Editing Reporter gene

{{short description|Technique in molecular biology}}
{{Missing information|the history of the subject|date=January 2012}}

[[Image:Reporter gene.png|thumb|295px|A diagram of a how a reporter gene is used to study a regulatory sequence.]]
Reporter genes are molecular tools widely used in [[molecular biology]], [[genetics]], and [[biotechnology]] to study gene function, expression patterns, and regulatory mechanisms. These genes encode proteins that produce easily detectable signals, such as [[fluorescence]], [[luminescence]], or enzymatic activity, allowing researchers to monitor cellular processes in real-time. Reporter genes are often fused to [[regulatory sequence]]s of genes of interest, enabling scientists to analyze promoter activity, transcriptional regulation, and [[signal transduction]] pathways. Common '''reporter gene''' systems include [[green fluorescent protein]] (GFP), [[Β-Galactosidase|β-galactosidase]] (lacZ), [[luciferase]], and [[chloramphenicol acetyltransferase]] (CAT), each offering distinct advantages depending on the experimental application.<ref name=":5">{{Citation |last1=Debnath |first1=Mousumi |title=Reporter Gene |date=2010 |work=Molecular Diagnostics: Promises and Possibilities |pages=71–84 |editor-last=Debnath |editor-first=Mousumi |url=https://link.springer.com/chapter/10.1007/978-90-481-3261-4_5 |access-date=2025-04-04 |place=Dordrecht |publisher=Springer Netherlands |language=en |doi=10.1007/978-90-481-3261-4_5 |isbn=978-90-481-3261-4 |last2=Prasad |first2=Godavarthi B.K.S. |last3=Bisen |first3=Prakash S. |editor2-last=Prasad |editor2-first=Godavarthi B.K.S. |editor3-last=Bisen |editor3-first=Prakash S.|url-access=subscription }}</ref> Their versatility makes reporter genes invaluable in fields such as drug discovery, [[gene therapy]], and [[synthetic biology]].<ref name=":5" />

==Common Reporter Genes==
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 [[Prokaryote|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.<ref name=":5" />

Commonly used reporter genes that induce visually identifiable characteristics usually involve [[Fluorescence|fluorescent]] and [[Luminescence|luminescent]] proteins. Examples include the gene that encodes jellyfish [[green fluorescent protein]] (GFP), which causes [[Cell (biology)|cells]] that express it to glow green under blue or ultraviolet light, the enzyme [[luciferase]], which catalyzes a reaction with [[luciferin]] to produce light,<ref>{{Cite journal |last1=van Thor |first1=Jasper J. |last2=Gensch |first2=Thomas |last3=Hellingwerf |first3=Klaas J. |last4=Johnson |first4=Louise N. |date=January 2002 |title=Phototransformation of green fluorescent protein with UV and visible light leads to decarboxylation of glutamate 222 |url=https://www.nature.com/articles/nsb739 |journal=Nature Structural Biology |language=en |volume=9 |issue=1 |pages=37–41 |doi=10.1038/nsb739 |pmid=11740505 |issn=1545-9985|url-access=subscription }}</ref> and the red fluorescent protein from the gene [[dsRed]].<ref name=":1">{{Cite journal |last1=Soboleski |first1=Mark R. |last2=Oaks |first2=Jason |last3=Halford |first3=William P. |date=2005 |title=Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells |journal=The FASEB Journal |language=en |volume=19 |issue=3 |pages=440–442 |doi=10.1096/fj.04-3180fje |doi-access=free |issn=1530-6860 |pmc=1242169 |pmid=15640280}}</ref><ref name=":2">{{Cite journal |last=Smale |first=Stephen T. |date=2010-05-01 |title=Luciferase Assay |url=https://cshprotocols.cshlp.org/content/2010/5/pdb.prot5421 |journal=Cold Spring Harbor Protocols |language=en |volume=2010 |issue=5 |pages=pdb.prot5421 |doi=10.1101/pdb.prot5421 |issn=1940-3402 |pmid=20439408|url-access=subscription }}</ref><ref>{{Cite journal |last1=Jach |first1=Guido |last2=Binot |first2=Elke |last3=Frings |first3=Sabine |last4=Luxa |first4=Kerstin |last5=Schell |first5=Jeff |date=2001 |title=Use of red fluorescent protein from Discosoma sp. (dsRED) as a reporter for plant gene expression |url=https://onlinelibrary.wiley.com/doi/10.1046/j.1365-313X.2001.01153.x |journal=The Plant Journal |language=en |volume=28 |issue=4 |pages=483–491 |doi=10.1046/j.1365-313X.2001.01153.x |pmid=11737785 |issn=1365-313X|url-access=subscription }}</ref><ref>{{Cite journal |last1=Zhang |first1=Qixiang |last2=Walawage |first2=Sriema L. |last3=Tricoli |first3=David M. |last4=Dandekar |first4=Abhaya M. |last5=Leslie |first5=Charles A. |date=2015-05-01 |title=A red fluorescent protein (DsRED) from Discosoma sp. as a reporter for gene expression in walnut somatic embryos |url=https://link.springer.com/article/10.1007/s00299-015-1749-1 |journal=Plant Cell Reports |language=en |volume=34 |issue=5 |pages=861–869 |doi=10.1007/s00299-015-1749-1 |bibcode=2015PCelR..34..861Z |issn=1432-203X|url-access=subscription }}</ref><ref name=":6">{{Cite journal |last1=Mikkelsen |first1=Lisbeth |last2=Sarrocco |first2=Sabrina |last3=Lübeck |first3=Mette |last4=Jensen |first4=Dan Funck |date=2003-06-01 |title=Expression of the red fluorescent protein DsRed-Express in filamentous ascomycete fungi |url=https://academic.oup.com/femsle/article-abstract/223/1/135/515928?redirectedFrom=fulltext |journal=FEMS Microbiology Letters |volume=223 |issue=1 |pages=135–139 |doi=10.1016/S0378-1097(03)00355-0 |pmid=12799012 |issn=0378-1097}}</ref> The [[GUS reporter system|GUS]] gene has been commonly used in plants, but luciferase and [[Green fluorescent protein|GFP]] are becoming more common.<ref name=":7">{{Citation |last1=Hull |first1=Gillian A. |title=The β-Glucuronidase (gus) Reporter Gene System |date=1995 |work=Plant Gene Transfer and Expression Protocols |pages=125–141 |editor-last=Jones |editor-first=Heddwyn |url=https://link.springer.com/protocol/10.1385/0-89603-321-X:125 |access-date=2025-04-04 |place=Totowa, NJ |publisher=Springer New York |language=en |doi=10.1385/0-89603-321-x:125 |isbn=978-1-59259-536-5 |last2=Devic |first2=Martine|pmid=8563799 |url-access=subscription }}</ref><ref name=":8">{{Cite journal |last1=Koo |first1=Jachoon |last2=Kim |first2=Yumi |last3=Kim |first3=Jeongsik |last4=Yeom |first4=Miji |last5=Lee |first5=In Chul |last6=Nam |first6=Hong Gil |date=2007-08-01 |title=A GUS/Luciferase Fusion Reporter for Plant Gene Trapping and for Assay of Promoter Activity with Luciferin-Dependent Control of the Reporter Protein Stability |url=https://academic.oup.com/pcp/article-abstract/48/8/1121/1846797?redirectedFrom=fulltext |journal=Plant and Cell Physiology |volume=48 |issue=8 |pages=1121–1131 |doi=10.1093/pcp/pcm081 |pmid=17597079 |issn=0032-0781}}</ref>

A common reporter in bacteria is the [[Escherichia coli|''E. coli'']] [[Lac operon|''lacZ'']] gene, which encodes the protein [[beta-galactosidase]].<ref name=":5" /> 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]].<ref name=":10">{{Cite journal |last=Smale |first=Stephen T. |date=2010-05-01 |title=Chloramphenicol Acetyltransferase Assay |url=https://cshprotocols.cshlp.org/content/2010/5/pdb.prot5422 |journal=Cold Spring Harbor Protocols |language=en |volume=2010 |issue=5 |pages=pdb.prot5422 |doi=10.1101/pdb.prot5422 |issn=1940-3402 |pmid=20439409|url-access=subscription }}</ref>

== History and Discovery ==

=== Discovery of Types of Reporter Genes ===
{| class="wikitable"
|-
!Year
! Gene name
! Gene product
!Significance
! Assay
! Ref.
|-
|1961
| [[lac operon|''lacZ'']]
| [[Beta-galactosidase|β-galactosidase]]
|[[François Jacob]] and [[Jacques Monod]] were awarded a [[Nobel Prize]] in 1965 for their work.
| Enzyme assay, Histochemical (X-gal)
|<ref name=":9" /><ref>{{Cite web |title=Nobel Prize in Physiology or Medicine 1965 |url=https://www.nobelprize.org/prizes/medicine/1965/summary/ |access-date=2025-04-04 |website=NobelPrize.org |language=en-US}}</ref>
|-
|1962
| ''rfp''
|[[Red fluorescent protein]]
|
|[[microscope|Microscopical]], [[Spectrophotometry]]
|<ref name="Nordgren2014">{{Cite journal
 | pmid = 24382456
| year = 2014
| last1 = Nordgren
| first1 = I. K.
| title = A bidirectional fluorescent two-hybrid system for monitoring protein-protein interactions
| journal = Molecular BioSystems
| volume = 10
| issue = 3
| pages = 485–90
| last2 = Tavassoli
| first2 = A
| doi = 10.1039/c3mb70438f
| doi-access = free
| s2cid = 12713372
}}</ref>
|-
|1979
| ''cat''
| [[Chloramphenicol acetyltransferase]]
|Used for measuring gene expression in [[Eukaryote|eukaryotic]] cells.
| [[Chloramphenicol]] acetylation
|<ref name=":10" />
|-
|1985
|''luc''
|[[Luciferase|Luciferase enzyme]]
|Provided a sensitive [[Bioluminescence|bioluminescent]] reporter for gene expression studies.
|[[Bioluminescence]]
|<ref name=":2" />
|-
|1987
|''gus''
|[[Β-Glucuronidase|B-Glucuronidase]]
|Became a widely used reporter gene in plant biology due to its high stability and easy detection in [[Histology|histochemical]] assays. Enabled visualization of gene expression patterns in plant tissues.
|Histochemical, [[Fluorescence spectroscopy|Fluorometric]]
|<ref>{{Cite journal |last1=Jefferson |first1=R. A. |last2=Kavanagh |first2=T. A. |last3=Bevan |first3=M. W. |date=December 1987 |title=GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. |journal=The EMBO Journal |volume=6 |issue=13 |pages=3901–3907 |doi=10.1002/j.1460-2075.1987.tb02730.x |issn=0261-4189 |pmc=553867 |pmid=3327686}}</ref>
|-
|1994
| ''gfp''    
| [[Green fluorescent protein]]
|Enabled real-time visualization of gene expression in live cells.
| [[Fluorescence in the life sciences|Fluorescence microscopy]]
|<ref name=":1" />
|}

==Transformation and Transfection Assays==
Many methods of [[transfection]] and [[Transformation (genetics)|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. 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 [[Promotor (biology)|promoter]] (DNA regions that initiates gene transcription) independent from that of the introduced gene of interest; the reporter gene can be expressed [[Gene expression|constitutively]] ("always on") or [[Gene expression|inducibly]]. This independence is advantageous when the gene of interest is expressed under specific or hard-to-access conditions.<ref name=":5" />

Reporter genes employ diverse mechanisms to visualize or quantify gene activity:

* '''Enzymatic reporters''' (e.g., ''LacZ'') encode enzymes that catalyze reactions yielding a visible product. For example, β-galactosidase (encoded by ''LacZ'') cleaves X-gal to produce a blue color, allowing easy identification of successful gene disruption (white colonies) versus intact genes (blue colonies).<ref name=":6" />
* '''Bioluminescent reporters''' (e.g., luciferase) produce light via chemical reactions, enabling live-cell imaging and promoter studies without external light sources.<ref name=":7" />
* '''Colorimetric reporters''' (e.g., ''CAT'') generate detectable color changes when enzymes react with substrates, measurable via spectrophotometry or TLC.<ref name=":8" />
* '''Selectable markers''' (e.g., ''Neo'') confer antibiotic resistance (e.g., to G418), ensuring only transformed cells survive in selective media.<ref name=":9">{{Cite journal |last=Smale |first=Stephen T. |date=2010-05-01 |title=β-Galactosidase Assay |url=https://cshprotocols.cshlp.org/content/2010/5/pdb.prot5423 |journal=Cold Spring Harbor Protocols |language=en |volume=2010 |issue=5 |pages=pdb.prot5423 |doi=10.1101/pdb.prot5423 |issn=1940-3402 |pmid=20439410|url-access=subscription }}</ref><ref>{{Cite journal |last1=Franke |first1=C A |last2=Rice |first2=C M |last3=Strauss |first3=J H |last4=Hruby |first4=D E |date=August 1985 |title=Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants. |journal=Molecular and Cellular Biology |language=en |volume=5 |issue=8 |pages=1918–1924 |doi=10.1128/MCB.5.8.1918 |issn=0270-7306 |pmc=366908 |pmid=3018537}}</ref>

In the case of selectable-marker reporters such as ''CAT'', the transfected population can be grown on a chloramphenicol-containing substrate. Only cells with the ''CAT'' gene survive, confirming successful transformation.<ref name=":10" />

==Gene expression assays==
Reporter genes can be used to assay for the expression of a gene of interest that is normally difficult to quantitatively assay.<ref name=":5" /> 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.<ref name=":5" /><ref>Archived at [https://ghostarchive.org/varchive/youtube/20211211/PD_6JU3NayE Ghostarchive]{{cbignore}} and the [https://web.archive.org/web/20200614175119/https://www.youtube.com/watch?v=PD_6JU3NayE&gl=US&hl=en Wayback Machine]{{cbignore}}: {{Cite web|url=https://www.youtube.com/watch?v=PD_6JU3NayE|title=Introduction to Reporter Gene Assays|last=Promega Corporation|first=Promega Corporation|date=October 22, 2014|website=YouTube|access-date=March 21, 2020}}{{cbignore}}</ref>

To activate reporter genes, they can be expressed [[Gene expression|constitutively]], where they are directly attached to the gene of interest to create a [[gene fusion]].<ref>{{Cite book|last1=de Jong|first1=Hidde|last2=Geiselmann|first2=Johannes|title=Hybrid Systems Biology |chapter=Fluorescent Reporter Genes and the Analysis of Bacterial Regulatory Networks |date=2015|editor-last=Maler|editor-first=Oded|editor2-last=Halász|editor2-first=Ádám|editor3-last=Dang|editor3-first=Thao|editor4-last=Piazza|editor4-first=Carla|volume=7699|series=Lecture Notes in Computer Science|language=en|publisher=Springer International Publishing|pages=27–50|doi=10.1007/978-3-319-27656-4_2|isbn=978-3-319-27656-4}}</ref> This method is an example of using [[Cis-regulatory element|''cis''-acting]] elements where the two genes are under the same promoter elements and are [[Transcription (genetics)|transcribed]] into a single [[messenger RNA]] molecule. The [[mRNA]] is then [[Translation (biology)|translated]] into protein. It is important that both proteins be able to properly [[protein folding|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.<ref>{{Cite journal|last1=Spector|first1=David L.|last2=Goldman|first2=Robert D.|date=2006-12-01|title=Constructing and Expressing GFP Fusion Proteins|journal=Cold Spring Harbor Protocols|volume=2006|issue=7|pages=pdb.prot4649|doi=10.1101/pdb.prot4649|pmid=22484672}}</ref><ref>{{Cite journal|last1=Chen|first1=Xiaoying|last2=Zaro|first2=Jennica|last3=Shen|first3=Wei-Chiang|date=2013-10-15|title=Fusion Protein Linkers: Property, Design and Functionality|journal=Advanced Drug Delivery Reviews|volume=65|issue=10|pages=1357–1369|doi=10.1016/j.addr.2012.09.039|issn=0169-409X|pmc=3726540|pmid=23026637}}</ref> Reporter genes can also be expressed by [[Gene expression|induction]] during growth. In these cases, [[Trans-acting|''trans''-acting]] elements, such as [[transcription factor]]s are used to express the reporter gene.<ref>{{Citation|last1=Hanko|first1=Erik K. R.|chapter=Chapter Nine - Design, cloning and characterization of transcription factor-based inducible gene expression systems|date=2019-01-01|url=http://www.sciencedirect.com/science/article/pii/S0076687919300370|series=Methods in Enzymology|volume=621|pages=153–169|editor-last=Shukla|editor-first=Arun K.|title=Chemical and Synthetic Biology Approaches To Understand Cellular Functions - Part A|publisher=Academic Press|access-date=2019-12-16|last2=Minton|first2=Nigel P.|last3=Malys|first3=Naglis|doi=10.1016/bs.mie.2019.02.018|pmid=31128776|isbn=978-0-12-818117-1 |s2cid=91744525|url-access=subscription}}</ref><ref>{{Cite journal|last1=Kallunki|first1=Tuula|last2=Barisic|first2=Marin|last3=Jäättelä|first3=Marja|last4=Liu|first4=Bin|date=2019-07-30|title=How to Choose the Right Inducible Gene Expression System for Mammalian Studies?|journal=Cells|volume=8|issue=8|page=796|doi=10.3390/cells8080796|issn=2073-4409|pmc=6721553|pmid=31366153|doi-access=free}}</ref>

Reporter gene assay have been increasingly used in [[High-throughput screening|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.<ref>{{cite journal | title = A coincidence reporter-gene system for high throughput screening. |author1 = Cheng, K.C.| author2 = Inglese, J. | journal = Nature Methods|date = 2012 |volume = 9|issue = 10|page = 937|pmid = 23018994 | doi = 10.1038/nmeth.2170 | pmc=4970863}}</ref><ref>{{cite journal | title = Chemogenomic profiling of endogenous PARK2 expression using a genome-edited coincidence reporter. |author1 = Hasson, S.A.|author2 = Fogel, A.I.|author3 = Wang, C.| author4 = MacArthur, R.|author5 = Guha, R.|author6 = Heman-Ackahc, S.|author7 = Martin, S.| author8 = Youle, R.J.|author9 = Inglese, J. | journal = ACS Chem. Biol.|date = 2015 |volume = 10|issue = 5|pages = 1188–1197|pmid = 25689131| doi = 10.1021/cb5010417| pmc=9927027 | s2cid=20139739 }}</ref>

==Promoter assays==
Reporter genes can be used to assay for the activity of a particular promoter in a cell or organism.<ref>{{Cite journal|last1=Jugder|first1=Bat-Erdene|last2=Welch|first2=Jeffrey|last3=Braidy|first3=Nady|last4=Marquis|first4=Christopher P.|date=2016-07-26|title=Construction and use of aCupriavidus necatorH16 soluble hydrogenase promoter (PSH) fusion togfp(green fluorescent protein)|journal=PeerJ|language=en|volume=4|pages=e2269|doi=10.7717/peerj.2269|issn=2167-8359|pmc=4974937|pmid=27547572 |doi-access=free }}</ref> 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.<ref>{{Cite book|last1=Solberg|first1=Nina|last2=Krauss|first2=Stefan|chapter=Luciferase Assay to Study the Activity of a Cloned Promoter DNA Fragment |date=2013|series=Methods in Molecular Biology|volume=977|pages=65–78|doi=10.1007/978-1-62703-284-1_6|issn=1940-6029|pmid=23436354|title=Gene Regulation|isbn=978-1-62703-283-4}}</ref>

== Limitations and Advancements ==
While reporter gene technology has become an essential component of molecular biology, its application still has limitations. One primary concern is the influence of genomic context on reporter expression. Reporter genes integrated into the [[genome]] can be subject to [[position-effect variegation]], where the surrounding chromatin structure influences transcriptional activity. This can lead to inconsistent expression and complicate the interpretation of results, especially in stable [[Cell lineage|cell lines]] and transgenic organisms.<ref>{{Cite journal |last1=Kulaeva |first1=Olga I. |last2=Gaykalova |first2=Daria A. |last3=Studitsky |first3=Vasily M. |date=2007-05-01 |title=Transcription through chromatin by RNA polymerase II: Histone displacement and exchange |journal=Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis |series=Chromatin: Repair, Remodeling and Regulation |volume=618 |issue=1 |pages=116–129 |doi=10.1016/j.mrfmmm.2006.05.040 |issn=0027-5107 |pmc=1924643 |pmid=17313961|bibcode=2007MRFMM.618..116K }}</ref> Additionally, reporter expression may not always accurately reflect the activity of the endogenous gene of interest due to differences in [[post-transcriptional regulation]], [[Messenger RNA|mRNA]] stability, or [[translational efficiency]].<ref>{{Cite journal |last1=Kitsis |first1=R. N. |last2=Leinwand |first2=L. A. |date=1992 |title=Discordance between gene regulation in vitro and in vivo |journal=Gene Expression |volume=2 |issue=4 |pages=313–318 |issn=1052-2166 |pmc=6057365 |pmid=1472867}}</ref>

Another common limitation is the cellular burden that reporter expression may impose. High levels of reporter protein production, such as fluorescent proteins or luciferases, can divert cellular resources, potentially impacting normal metabolism or physiology. This is particularly problematic in sensitive systems like [[stem cell]]s or [[primary cell culture]]s, where even subtle changes in [[metabolism]] can influence cell behavior.<ref name=":0">{{Cite journal |last1=Yang |first1=Jinfeng |last2=Wang |first2=Nan |last3=Chen |first3=Deying |last4=Yu |first4=Jiong |last5=Pan |first5=Qiaoling |last6=Wang |first6=Dan |last7=Liu |first7=Jingqi |last8=Shi |first8=Xiaowei |last9=Dong |first9=Xiaotian |last10=Cao |first10=Hongcui |last11=Li |first11=Liang |last12=Li |first12=Lanjuan |date=2017 |title=The Impact of GFP Reporter Gene Transduction and Expression on Metabolomics of Placental Mesenchymal Stem Cells Determined by UHPLC-Q/TOF-MS |journal=Stem Cells International |language=en |volume=2017 |issue=1 |page=3167985 |doi=10.1155/2017/3167985 |doi-access=free |issn=1687-9678 |pmc=5694582 |pmid=29230249}}</ref> Additionally, some reporter systems, like luciferase assays, require the addition of exogenous substrates (e.g., luciferin), adds complexity and may reduce reproducibility, particularly in live animal models where substrate availability can vary.<ref name=":0" />

To address these challenges, several innovations have improved the reliability and flexibility of reporter gene technologies. One advancement involves the use of the [[2A peptides|2A peptide]], which allows the co-expression of multiple proteins from a single transcript without requiring a direct fusion. This approach enables the simultaneous expression of a gene of interest and a reporter while preserving the function of both.<ref>{{Cite journal |last1=Szymczak |first1=Andrea L. |last2=Workman |first2=Creg J. |last3=Wang |first3=Yao |last4=Vignali |first4=Kate M. |last5=Dilioglou |first5=Smaroula |last6=Vanin |first6=Elio F. |last7=Vignali |first7=Dario A. A. |date=May 2004 |title=Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide–based retroviral vector |url=https://www.nature.com/articles/nbt957 |journal=Nature Biotechnology |language=en |volume=22 |issue=5 |pages=589–594 |doi=10.1038/nbt957 |pmid=15064769 |issn=1546-1696|url-access=subscription }}</ref> Additionally, split-reporter systems, which produce a functional signal only when two proteins of interest interact, have become widely used in studies of protein–protein interactions due to their low background activity and high specificity.<ref name=":11">{{Cite journal |last=Kerppola |first=Tom K. |date=2008 |title=Bimolecular Fluorescence Complementation (BiFC) Analysis as a Probe of Protein Interactions in Living Cells |journal=Annual Review of Biophysics |language=en |volume=37  |pages=465–487 |doi=10.1146/annurev.biophys.37.032807.125842 |issn=1936-122X |pmc=2829326 |pmid=18573091}}</ref>

== Current Applications ==
'''''Common Research Applications'''''

The most commonly used application used for Reporter genes has been for the identification of cis and trans acting elements. Through fusion to the promoter region of possible trans-cis acting elements, the change in fluorescence is measured and allows for tracking into [https://www.promega.com/resources/guides/cell-biology/bioluminescent-reporters/ transcriptional activity].<ref name=":12">{{Cite web |title=Bioluminescent Reporters {{!}} Reporter Gene Applications {{!}} An Introduction to Reporter Genes |url=https://www.promega.com/resources/guides/cell-biology/bioluminescent-reporters/ |access-date=2025-04-04 |website=www.promega.com}}</ref> This provides useful information into understanding the pathways these elements are involved in and its regulatory uses for [https://www.promega.com/resources/guides/cell-biology/bioluminescent-reporters/ cell development and growth].<ref name=":12" /> [[doi:10.1111/j.1365-2567.2010.03372.x|Immune responses]] are also a commonly used application of reporter genes and have benefited greatly through their use. They have allowed for further understanding in cell proliferation and differentiation into [[doi:10.1111/j.1365-2567.2010.03372.x|B-cells and T-cells]] during immune responses and have contributed to understanding activation through tracking cytokine signaling pathways.<ref>{{Cite journal |last1=Croxford |first1=Andrew L. |last2=Buch |first2=Thorsten |date=2011 |title=Cytokine reporter mice in immunological research: perspectives and lessons learned |journal=Immunology |language=en |volume=132 |issue=1 |pages=1–8 |doi=10.1111/j.1365-2567.2010.03372.x |issn=1365-2567 |pmc=3015069 |pmid=21070235}}</ref>

The development of [https://www.biocompare.com/26140-Reporter-Cell-Lines/#:~:text=Reporter%20cell%20lines%20are%20stable,consideration%20when%20selecting%20your%20product. reporter cell lines] have also emerged with the discovery and use of reporter genes.<ref name=":12" /> The cell lines are labelled with reporter genes to allow for fluorescent detection to help with identification into proteins used in cellular pathways and identification into [https://www.biocompare.com/26140-Reporter-Cell-Lines/#:~:text=Reporter%20cell%20lines%20are%20stable,consideration%20when%20selecting%20your%20product. protein localization].<ref name=":11" /> This has allowed for a simple way to study protein progression that doesn't permit further experimentation for introduction and fusion of a reporter gene as the reporter gene is already present in the cell line.

'''''Common Medical Applications'''''

Tracking expression has allowed for multiple investigations into the progression of [https://pmc.ncbi.nlm.nih.gov/articles/PMC5996353/ diseased cells].<ref name=":3">{{Cite journal |last=Li |first=Mengting |last2=Wang |first2=Yichun |last3=Liu |first3=Mei |last4=Lan |first4=Xiaoli |date=2018-04-18 |title=Multimodality reporter gene imaging: Construction strategies and application |url=https://www.thno.org/v08p2954.htm |journal=Theranostics |language=en |volume=8 |issue=11 |pages=2954–2973 |doi=10.7150/thno.24108 |issn=1838-7640 |pmc=5996353 |pmid=29896296}}</ref> Reporter genes have shown to provide critical insight into genes upregulated in cancer regulatory pathways as well as the identification into [https://pmc.ncbi.nlm.nih.gov/articles/PMC5996353/ oncogenes and tumor suppressor genes]. These have been used for further research into the development of therapeutics to stop further disease progression and metastasis.<ref name=":3" /> Gene therapy has also been tracked through the use of reporter genes. This allows for the monitoring of [https://pmc.ncbi.nlm.nih.gov/articles/PMC8517204/ gene therapy vectors] to see if they are achieving intended results as well as to monitor patient safety for short and long term periods.<ref>{{Cite journal |last1=Taghian |first1=Toloo |last2=Batista |first2=Ana Rita |last3=Kamper |first3=Sarah |last4=Caldwell |first4=Michael |last5=Lilley |first5=Laura |last6=Li |first6=Hao |last7=Rodriguez |first7=Paola |last8=Mesa |first8=Katerina |last9=Zheng |first9=Shaokuan |last10=King |first10=Robert M. |last11=Gounis |first11=Matthew J. |last12=Todeasa |first12=Sophia |last13=Maguire |first13=Anne |last14=Martin |first14=Douglas R. |last15=Sena-Esteves |first15=Miguel |date=2021-12-10 |title=Real-time MR tracking of AAV gene therapy with βgal-responsive MR probe in a murine model of GM1-gangliosidosis |journal=Molecular Therapy Methods & Clinical Development |language=English |volume=23 |pages=128–134 |doi=10.1016/j.omtm.2021.08.003 |issn=2329-0501 |pmc=8517204 |pmid=34703836}}</ref> Therapeutics developed have benefited from the use of reporter genres such as a dual-reporter system developed for [[CRISPR/Cas9-mediated genome editing|CRISPR/Cas9]] models to monitor progression and success and benefits of being gene editing tools.<ref>{{Cite journal |last1=Liu |first1=Wen-Hsin |last2=Völse |first2=Kerstin |last3=Senft |first3=Daniela |last4=Jeremias |first4=Irmela |date=2021-06-16 |title=A reporter system for enriching CRISPR/Cas9 knockout cells in technically challenging settings like patient models |journal=Scientific Reports |language=en |volume=11 |issue=1 |page=12649 |doi=10.1038/s41598-021-91760-9 |issn=2045-2322 |pmc=8209181 |pmid=34135367}}</ref>

==Further Applications==
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]]''.The yeast [[Two-hybrid screening|two-hybrid (Y2H) system]], developed in the late 1980s and early 1990s, was an immense advancement in the use of reporter genes to study [[Protein–protein interaction|protein-protein interactions]] ''[[in vivo]]''.<ref>{{Cite journal |last1=Brückner |first1=Anna |last2=Polge |first2=Cécile |last3=Lentze |first3=Nicolas |last4=Auerbach |first4=Daniel |last5=Schlattner |first5=Uwe |date=2009-06-18 |title=Yeast Two-Hybrid, a Powerful Tool for Systems Biology |journal=International Journal of Molecular Sciences |language=en |volume=10 |issue=6 |pages=2763–2788 |doi=10.3390/ijms10062763 |doi-access=free |issn=1422-0067 |pmc=2705515 |pmid=19582228}}</ref>  This technique takes advantage of transcription factors' modular nature, which often consists of separate [[DNA-binding domain|DNA-binding]] and [[Transactivation domain|activation domains]]. By genetically fusing two proteins of interest to these domains, researchers can detect physical interactions between them through the activation of a downstream reporter gene. Due to the simple genetic nature of the Y2H system, this technique significantly increased the accessibility of [[Protein–protein interaction|protein-protein interaction]] studies without the requirement of [[protein purification]] or complex biochemical [[assay]]s. Experimental Y2H data have played a pivotal role in building large-scale synthetic human [[interactome]]s and in dissecting mechanisms in human disease.<ref>{{Cite journal |last1=Gandhi |first1=T. K. B. |last2=Zhong |first2=Jun |last3=Mathivanan |first3=Suresh |last4=Karthick |first4=L. |last5=Chandrika |first5=K. N. |last6=Mohan |first6=S. Sujatha |last7=Sharma |first7=Salil |last8=Pinkert |first8=Stefan |last9=Nagaraju |first9=Shilpa |last10=Periaswamy |first10=Balamurugan |last11=Mishra |first11=Goparani |last12=Nandakumar |first12=Kannabiran |last13=Shen |first13=Beiyi |last14=Deshpande |first14=Nandan |last15=Nayak |first15=Rashmi |date=March 2006 |title=Analysis of the human protein interactome and comparison with yeast, worm and fly interaction datasets |url=https://www.nature.com/articles/ng1747 |journal=Nature Genetics |language=en |volume=38 |issue=3 |pages=285–293 |doi=10.1038/ng1747 |pmid=16501559 |issn=1546-1718|url-access=subscription }}</ref><ref>{{Cite journal |last1=Lim |first1=Janghoo |last2=Hao |first2=Tong |last3=Shaw |first3=Chad |last4=Patel |first4=Akash J. |last5=Szabó |first5=Gábor |last6=Rual |first6=Jean-François |last7=Fisk |first7=C. Joseph |last8=Li |first8=Ning |last9=Smolyar |first9=Alex |last10=Hill |first10=David E. |last11=Barabási |first11=Albert-László |last12=Vidal |first12=Marc |last13=Zoghbi |first13=Huda Y. |date=2006-05-19 |title=A Protein–Protein Interaction Network for Human Inherited Ataxias and Disorders of Purkinje Cell Degeneration |url=https://linkinghub.elsevier.com/retrieve/pii/S0092867406004399 |journal=Cell |language=English |volume=125 |issue=4 |pages=801–814 |doi=10.1016/j.cell.2006.03.032 |issn=0092-8674 |pmid=16713569}}</ref>

However, there are still some limitations. Y2H sometimes detects interactions that don't occur naturally or fails to detect weak or transient interactions, and because it occurs in an artificial setting, Y2H is missing key factors like [[post-translational modification]]s and [[cellular compartment|compartmentalization]]. For example, Y2H has been shown to generate false positives due to indirect interactions mediated by host proteins, as demonstrated in studies of cyanobacterial PipX interactions where the self-interaction of PipX was found to be dependent on PII homologues from the host organism rather than a direct interaction.<ref>{{Cite journal |last1=Feuer |first1=Erez |last2=Zimran |first2=Gil |last3=Shpilman |first3=Michal |last4=Mosquna |first4=Assaf |date=2022-08-19 |title=A Modified Yeast Two-Hybrid Platform Enables Dynamic Control of Expression Intensities to Unmask Properties of Protein–Protein Interactions |journal=ACS Synthetic Biology |volume=11 |issue=8 |pages=2589–2598 |doi=10.1021/acssynbio.2c00192 |pmc=9442787 |pmid=35895499}}</ref>

[[Massive parallel sequencing|Massively parallel reporter assays]] (MPRAs) and [[machine learning]] are newer ways we study gene regulation which utilize reporter genes. One major use is in synthetic biology and gene therapy, where researchers can design better regulatory elements to control gene expression.<ref name=":4">{{Cite journal |last1=Fleur |first1=Alyssa La |last2=Shi |first2=Yongsheng |last3=Seelig |first3=Georg |date=2024-09-01 |title=Decoding biology with massively parallel reporter assays and machine learning |url=https://genesdev.cshlp.org/content/38/17-20/843 |journal=Genes & Development |language=en |volume=38 |issue=17–20 |pages=843–865 |doi=10.1101/gad.351800.124 |issn=0890-9369 |pmc=11535156 |pmid=39362779}}</ref> For example, deep learning models trained on MPRA data have been used to optimize 5' untranslated regions (UTRs) for mRNA translation, enabling tailored designs that enhance gene-editing efficiency in the therapeutic context. This could make mRNA-based treatments more effective, as MPRAs also help identify how genetic variants affect gene expression, which is used in precision medicine and developing personalized treatments.<ref name=":4" />

Machine learning models trained on MPRA data can predict how different sequences impact gene activity, making it easier to design reporter genes that respond in specific ways. Combining MPRAs with next-gen sequencing also makes reporter gene experiments faster and more scalable. These advances could even improve mRNA-based vaccines and therapeutics by optimizing [[untranslated region]]s (UTRs) to boost stability and translation. For instance, modular MPRAs have uncovered context-specific regulatory sequences linked to type 2 diabetes, revealing enhancer-promoter interactions dependent on cell-specific transcription factors like HNF1.<ref>{{Citation |last1=Tovar |first1=Adelaide |title=Using a modular massively parallel reporter assay to discover context-specific regulatory grammars in type 2 diabetes |date=2023-10-10 |url=https://www.biorxiv.org/content/10.1101/2023.10.08.561391v1 |access-date=2025-04-04 |publisher=bioRxiv |language=en |doi=10.1101/2023.10.08.561391 |pmc=10592691 |pmid=37873175 |last2=Kyono |first2=Yasuhiro |last3=Nishino |first3=Kirsten |last4=Bose |first4=Maya |last5=Varshney |first5=Arushi |last6=Parker |first6=Stephen C. J. |last7=Kitzman |first7=Jacob O.|journal=bioRxiv: The Preprint Server for Biology }}</ref> Similarly, MPRA screens of cardiac enhancer variants have pinpointed functional noncoding sequences influencing QT interval variability, directly linking genetic variation to disease-associated gene dysregulation.<ref>{{Citation |last1=Lee |first1=Dongwon |title=Massively parallel reporter assays identify functional enhancer variants at QT interval GWAS loci |date=2025-03-12 |url=https://www.biorxiv.org/content/10.1101/2025.03.11.642686v1 |access-date=2025-04-04 |publisher=bioRxiv |language=en |doi=10.1101/2025.03.11.642686 |pmc=11952420 |pmid=40161821 |last2=Gunamalai |first2=Lavanya |last3=Kannan |first3=Jeerthi |last4=Vickery |first4=Kyla |last5=Yaacov |first5=Or |last6=Onuchic-Whitford |first6=Ana C. |last7=Chakravarti |first7=Aravinda |last8=Kapoor |first8=Ashish|journal=bioRxiv: The Preprint Server for Biology }}</ref>

==See also==
* [[Bioluminescence imaging]]
* [[Gene expression]]
* [[Gene knock-in]]
* [[Gene regulatory network]]
* [[GUS reporter system]]
* [[Molecular cloning]]
* [[Promoter (genetics)]]
* [[Selectable marker]]
* [[Synthetic biology]]
* [[Transcription factor]]
* [[Transfection]]

==References==
{{reflist}}

==External links==
*[http://www.reportergene.com Research highlights] and updated information on reporter genes.
*[http://www.cshprotocols.org/cgi/content/full/2007/8/pdb.prot4725 Staining Whole Mouse Embryos for β-Galactosidase (lacZ) Activity]

[[Category:Biochemistry detection methods]]
[[Category:Genetics techniques]]
[[Category:Molecular biology]]

[[es:Gen reportero]]