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Reporter gene

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A diagram of a how a reporter gene is used to study a regulatory sequence.

In molecular biology, a reporter gene (often simply reporter) is a gene that researchers attach to a regulatory sequence of another gene of interest in bacteria, cell culture, animals or plants. Such genes are called reporters because the characteristics they confer on organisms expressing them are easily identified and measured, or because they are selectable markers. Reporter genes are often used as an indication of whether a certain gene has been taken up by or expressed in the cell or organism population.

Common reporter genes

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To introduce a reporter gene into an organism, scientists place the reporter gene and the gene of interest in the same DNA construct to be inserted into the cell or organism. For bacteria or prokaryotic cells in culture, this is usually in the form of a circular DNA molecule called a plasmid. For viruses, this is known as a viral vector. It is important to use a reporter gene that is not natively expressed in the cell or organism under study, since the expression of the reporter is being used as a marker for successful uptake of the gene of interest.[1]

Commonly used reporter genes that induce visually identifiable characteristics usually involve fluorescent and luminescent proteins. Examples include the gene that encodes jellyfish green fluorescent protein (GFP), which causes cells that express it to glow green under blue or ultraviolet light,[2] the enzyme luciferase, which catalyzes a reaction with luciferin to produce light, and the red fluorescent protein from the gene dsRed [fr].[3][4][5][6][7] The GUS gene has been commonly used in plants but luciferase and GFP are becoming more common.[8][9]

A common reporter in bacteria is the E. coli lacZ gene, which encodes the protein beta-galactosidase.[10] This enzyme causes bacteria expressing the gene to appear blue when grown on a medium that contains the substrate analog X-gal. An example of a selectable marker which is also a reporter in bacteria is the chloramphenicol acetyltransferase (CAT) gene, which confers resistance to the antibiotic chloramphenicol.[11]

Gene name Gene product Assay Ref.
lacZ β-galactosidase Enzyme assay, Histochemical [10]
cat Chloramphenicol acetyltransferase Chloramphenicol acetylation [11]
gfp Green fluorescent protein Fluorescent [3]
rfp Red fluorescent protein Microscopical, Spectrophotometry [12]
luc Luciferase enzyme Bioluminescence [4]

Transformation and transfection assays

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Many methods of transfection and transformation – two ways of expressing a foreign or modified gene in an organism – are effective in only a small percentage of a population subjected to the techniques.[13][14] Thus, a method for identifying those few successful gene uptake events is necessary. Reporter genes used in this way are normally expressed under their own promoter (DNA regions that initiates gene transcription) independent from that of the introduced gene of interest; the reporter gene can be expressed constitutively (that is, it is "always on") or inducibly with an external intervention such as the introduction of Isopropyl β-D-1-thiogalactopyranoside (IPTG) in the β-galactosidase system.[10] As a result, the reporter gene's expression is independent of the gene of interest's expression, which is an advantage when the gene of interest is only expressed under certain specific conditions or in tissues that are difficult to access.[1]

In the case of selectable-marker reporters such as CAT, the transfected population of bacteria can be grown on a substrate that contains chloramphenicol. Only those cells that have successfully taken up the construct containing the CAT gene will survive and multiply under these conditions.[11]

Gene expression assays

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Reporter genes can be used to assay for the expression of a gene of interest that is normally difficult to quantitatively assay.[1] Reporter genes can produce a protein that has little obvious or immediate effect on the cell culture or organism. They are ideally not present in the native genome to be able to isolate reporter gene expression as a result of the gene of interest's expression.[1][15]

To activate reporter genes, they can be expressed constitutively, where they are directly attached to the gene of interest to create a gene fusion.[16] This method is an example of using cis-acting elements where the two genes are under the same promoter elements and are transcribed into a single messenger RNA molecule. The mRNA is then translated into protein. It is important that both proteins be able to properly fold into their active conformations and interact with their substrates despite being fused. In building the DNA construct, a segment of DNA coding for a flexible polypeptide linker region is usually included so that the reporter and the gene product will only minimally interfere with one another.[17][18] Reporter genes can also be expressed by induction during growth. In these cases, trans-acting elements, such as transcription factors are used to express the reporter gene.[19][20]

Reporter gene assay have been increasingly used in high throughput screening (HTS) to identify small molecule inhibitors and activators of protein targets and pathways for drug discovery and chemical biology. Because the reporter enzymes themselves (e.g. firefly luciferase) can be direct targets of small molecules and confound the interpretation of HTS data, novel coincidence reporter designs incorporating artifact suppression have been developed.[21][22]

Promoter assays

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Reporter genes can be used to assay for the activity of a particular promoter in a cell or organism.[23] In this case there is no separate "gene of interest"; the reporter gene is simply placed under the control of the target promoter and the reporter gene product's activity is quantitatively measured. The results are normally reported relative to the activity under a "consensus" promoter known to induce strong gene expression.[24]

Further uses

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A more complex use of reporter genes on a large scale is in two-hybrid screening, which aims to identify proteins that natively interact with one another in vivo.[25]

See also

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References

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  1. ^ a b c d Debnath, Mousumi; Prasad, Godavarthi B.K.S.; Bisen, Prakash S. (2010), Debnath, Mousumi; Prasad, Godavarthi B.K.S.; Bisen, Prakash S. (eds.), "Reporter Gene", Molecular Diagnostics: Promises and Possibilities, Springer Netherlands, pp. 71–84, doi:10.1007/978-90-481-3261-4_5, ISBN 978-90-481-3261-4
  2. ^ van Thor, Jasper J.; Gensch, Thomas; Hellingwerf, Klaas J.; Johnson, Louise N. (Jan 2002). "Phototransformation of green fluorescent protein with UV and visible light leads to decarboxylation of glutamate 222". Nature Structural Biology. 9 (1): 37–41. doi:10.1038/nsb739. ISSN 1072-8368. PMID 11740505.
  3. ^ a b Soboleski, Mark R.; Oaks, Jason; Halford, William P. (March 2005). "Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells". The FASEB Journal. 19 (3): 440–442. doi:10.1096/fj.04-3180fje. ISSN 0892-6638. PMC 1242169. PMID 15640280.
  4. ^ a b Smale, S. T. (2010-05-01). "Luciferase Assay". Cold Spring Harbor Protocols. 2010 (5): pdb.prot5421. doi:10.1101/pdb.prot5421. ISSN 1559-6095. PMID 20439408.
  5. ^ Jach, Guido; Binot, Elke; Frings, Sabine; Luxa, Kerstin; Schell, Jeff (2001). "Use of red fluorescent protein from Discosoma sp. (dsRED) as a reporter for plant gene expression". The Plant Journal. 28 (4): 483–491. doi:10.1046/j.1365-313X.2001.01153.x. ISSN 1365-313X. PMID 11737785.
  6. ^ Zhang, Qixiang; Walawage, Sriema L.; Tricoli, David M.; Dandekar, Abhaya M.; Leslie, Charles A. (May 2015). "A red fluorescent protein (DsRED) from Discosoma sp. as a reporter for gene expression in walnut somatic embryos". Plant Cell Reports. 34 (5): 861–869. doi:10.1007/s00299-015-1749-1. ISSN 1432-203X. PMID 25627255. S2CID 9184712.
  7. ^ Mikkelsen, Lisbeth; Sarrocco, Sabrina; Lübeck, Mette; Jensen, Dan Funck (2003-06-01). "Expression of the red fluorescent protein DsRed-Express in filamentous ascomycete fungi". FEMS Microbiology Letters. 223 (1): 135–139. doi:10.1016/S0378-1097(03)00355-0. ISSN 0378-1097. PMID 12799012.
  8. ^ Hull, Gillian A.; Devic, Martine (1995), Jones, Heddwyn (ed.), "The β-Glucuronidase (Gus) Reporter Gene System Gene: Fusions; Spectrophotometric, Fluorometric, and Histochemical Detection", Plant Gene Transfer and Expression Protocols, Methods in Molecular Biology, vol. 49, Springer New York, pp. 125–141, doi:10.1385/0-89603-321-x:125, ISBN 978-1-59259-536-5, PMID 8563799
  9. ^ Koo, J.; Kim, Y.; Kim, J.; Yeom, M.; Lee, I. C.; Nam, H. G. (2007). "A GUS/Luciferase Fusion Reporter for Plant Gene Trapping and for Assay of Promoter Activity with Luciferin-Dependent Control of the Reporter Protein Stability". Plant and Cell Physiology. 48 (8): 1121–1131. doi:10.1093/pcp/pcm081. PMID 17597079.
  10. ^ a b c Smale, S. T. (2010-05-01). "-Galactosidase Assay". Cold Spring Harbor Protocols. 2010 (5): pdb.prot5423. doi:10.1101/pdb.prot5423. ISSN 1559-6095. PMID 20439410.
  11. ^ a b c Smale, S. T. (2010-05-01). "Chloramphenicol Acetyltransferase Assay". Cold Spring Harbor Protocols. 2010 (5): pdb.prot5422. doi:10.1101/pdb.prot5422. ISSN 1559-6095. PMID 20439409.
  12. ^ Nordgren, I. K.; Tavassoli, A (2014). "A bidirectional fluorescent two-hybrid system for monitoring protein-protein interactions". Molecular BioSystems. 10 (3): 485–90. doi:10.1039/c3mb70438f. PMID 24382456. S2CID 12713372.
  13. ^ Hanahan, Douglas; Jessee, Joel; Bloom, Fredric R. (1991-01-01), "[4] Plasmid transformation of Escherichia coli and other bacteria", Bacterial Genetic Systems, Methods in Enzymology, vol. 204, Academic Press, pp. 63–113, doi:10.1016/0076-6879(91)04006-a, ISBN 9780121821050, PMID 1943786
  14. ^ Hanahan, Douglas (1983-06-05). "Studies on transformation of Escherichia coli with plasmids". Journal of Molecular Biology. 166 (4): 557–580. CiteSeerX 10.1.1.460.2021. doi:10.1016/S0022-2836(83)80284-8. ISSN 0022-2836. PMID 6345791.
  15. ^ Archived at Ghostarchive and the Wayback Machine: Promega Corporation, Promega Corporation (October 22, 2014). "Introduction to Reporter Gene Assays". YouTube. Retrieved March 21, 2020.
  16. ^ de Jong, Hidde; Geiselmann, Johannes (2015). "Fluorescent Reporter Genes and the Analysis of Bacterial Regulatory Networks". In Maler, Oded; Halász, Ádám; Dang, Thao; Piazza, Carla (eds.). Hybrid Systems Biology. Lecture Notes in Computer Science. Vol. 7699. Springer International Publishing. pp. 27–50. doi:10.1007/978-3-319-27656-4_2. ISBN 978-3-319-27656-4.
  17. ^ Spector, David L.; Goldman, Robert D. (2006-12-01). "Constructing and Expressing GFP Fusion Proteins". Cold Spring Harbor Protocols. 2006 (7): pdb.prot4649. doi:10.1101/pdb.prot4649. PMID 22484672.
  18. ^ Chen, Xiaoying; Zaro, Jennica; Shen, Wei-Chiang (2013-10-15). "Fusion Protein Linkers: Property, Design and Functionality". Advanced Drug Delivery Reviews. 65 (10): 1357–1369. doi:10.1016/j.addr.2012.09.039. ISSN 0169-409X. PMC 3726540. PMID 23026637.
  19. ^ Hanko, Erik K. R.; Minton, Nigel P.; Malys, Naglis (2019-01-01), "Chapter Nine - Design, cloning and characterization of transcription factor-based inducible gene expression systems", in Shukla, Arun K. (ed.), Chemical and Synthetic Biology Approaches To Understand Cellular Functions - Part A, Methods in Enzymology, vol. 621, Academic Press, pp. 153–169, doi:10.1016/bs.mie.2019.02.018, ISBN 978-0-12-818117-1, PMID 31128776, S2CID 91744525, retrieved 2019-12-16
  20. ^ Kallunki, Tuula; Barisic, Marin; Jäättelä, Marja; Liu, Bin (2019-07-30). "How to Choose the Right Inducible Gene Expression System for Mammalian Studies?". Cells. 8 (8): 796. doi:10.3390/cells8080796. ISSN 2073-4409. PMC 6721553. PMID 31366153.
  21. ^ Cheng, K.C.; Inglese, J. (2012). "A coincidence reporter-gene system for high throughput screening". Nature Methods. 9 (10): 937. doi:10.1038/nmeth.2170. PMC 4970863. PMID 23018994.
  22. ^ Hasson, S.A.; Fogel, A.I.; Wang, C.; MacArthur, R.; Guha, R.; Heman-Ackahc, S.; Martin, S.; Youle, R.J.; Inglese, J. (2015). "Chemogenomic profiling of endogenous PARK2 expression using a genome-edited coincidence reporter". ACS Chem. Biol. 10 (5): 1188–1197. doi:10.1021/cb5010417. PMC 9927027. PMID 25689131. S2CID 20139739.
  23. ^ Jugder, Bat-Erdene; Welch, Jeffrey; Braidy, Nady; Marquis, Christopher P. (2016-07-26). "Construction and use of aCupriavidus necatorH16 soluble hydrogenase promoter (PSH) fusion togfp(green fluorescent protein)". PeerJ. 4: e2269. doi:10.7717/peerj.2269. ISSN 2167-8359. PMC 4974937. PMID 27547572.
  24. ^ Solberg, Nina; Krauss, Stefan (2013). "Luciferase Assay to Study the Activity of a Cloned Promoter DNA Fragment". Gene Regulation. Methods in Molecular Biology. Vol. 977. pp. 65–78. doi:10.1007/978-1-62703-284-1_6. ISBN 978-1-62703-283-4. ISSN 1940-6029. PMID 23436354.
  25. ^ Brückner, Anna; Polge, Cécile; Lentze, Nicolas; Auerbach, Daniel; Schlattner, Uwe (2009-06-18). "Yeast Two-Hybrid, a Powerful Tool for Systems Biology". International Journal of Molecular Sciences. 10 (6): 2763–2788. doi:10.3390/ijms10062763. ISSN 1422-0067. PMC 2705515. PMID 19582228.
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