2759–2769 | date = Jun 2010 | pmid = 20550887 | pmc = 2884236 | doi = 10.1016/j.bpj.2010.03.032 | bibcode = 2010BpJ....98.2759K }}</ref> Similarly, GFP can be used as an indicator of protein expression in heterologous systems. In this scenario, fusion proteins containing GFP are introduced indirectly, using RNA of the construct, or directly, with the tagged protein itself. This method is useful for studying structural and functional characteristics of the tagged protein on a macromolecular or single-molecule scale with fluorescence microscopy.

The [[Vertico SMI]] microscope using the SPDM Phymod technology uses the so-called "reversible photobleaching" effect of fluorescent dyes like GFP and its derivatives to localize them as single molecules in an optical resolution of 10&nbsp;nm. This can also be performed as a co-localization of two GFP derivatives (2CLM).<ref name="pmid19548231">{{cite journal | vauthors = Gunkel M, Erdel F, Rippe K, Lemmer P, Kaufmann R, Hörmann C, Amberger R, Cremer C | title = Dual color localization microscopy of cellular nanostructures | journal = Biotechnology Journal | volume = 4 | issue = 6 | pages = 927–38 | date = Jun 2009 | pmid = 19548231 | doi = 10.1002/biot.200900005 | s2cid = 18162278 | url = https://hal.archives-ouvertes.fr/hal-00494027/document }}</ref>

Another powerful use of GFP is to express the protein in small sets of specific cells. This allows researchers to optically detect specific types of cells ''[[in vitro]]'' (in a dish), or even ''[[in vivo]]'' (in the living organism).<ref name=Chudakov_2005>{{cite journal | vauthors = Chudakov DM, Lukyanov S, Lukyanov KA | title = Fluorescent proteins as a toolkit for in vivo imaging | journal = Trends in Biotechnology | volume = 23 | issue = 12 | pages = 605–13 | date = Dec 2005 | pmid = 16269193 | doi = 10.1016/j.tibtech.2005.10.005 }}</ref> GFP is considered to be a reliable reporter of gene expression in eukaryotic cells when the fluorescence is measured by flow cytometry.<ref>{{cite journal | vauthors = Soboleski MR, Oaks J, Halford WP | title = Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells | journal = FASEB Journal | volume = 19 | issue = 3 | pages = 440–442 | date = March 2005 | pmid = 15640280 | pmc = 1242169 | doi = 10.1096/fj.04-3180fje | doi-access = free }}</ref> Genetically combining several spectral variants of GFP is a useful trick for the analysis of brain circuitry ([[Brainbow]]).<ref name="pmid17972876">{{cite journal | vauthors = Livet J, Weissman TA, Kang H, Draft RW, Lu J, Bennis RA, Sanes JR, Lichtman JW | title = Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system | journal = Nature | volume = 450 | issue = 7166 | pages = 56–62 | date = Nov 2007 | pmid = 17972876 | doi = 10.1038/nature06293 | bibcode = 2007Natur.450...56L | s2cid = 4402093 }}</ref> Other interesting uses of fluorescent proteins in the literature include using FPs as sensors of [[neuron]] [[membrane potential]],<ref name="pmid18679801">{{cite journal | vauthors = Baker BJ, Mutoh H, Dimitrov D, Akemann W, Perron A, Iwamoto Y, Jin L, Cohen LB, Isacoff EY, Pieribone VA, Hughes T, Knöpfel T | title = Genetically encoded fluorescent sensors of membrane potential | journal = Brain Cell Biology | volume = 36 | issue = 1–4 | pages = 53–67 | date = Aug 2008 | pmid = 18679801 | pmc = 2775812 | doi = 10.1007/s11068-008-9026-7 }}</ref> tracking of [[AMPA]] receptors on cell membranes,<ref name="pmid16364901">{{cite journal | vauthors = Adesnik H, Nicoll RA, England PM | title = Photoinactivation of native AMPA receptors reveals their real-time trafficking | journal = Neuron | volume = 48 | issue = 6 | pages = 977–85 | date = Dec 2005 | pmid = 16364901 | doi = 10.1016/j.neuron.2005.11.030 | doi-access = free }}</ref> [[viral entry]] and the infection of individual [[influenza]] viruses and lentiviral viruses,<ref name="pmid12883000">{{cite journal | vauthors = Lakadamyali M, Rust MJ, Babcock HP, Zhuang X | title = Visualizing infection of individual influenza viruses | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 100 | issue = 16 | pages = 9280–5 | date = Aug 2003 | pmid = 12883000 | pmc = 170909 | doi = 10.1073/pnas.0832269100 | bibcode = 2003PNAS..100.9280L | doi-access = free }}</ref><ref name="pmid18480844">{{cite journal | vauthors = Joo KI, Wang P | title = Visualization of targeted transduction by engineered lentiviral vectors | journal = Gene Therapy | volume = 15 | issue = 20 | pages = 1384–96 | date = Oct 2008 | pmid = 18480844 | pmc = 2575058 | doi = 10.1038/gt.2008.87 }}</ref> etc.

It has also been found that new lines of transgenic GFP rats can be relevant for gene therapy as well as regenerative medicine.<ref name="pmid20094912">{{cite journal | vauthors = Remy S, Tesson L, Usal C, Menoret S, Bonnamain V, Nerriere-Daguin V, Rossignol J, Boyer C, Nguyen TH, Naveilhan P, Lescaudron L, Anegon I | title = New lines of GFP transgenic rats relevant for regenerative medicine and gene therapy | journal = Transgenic Research | volume = 19 | issue = 5 | pages = 745–63 | date = Oct 2010 | pmid = 20094912 | doi = 10.1007/s11248-009-9352-2 | s2cid = 42499768 }}</ref> By using "high-expresser" GFP, transgenic rats display high expression in most tissues, and many cells that have not been characterized or have been only poorly characterized in previous GFP-transgenic rats.

GFP has been shown to be useful in [[cryobiology]] as a [[viability assay]]. Correlation of viability as measured by [[trypan blue]] assays were 0.97.<ref>{{cite journal | vauthors = Elliott G, McGrath J, Crockett-Torabi E | title = Green fluorescent protein: A novel viability assay for cryobiological applications | journal = Cryobiology | volume = 40 | issue = 4 | pages = 360–369 | date = Jun 2000 | pmid = 10924267 | doi = 10.1006/cryo.2000.2258 }}</ref> Another application is the use of GFP co-transfection as internal control for transfection efficiency in mammalian cells.<ref name="pmid20064974">{{cite journal | vauthors = Fakhrudin N, Ladurner A, Atanasov AG, Heiss EH, Baumgartner L, Markt P, Schuster D, Ellmerer EP, Wolber G, Rollinger JM, Stuppner H, Dirsch VM | title = Computer-aided discovery, validation, and mechanistic characterization of novel neolignan activators of peroxisome proliferator-activated receptor gamma | journal = Molecular Pharmacology | volume = 77 | issue = 4 | pages = 559–66 | date = Apr 2010 | pmid = 20064974 | pmc = 3523390 | doi = 10.1124/mol.109.062141 }}</ref>

A novel possible use of GFP includes using it as a sensitive monitor of intracellular processes via an eGFP laser system made out of a human embryonic kidney cell line. The first engineered living laser is made by an eGFP expressing cell inside a reflective optical cavity and hitting it with pulses of blue light. At a certain pulse threshold, the eGFP's optical output becomes brighter and completely uniform in color of pure green with a wavelength of 516&nbsp;nm. Before being emitted as laser light, the light bounces back and forth within the resonator cavity and passes the cell numerous times. By studying the changes in optical activity, researchers may better understand cellular processes.<ref>{{cite journal | vauthors = Gather MC, Yun SH | s2cid = 54971962 | title = Single-cell biological lasers | date = 2011 | journal = Nature Photonics | volume = 5 | issue = 7 | pages = 406–410 | doi = 10.1038/nphoton.2011.99 | bibcode = 2011NaPho...5..406G }}</ref><ref>{{cite journal | vauthors = Matson J | date = 2011 | title = Green Fluorescent Protein Makes for Living Lasers | journal = Scientific American | url = http://www.scientificamerican.com/article.cfm?id=biological-laser-cell | access-date = 2011-06-13 }}</ref>

GFP is used widely in cancer research to label and track cancer cells. GFP-labelled cancer cells have been used to model metastasis, the process by which cancer cells spread to distant organs.<ref>{{cite journal | vauthors = Kouros-Mehr H, Bechis SK, Slorach EM, Littlepage LE, Egeblad M, Ewald AJ, Pai SY, Ho IC, Werb Z | title = GATA-3 links tumor differentiation and dissemination in a luminal breast cancer model | journal = Cancer Cell | volume = 13 | issue = 2 | pages = 141–52 | date = Feb 2008 | pmid = 18242514 | pmc = 2262951 | doi = 10.1016/j.ccr.2008.01.011 }}</ref>

=== Split GFP ===
GFP can be used to analyse the colocalization of proteins. This is achieved by "splitting" the protein into two fragments which are able to self-assemble, and then fusing each of these to the two proteins of interest. Alone, these incomplete GFP fragments are unable to fluoresce. However, if the two proteins of interest colocalize, then the two GFP fragments assemble together to form a GFP-like structure which is able to fluoresce. Therefore, by measuring the level of fluorescence it is possible to determine whether the two proteins of interest colocalize.<ref>{{cite journal | vauthors = Cabantous S, Terwilliger TC, Waldo GS | title = Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein | journal = Nature Biotechnology | volume = 23 | issue = 1 | pages = 102–7 | date = January 2005 | pmid = 15580262 | doi = 10.1038/nbt1044 | s2cid = 25833063 }}</ref>

=== Macro-photography ===
Macro-scale biological processes, such as the spread of virus infections, can be followed using GFP labeling.<ref>{{cite journal | vauthors = Rodman MK, Yadav NS, Artus NN | title = Progression of geminivirus-induced transgene silencing is associated with transgene methylation | journal = The New Phytologist | volume = 155 | issue = 3 | pages = 461–468 | date = September 2002 | pmid = 33873315 | doi = 10.1046/j.1469-8137.2002.00467.x | bibcode = 2002NewPh.155..461R }}</ref>  In the past, mutagenic ultra violet light (UV) has been used to illuminate living organisms  (e.g., see<ref>{{cite journal | vauthors = Zhu YJ, Agbayani R, Moore PH | title = Green fluorescent protein as a visual selection marker for papaya (Carica papaya L.) transformation | journal = Plant Cell Reports | volume = 22 | issue = 9 | pages = 660–7 | date = Apr 2004 | pmid = 14749892 | doi = 10.1007/s00299-004-0755-5 | s2cid = 23198182 }}</ref>) to detect and photograph the GFP expression. Recently, a technique using non-mutagenic LED lights<ref name="pmid10406127">{{cite journal | vauthors = Niwa Y, Hirano T, Yoshimoto K, Shimizu M, Kobayashi H | s2cid = 292648 | title = Non-invasive quantitative detection and applications of non-toxic, S65T-type green fluorescent protein in living plants | journal = The Plant Journal | volume = 18 | issue = 4 | pages = 455–63 | year = 1999 | pmid = 10406127 | doi = 10.1046/j.1365-313X.1999.00464.x | doi-access = free }}</ref> have been developed for macro-photography.<ref>{{cite journal | vauthors = Baker SS, Vidican CB, Cameron DS, Greib HG, Jarocki CC, Setaputri AW, Spicuzza CH, Burr AA, Waqas MA, Tolbert DA | title = An epifluorescent attachment improves whole-plant digital photography of Arabidopsis thaliana expressing red-shifted green fluorescent protein | journal = AoB Plants | volume = 2012 | pages = pls003 | date = 2012-01-01 | pmid = 22479674 | pmc = 3296078 | doi = 10.1093/aobpla/pls003 }}</ref>  The technique uses an epifluorescence camera attachment<ref>{{cite web | url = http://planted.botany.org/index.php?P=FullRecord&ID=570 | title = PlantEdDL - Using SRL digital cameras in quantitative investigations of plants expressing green fluorescent protein (GFP) | website = planted.botany.org | access-date = 2016-03-23 }}</ref> based on the same principle used in the construction of [[Fluorescence microscope|epifluorescence microscopes]].

=== Transgenic pets ===
[[Image:GFP Mice 01.jpg|right|thumb|Mice expressing GFP under UV light (left & right), compared to normal mouse (center)]]
[[Alba (rabbit)|Alba]], a green-fluorescent rabbit, was created by a French laboratory commissioned by [[Eduardo Kac]] using GFP for purposes of art and social commentary.<ref>{{cite web|url= http://www.ekac.org/gfpbunny.html#gfpbunnyanchor | vauthors = Kac E |title=GFP Bunny}}</ref> The US company Yorktown Technologies markets to aquarium shops green fluorescent [[zebrafish]] ([[GloFish]]) that were initially developed to detect pollution in waterways. NeonPets, a US-based company has marketed green fluorescent mice to the pet industry as NeonMice.<ref>{{cite web |url=http://neonmice.com/ |archive-url=https://web.archive.org/web/20090214004617/http://www.neonmice.com/ |archive-date=February 14, 2009 |title=Glow-In-The Dark NeonMice |url-status=dead |access-date=August 30, 2016}}</ref> Green fluorescent pigs, known as Noels, were bred by a group of researchers led by Wu Shinn-Chih at the Department of Animal Science and Technology at [[National Taiwan University]].<ref>[http://news.bbc.co.uk/1/hi/world/asia-pacific/4605202.stm Scientists in Taiwan breed fluorescent green pigs]</ref> A Japanese-American Team created green-fluorescent [[cat]]s as proof of concept to use them potentially as model organisms for diseases, particularly [[HIV]].<ref name=Wongsrikeao_2011>{{cite journal|author-link5=Eric Poeschla | vauthors = Wongsrikeao P, Saenz D, Rinkoski T, Otoi T, Poeschla E | title = Antiviral restriction factor transgenesis in the domestic cat | journal = Nature Methods | volume = 8 | issue = 10 | pages = 853–9 | date = 2011 | pmid = 21909101 | pmc = 4006694 | doi = 10.1038/nmeth.1703 }}</ref> In 2009 a South Korean team from Seoul National University bred the first transgenic [[Ruppy|beagles]] with fibroblast cells from sea anemones. The dogs give off a red fluorescent light, and they are meant to allow scientists to study the genes that cause human diseases like [[narcolepsy]] and [[blindness]].<ref>{{Cite web | vauthors = Callaway E | date = 23 April 2009 | url=https://www.newscientist.com/article/dn17003-fluorescent-puppy-is-worlds-first-transgenic-dog.html#.U3O8tvldV8G | title=Fluorescent puppy is world's first transgenic dog | work = New Scientist }}</ref>

=== Art ===

[[Julian Voss-Andreae]], a German-born artist specializing in "protein sculptures,"<ref>{{cite journal | vauthors = Voss-Andreae J | date = 2005 | title = Protein Sculptures: Life's Building Blocks Inspire Art | journal = Leonardo | volume = 38 | pages = 41–45 | doi = 10.1162/leon.2005.38.1.41| s2cid = 57558522 }}</ref> created sculptures based on the structure of GFP, including the {{convert|1.70|m|ftin|abbr=off}}  tall "Green Fluorescent Protein" (2004)<ref>{{cite journal | vauthors = Pawlak A  | date = 2005 | title = Inspirierende Proteine | journal = Physik Journal | volume = 4 | pages = 12 | url = https://pro-physik.de/zeitschriften/download/16101 }}</ref> and the {{convert|1.40|m|ftin|abbr=off}} tall "Steel Jellyfish" (2006). The latter sculpture is located at the place of GFP's discovery by [[Osamu Shimomura|Shimomura]] in 1962, the [[University of Washington]]'s [[Friday Harbor Laboratories]].<ref>{{cite web | title = Julian Voss-Andreae Sculpture | url = http://www.julianvossandreae.com/ | access-date = 2007-06-14}}</ref>

[[Image:Steel Jellyfish (GFP).jpg|thumb|right|[[Julian Voss-Andreae]]'s GFP-based sculpture ''Steel Jellyfish'' (2006). The image shows the stainless-steel sculpture at [[Friday Harbor Laboratories]] on [[San Juan Island]] (Wash., USA), the place of GFP's discovery.]]