Editing Zebrafish (section)

===Genetics===

====Background genetics====
[[Inbred strains]] and traditional outbred stocks have not been developed for laboratory zebrafish, and the genetic variability of wild-type lines among institutions may contribute to the [[replication crisis]] in biomedical research.<ref>{{cite journal |vauthors=Crim MJ, Lawrence C |title=A fish is not a mouse: understanding differences in background genetics is critical for reproducibility |journal=Lab Animal |volume=50 |issue=1 |pages=19–25 |date=January 2021 |pmid=33268901 |doi=10.1038/s41684-020-00683-x |issn=0093-7355 |s2cid=227259359}}</ref> Genetic differences in wild-type lines among populations maintained at different research institutions have been demonstrated using both [[Single-nucleotide polymorphism]]s<ref>{{cite journal |vauthors=Whiteley AR, Bhat A, Martins EP, Mayden RL, Arunachalam M, Uusi-Heikkilä S, Ahmed AT, Shrestha J, Clark M, Stemple D, Bernatchez L |display-authors=6 |title=Population genomics of wild and laboratory zebrafish (''Danio rerio'') |journal=Molecular Ecology |volume=20 |issue=20 |pages=4259–4276 |date=October 2011 |pmid=21923777 |pmc=3627301 |doi=10.1111/j.1365-294X.2011.05272.x |bibcode=2011MolEc..20.4259W}}</ref> and [[microsatellite]] analysis.<ref>{{cite journal |vauthors=Coe TS, Hamilton PB, Griffiths AM, Hodgson DJ, Wahab MA, Tyler CR |title=Genetic variation in strains of zebrafish (''Danio rerio'') and the implications for ecotoxicology studies |journal=Ecotoxicology |volume=18 |issue=1 |pages=144–150 |date=January 2009 |pmid=18795247 |doi=10.1007/s10646-008-0267-0 |bibcode=2009Ecotx..18..144C |s2cid=18370151}}</ref>

====Gene expression====
Due to their fast and short life cycles and relatively large clutch sizes, ''D. rerio'' or zebrafish are a useful model for genetic studies. A common [[reverse genetics]] technique is to [[gene knockdown|reduce gene expression]] or modify [[Splicing (genetics)|splicing]] using [[Morpholino]] [[antisense]] technology. Morpholino [[oligonucleotide]]s (MO) are stable, synthetic [[macromolecule]]s that contain the same [[nucleoside|bases]] as DNA or RNA; by binding to complementary RNA sequences, they can reduce the [[gene expression|expression]] of specific genes or block other processes from occurring on RNA. MO can be injected into one cell of an embryo after the 32-cell stage, reducing gene expression in only cells descended from that cell. However, cells in the early embryo (less than 32 cells) are permeable to large molecules,<ref name=blast2>{{cite journal |vauthors=Kimmel CB, Law RD |title=Cell lineage of zebrafish blastomeres. I. Cleavage pattern and cytoplasmic bridges between cells |journal=Developmental Biology |volume=108 |issue=1 |pages=78–85 |date=March 1985 |pmid=3972182 |doi=10.1016/0012-1606(85)90010-7}}</ref><ref name=blast4>{{cite journal |vauthors=Kimmel CB, Law RD |title=Cell lineage of zebrafish blastomeres. III. Clonal analyses of the blastula and gastrula stages |journal=Developmental Biology |volume=108 |issue=1 |pages=94–101 |date=March 1985 |pmid=3972184 |doi=10.1016/0012-1606(85)90012-0}}</ref> allowing diffusion between cells. Guidelines for using Morpholinos in zebrafish describe appropriate control strategies.<ref>{{cite journal |vauthors=Stainier DY, Raz E, Lawson ND, Ekker SC, Burdine RD, Eisen JS, Ingham PW, Schulte-Merker S, Yelon D, Weinstein BM, Mullins MC, Wilson SW, Ramakrishnan L, Amacher SL, Neuhauss SC, Meng A, Mochizuki N, Panula P, Moens CB |display-authors=6 |title=Guidelines for morpholino use in zebrafish |journal=PLOS Genetics |volume=13 |issue=10 |pages=e1007000 |date=October 2017 |pmid=29049395 |pmc=5648102 |doi=10.1371/journal.pgen.1007000 |doi-access=free}}</ref> Morpholinos are commonly [[microinjection|microinjected]] in 500pL directly into 1–2 cell stage zebrafish embryos. The morpholino is able to integrate into most cells of the embryo.<ref>{{cite journal |vauthors=Rosen JN, Sweeney MF, Mably JD |title=Microinjection of zebrafish embryos to analyze gene function |journal=Journal of Visualized Experiments |issue=25 |date=March 2009 |pmid=19274045 |pmc=2762901 |doi=10.3791/1115}}</ref>

A known problem with gene knockdowns is that, because the genome underwent a [[genome#Genome evolution|duplication]] after the divergence of [[ray-finned fish]]es and [[lobe-finned fish]]es, it is not always easy to silence the activity of one of the two gene [[paralog]]s reliably due to [[Complementation (genetics)|complementation]] by the other paralog.<ref>{{cite journal |vauthors=Leong IU, Lan CC, Skinner JR, Shelling AN, Love DR |title=In vivo testing of microRNA-mediated gene knockdown in zebrafish |journal=Journal of Biomedicine & Biotechnology |volume=2012 |page=350352 |year=2012 |pmid=22500088 |pmc=3303736 |doi=10.1155/2012/350352 |publisher=Hindawi |doi-access=free}}</ref> Despite the complications of the zebrafish [[genome]], a number of commercially available global platforms exist for analysis of both gene expression by [[expression profiling|microarrays]] and promoter regulation using [[ChIP-on-chip]].<ref>{{cite journal |vauthors=Tan PK, Downey TJ, Spitznagel EL, Xu P, Fu D, Dimitrov DS, Lempicki RA, Raaka BM, Cam MC |display-authors=6 |title=Evaluation of gene expression measurements from commercial microarray platforms |journal=Nucleic Acids Research |volume=31 |issue=19 |pages=5676–5684 |date=October 2003 |pmid=14500831 |pmc=206463 |doi=10.1093/nar/gkg763}}</ref>

====Genome sequencing====
The [[Wellcome Trust Sanger Institute]] started the zebrafish genome sequencing project in 2001, and the full genome sequence of the Tuebingen reference strain is publicly available at the [[National Center for Biotechnology Information]] (NCBI)'s [https://www.ncbi.nlm.nih.gov/genome?term=danio%20rerio Zebrafish Genome Page]. The zebrafish reference genome sequence is annotated as part of the [[Ensembl]] [http://www.ensembl.org/Danio_rerio/Info/Index project], and is maintained by the [[Genome Reference Consortium]].<ref>{{cite web |title=Genome Reference Consortium |url=https://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/ |publisher=GRC |access-date=October 23, 2012 |archive-date=October 5, 2016 |archive-url=https://web.archive.org/web/20161005062544/https://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/ |url-status=live}}</ref>

In 2009, researchers at the [[Institute of Genomics and Integrative Biology]] in Delhi, India, announced the sequencing of the genome of a wild zebrafish strain, containing an estimated 1.7 billion genetic letters.<ref>[http://www.indianexpress.com/news/Decoding-the-Genome-Mystery/485122 "Decoding the Genome Mystery"] {{Webarchive|url=https://web.archive.org/web/20090715190646/http://www.indianexpress.com/news/Decoding-the-Genome-Mystery/485122 |date=2009-07-15 }}. ''[[Indian Express]]''. July 5, 2009. Retrieved February 5, 2013.</ref><ref>[http://genome.igib.res.in/ FishMap Zv8] {{Webarchive|url=https://web.archive.org/web/20180719084933/http://genome.igib.res.in/ |date=2018-07-19 }}. [[Institute of Genomics and Integrative Biology]] (IGIB). Retrieved June 7, 2012.</ref> The genome of the wild zebrafish was sequenced at 39-fold coverage. Comparative analysis with the zebrafish reference genome revealed over 5 million single nucleotide variations and over 1.6 million insertion deletion variations. The zebrafish reference genome sequence of 1.4GB and over 26,000 protein coding genes was published by Kerstin Howe ''et al.'' in 2013.<ref name=howe2013>{{cite journal |vauthors=Howe K, Clark MD, Torroja CF, Torrance J, Berthelot C, Muffato M, Collins JE, Humphray S, McLaren K, Matthews L, McLaren S, Sealy I, Caccamo M, Churcher C, Scott C, Barrett JC, Koch R, Rauch GJ, White S, Chow W, Kilian B, Quintais LT, Guerra-Assunção JA, Zhou Y, Gu Y, Yen J, Vogel JH, Eyre T, Redmond S, Banerjee R, Chi J, Fu B, Langley E, Maguire SF, Laird GK, Lloyd D, Kenyon E, Donaldson S, Sehra H, Almeida-King J, Loveland J, Trevanion S, Jones M, Quail M, Willey D, Hunt A, Burton J, Sims S, McLay K, Plumb B, Davis J, Clee C, Oliver K, Clark R, Riddle C, Elliot D, Eliott D, Threadgold G, Harden G, Ware D, Begum S, Mortimore B, Mortimer B, Kerry G, Heath P, Phillimore B, Tracey A, Corby N, Dunn M, Johnson C, Wood J, Clark S, Pelan S, Griffiths G, Smith M, Glithero R, Howden P, Barker N, Lloyd C, Stevens C, Harley J, Holt K, Panagiotidis G, Lovell J, Beasley H, Henderson C, Gordon D, Auger K, Wright D, Collins J, Raisen C, Dyer L, Leung K, Robertson L, Ambridge K, Leongamornlert D, McGuire S, Gilderthorp R, Griffiths C, Manthravadi D, Nichol S, Barker G, Whitehead S, Kay M, Brown J, Murnane C, Gray E, Humphries M, Sycamore N, Barker D, Saunders D, Wallis J, Babbage A, Hammond S, Mashreghi-Mohammadi M, Barr L, Martin S, Wray P, Ellington A, Matthews N, Ellwood M, Woodmansey R, Clark G, Cooper J, Cooper J, Tromans A, Grafham D, Skuce C, Pandian R, Andrews R, Harrison E, Kimberley A, Garnett J, Fosker N, Hall R, Garner P, Kelly D, Bird C, Palmer S, Gehring I, Berger A, Dooley CM, Ersan-Ürün Z, Eser C, Geiger H, Geisler M, Karotki L, Kirn A, Konantz J, Konantz M, Oberländer M, Rudolph-Geiger S, Teucke M, Lanz C, Raddatz G, Osoegawa K, Zhu B, Rapp A, Widaa S, Langford C, Yang F, Schuster SC, Carter NP, Harrow J, Ning Z, Herrero J, Searle SM, Enright A, Geisler R, Plasterk RH, Lee C, Westerfield M, de Jong PJ, Zon LI, Postlethwait JH, Nüsslein-Volhard C, Hubbard TJ, Roest Crollius H, Rogers J, Stemple DL |display-authors=6 |title=The zebrafish reference genome sequence and its relationship to the human genome |journal=[[Nature (journal)|Nature]] |volume=496 |issue=7446 |pages=498–503 |date=April 2013 |pmid=23594743 |pmc=3703927 |doi=10.1038/nature12111 |bibcode=2013Natur.496..498H}}</ref>

====Mitochondrial DNA====
In October 2001, researchers from the [[University of Oklahoma]] published ''D. rerio's'' complete [[mitochondrial DNA]] sequence.<ref name=2001Journal>{{cite journal |vauthors=Broughton RE, Milam JE, Roe BA |title=The complete sequence of the zebrafish (''Danio rerio'') mitochondrial genome and evolutionary patterns in vertebrate mitochondrial DNA |journal=Genome Research |volume=11 |issue=11 |pages=1958–1967 |date=November 2001 |pmid=11691861 |pmc=311132 |doi=10.1101/gr.156801}}</ref> Its length is 16,596 base pairs. This is within 100 base pairs of other related species of fish, and it is notably only 18 pairs longer than the goldfish (''Carassius auratus'') and 21 longer than the [[carp]] (''Cyprinus carpio''). Its gene order and content are identical to the common [[vertebrate]] form of mitochondrial DNA. It contains 13 [[protein]]-coding genes and a noncoding control region containing the [[origin of replication]] for the heavy strand. In between a grouping of five [[tRNA]] genes, a sequence resembling vertebrate origin of light strand replication is found. It is difficult to draw evolutionary conclusions because it is difficult to determine whether base pair changes have adaptive significance via comparisons with other vertebrates' [[nucleotide]] sequences.<ref name=2001Journal/>

====Developmental genetics====
[[T-box]]es and [[homeobox]]es are vital in ''Danio'' similarly to other vertebrates.<ref name="Schier-Talbot-2005">{{cite journal |last1=Schier |first1=Alexander F. |last2=Talbot |first2=William S. |title=Molecular Genetics of Axis Formation in Zebrafish |journal=[[Annual Review of Genetics]] |publisher=[[Annual Reviews (publisher)|Annual Reviews]] |volume=39 |issue=1 |date=2005-12-01 |issn=0066-4197 |doi=10.1146/annurev.genet.37.110801.143752 |pages=561–613 |pmid=16285872}}</ref><ref name="Naiche-et-al-2005">{{cite journal |last1=Naiche |first1=L.A. |last2=Harrelson |first2=Zachary |last3=Kelly |first3=Robert G. |last4=Papaioannou |first4=Virginia E. |title=T-Box Genes in Vertebrate Development |journal=[[Annual Review of Genetics]] |publisher=[[Annual Reviews (publisher)|Annual Reviews]] |volume=39 |issue=1 |date=2005-12-01 |issn=0066-4197 |doi=10.1146/annurev.genet.39.073003.105925 |pages=219–239 |pmid=16285859}}</ref> The Bruce et al. team are known for this area, and in Bruce et al. 2003 & Bruce et al. 2005 uncover the role of two of these elements in [[oocyte]]s of this species.<ref name="Schier-Talbot-2005" /><ref name="Naiche-et-al-2005" /> By interfering via a [[dominance (genetics)|dominant]] nonfunctional [[allele]] and a [[morpholino]] they find the T-box transcription activator [[Eomesodermin]] and its target ''[[mtx2]]'' – a [[transcription factor]] – are vital to [[epiboly]].<ref name="Schier-Talbot-2005" /><ref name="Naiche-et-al-2005" /> (In Bruce et al. 2003 they failed to support the possibility that Eomesodermin behaves like [[Vegt (development)|Vegt]].<ref name="Schier-Talbot-2005" /> Neither they nor anyone else has been able to locate any [[mutation]] which – in the mother – will prevent initiation of the [[mesoderm]] or [[endoderm]] development processes in this species.)<ref name="Schier-Talbot-2005" />

====Pigmentation genes====
In 1999, the ''nacre'' mutation was identified in the zebrafish ortholog of the mammalian ''MITF'' transcription factor.<ref>{{cite journal |vauthors=Lister JA, Robertson CP, Lepage T, Johnson SL, Raible DW |title=nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate |journal=Development |volume=126 |issue=17 |pages=3757–3767 |date=September 1999 |pmid=10433906 |doi=10.1242/dev.126.17.3757}}</ref> Mutations in human ''[[MITF]]'' result in eye defects and loss of pigment, a type of [[Waardenburg Syndrome]]. In December 2005, a study of the ''golden'' strain identified the gene responsible for its unusual pigmentation as [[SLC24A5]], a [[solute]] carrier that appeared to be required for [[melanin]] production, and confirmed its function with a Morpholino knockdown. The [[Orthologue|orthologous]] gene was then characterized in humans and a one base pair difference was found to strongly segregate fair-skinned Europeans and dark-skinned Africans.<ref>{{cite journal |vauthors=Lamason RL, Mohideen MA, Mest JR, Wong AC, Norton HL, Aros MC, Jurynec MJ, Mao X, Humphreville VR, Humbert JE, Sinha S, Moore JL, Jagadeeswaran P, Zhao W, Ning G, Makalowska I, McKeigue PM, O'donnell D, Kittles R, Parra EJ, Mangini NJ, Grunwald DJ, Shriver MD, Canfield VA, Cheng KC |display-authors=6 |title=SLC24A5, a putative cation exchanger, affects pigmentation in zebrafish and humans |journal=Science |volume=310 |issue=5755 |pages=1782–1786 |date=December 2005 |pmid=16357253 |doi=10.1126/science.1116238 |s2cid=2245002 |bibcode=2005Sci...310.1782L}}</ref> <!-- this article is on fish, not on studies...This study featured on the cover of the [[academic journal]] [[Science (journal)|''Science'']] and demonstrates the power of zebrafish as a model organism in the relatively new field of [[comparative genomics]].--> Zebrafish with the ''nacre'' mutation have since been bred with fish with a ''roy orbison (roy)'' mutation to make Casper strain fish that have no melanophores or iridophores, and are transparent into adulthood. These fish are characterized by uniformly pigmented eyes and translucent skin.<ref name=zviv>{{cite journal |vauthors=White RM, Sessa A, Burke C, Bowman T, LeBlanc J, Ceol C, Bourque C, Dovey M, Goessling W, Burns CE, Zon LI |display-authors=6 |title=Transparent adult zebrafish as a tool for in vivo transplantation analysis |journal=Cell Stem Cell |volume=2 |issue=2 |pages=183–189 |date=February 2008 |pmid=18371439 |pmc=2292119 |doi=10.1016/j.stem.2007.11.002}}</ref><ref>{{Cite web |url=https://www.livescience.com/2267-scientists-create-fish-watch-cancer-grow.html |title=Scientists Create See-Through Fish, Watch Cancer Grow |author1=Jeanna Bryner |date=February 6, 2008 |website=livescience.com |access-date=January 23, 2022 |archive-date=May 19, 2024 |archive-url=https://web.archive.org/web/20240519141106/https://www.livescience.com/2267-scientists-create-fish-watch-cancer-grow.html |url-status=live}}</ref>

====Transgenesis====
[[Transgene]]sis is a popular approach to study the function of genes in zebrafish. Construction of transgenic zebrafish is rather easy by a method using the ''Tol2'' transposon system. ''Tol2'' element which encodes a gene for a fully functional transposase capable of catalyzing transposition in the zebrafish germ lineage. ''Tol2'' is the only natural DNA transposable element in vertebrates from which an autonomous member has been identified.<ref>{{cite journal |vauthors=Kawakami K, Takeda H, Kawakami N, Kobayashi M, Matsuda N, Mishina M |title=A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish |journal=Developmental Cell |volume=7 |issue=1 |pages=133–144 |date=July 2004 |pmid=15239961 |doi=10.1016/j.devcel.2004.06.005 |doi-access=free}}</ref><ref>{{cite journal |vauthors=Parinov S, Kondrichin I, Korzh V, Emelyanov A |title=Tol2 transposon-mediated enhancer trap to identify developmentally regulated zebrafish genes in vivo |journal=Developmental Dynamics |volume=231 |issue=2 |pages=449–459 |date=October 2004 |pmid=15366023 |doi=10.1002/dvdy.20157 |doi-access=free}}</ref> Examples include the artificial interaction produced between [[Lymphoid enhancer-binding factor 1|LEF1]] and [[Catenin beta-1]]/β-catenin/''CTNNB1''. Dorsky et al. 2002 investigated the developmental role of [[Wnt signaling pathway|Wnt]] by transgenically expressing a Lef1/β-catenin reporter.<ref name="Barolo-Posakony-2002">{{cite journal |vauthors=Barolo S, Posakony JW |title=Three habits of highly effective signaling pathways: principles of transcriptional control by developmental cell signaling |journal=Genes & Development |volume=16 |issue=10 |pages=1167–1181 |date=May 2002 |pmid=12023297 |doi=10.1101/gad.976502 |publisher=[[Cold Spring Harbor Laboratory Press]] & [[The Genetics Society]] |s2cid=14376483 |doi-access=free}}</ref> The Tol2 transposon system was used to develop transgenic zebrafish as sensitive biosensors for heavy metal detection. This involved creating a transgenic zebrafish line expressing a fluorescent protein under the control of a heavy metal-responsive promoter, enabling the detection of low concentrations of cadmium (Cd2+) and zinc (Zn2+).<ref name="Herath_et_al_2024">{{cite journal |vauthors=Herath HM etal |title=Innovative transgenic zebrafish biosensor for heavy metal detection |journal=Environmental Pollution |page=125547 |date=2024 |volume=366 |doi=10.1016/j.envpol.2024.125547 |pmid=39694312}}</ref>


There are well-established protocols for editing zebrafish genes using [[CRISPR gene editing|CRISPR-Cas9]]<ref>{{Cite journal |last1=Vejnar |first1=Charles E. |last2=Moreno-Mateos |first2=Miguel A. |last3=Cifuentes |first3=Daniel |last4=Bazzini |first4=Ariel A. |last5=Giraldez |first5=Antonio J. |date=October 2016 |title=Optimized CRISPR–Cas9 System for Genome Editing in Zebrafish |url=http://www.cshprotocols.org/lookup/doi/10.1101/pdb.prot086850 |journal=Cold Spring Harbor Protocols |language=en |volume=2016 |issue=10 |pages=pdb.prot086850 |doi=10.1101/pdb.prot086850 |pmid=27698232 |issn=1940-3402 |access-date=2022-12-05 |archive-date=2024-05-19 |archive-url=https://web.archive.org/web/20240519141211/https://cshprotocols.cshlp.org/content/2016/10/pdb.prot086850 |url-status=live}}</ref> and this tool has been used to generate genetically modified models.

====Transparent adult bodies====
In 2008, researchers at [[Boston Children's Hospital]] developed a new strain of zebrafish, named Casper, whose adult bodies had transparent skin.<ref name=zviv/> This allows for detailed visualization of cellular activity, circulation, [[metastasis]] and many other phenomena.<ref name=zviv/> In 2019 researchers published a crossing of a ''prkdc<sup>-/-</sup>'' and a ''IL2rga<sup>-/-</sup>'' strain that produced transparent, immunodeficient offspring, lacking [[natural killer cell]]s as well as [[B cell|B]]- and [[T cell|T-cells]]. This strain can be adapted to {{convert|37|C|F}} warm water and the absence of an immune system makes the use of patient derived [[Xenotransplantation|xenografts]] possible.<ref>{{cite journal |vauthors=Yan C, Brunson DC, Tang Q, Do D, Iftimia NA, Moore JC, Hayes MN, Welker AM, Garcia EG, Dubash TD, Hong X, Drapkin BJ, Myers DT, Phat S, Volorio A, Marvin DL, Ligorio M, Dershowitz L, McCarthy KM, Karabacak MN, Fletcher JA, Sgroi DC, Iafrate JA, Maheswaran S, Dyson NJ, Haber DA, Rawls JF, Langenau DM |display-authors=6 |title=Visualizing Engrafted Human Cancer and Therapy Responses in Immunodeficient Zebrafish |journal=Cell |volume=177 |issue=7 |pages=1903–1914.e14 |date=June 2019 |pmid=31031007 |pmc=6570580 |doi=10.1016/j.cell.2019.04.004}}</ref> In January 2013, Japanese scientists genetically modified a transparent zebrafish specimen to produce a visible glow during periods of intense brain activity.<ref name=ithinkz>{{cite news |url=http://www.popsci.com/science/article/2013-01/watch-zebrafish-think-about-food |title=Researchers Capture A Zebrafish's Thought Process On Video |website=Popular Science |date=January 31, 2013 |access-date=February 4, 2013 |archive-date=October 3, 2016 |archive-url=https://web.archive.org/web/20161003135509/http://www.popsci.com/science/article/2013-01/watch-zebrafish-think-about-food |url-status=live}}</ref>

In January 2007, Chinese researchers at [[Fudan University]] genetically modified zebrafish to detect [[oestrogen]] pollution in lakes and rivers, which is linked to male infertility. The researchers cloned oestrogen-sensitive genes and injected them into the fertile eggs of zebrafish. The modified fish turned green if placed into water that was polluted by oestrogen.<ref name=ChinaOest>[https://web.archive.org/web/20080225200714/http://news.xinhuanet.com/english/2007-01/12/content_5597696.htm "Fudan scientists turn fish into estrogen alerts"]. [[Xinhua]]. January 12, 2007. Retrieved November 15, 2012.</ref>

====RNA splicing====
In 2015, researchers at [[Brown University]] discovered that 10% of zebrafish genes do not need to rely on the [[U2AF2]] [[protein]] to initiate [[RNA splicing]]. These genes have the DNA base pairs AC and TG as repeated sequences at the ends of each [[intron]]. On the 3'ss (3' splicing site), the base pairs [[adenine]] and [[cytosine]] alternate and repeat, and on the 5'ss (5' splicing site), their complements [[thymine]] and [[guanine]] alternate and repeat as well. They found that there was less reliance on U2AF2 protein than in humans, in which the protein is required for the splicing process to occur. The pattern of repeating base pairs around introns that alters RNA [[nucleic acid secondary structure|secondary structure]] was found in other [[teleost]]s, but not in [[tetrapod]]s. This indicates that an evolutionary change in tetrapods may have led to humans relying on the U2AF2 protein for RNA splicing while these genes in zebrafish undergo splicing regardless of the presence of the protein.<ref name="BrownRNA">{{cite journal |vauthors=Lin CL, Taggart AJ, Lim KH, Cygan KJ, Ferraris L, Creton R, Huang YT, Fairbrother WG |display-authors=6 |title=RNA structure replaces the need for U2AF2 in splicing |journal=Genome Research |volume=26 |issue=1 |pages=12–23 |date=January 2016 |pmid=26566657 |pmc=4691745 |doi=10.1101/gr.181008.114}}</ref>

====Orthology====
''D. rerio'' has three [[transferrin]]s, all of which cluster closely with other [[vertebrate]]s.<ref name="Gabaldon-Koonin-2013">{{cite journal |vauthors=Gabaldón T, Koonin EV |title=Functional and evolutionary implications of gene orthology |journal=[[Nature Reviews Genetics]] |volume=14 |issue=5 |pages=360–366 |date=May 2013 |pmid=23552219 |pmc=5877793 |doi=10.1038/nrg3456}}</ref>