GENETICS | REVIEW

GENETIC TOOLBOX

The Genomic and Genetic Toolbox of the TeleostMedaka (Oryzias latipes)

Stephan Kirchmaier Kiyoshi Narusedagger Joachim Wittbrodt1 and Felix LoosliDagger1

Centre for Organismal Studies University of Heidelberg INF 230 69120 Heidelberg Germany daggerLaboratory of BioresourcesNational Institute for Basic Biology Okazaki 444-8585 Aichi Japan and DaggerInstitute of Toxicology and Genetics Karlsruhe Institute

of Technology 76344 Eggenstein-Leopoldshafen Germany

ABSTRACT The Japanese medaka Oryzias latipes is a vertebrate teleost model with a long history of genetic research A number ofunique features and established resources distinguish medaka from other vertebrate model systems A large number of laboratorystrains from different locations are available Due to a high tolerance to inbreeding many highly inbred strains have been establishedthus providing a rich resource for genetic studies Furthermore closely related species native to different habitats in Southeast Asiapermit comparative evolutionary studies The transparency of embryos larvae and juveniles allows a detailed in vivo analysis ofdevelopment New tools to study diverse aspects of medaka biology are constantly being generated Thus medaka has become animportant vertebrate model organism to study development behavior and physiology In this review we provide a comprehensiveoverview of established genetic and molecular-genetic tools that render medaka fish a full-fledged vertebrate system

KEYWORDS teleost Oryzias latipes medaka genetics genomics

MEDAKA (Oryzias latipes) is a small freshwater fish ofthe family Adrianichthyidae in the order Beloni-

formes It is closely related to other members of the super-order Acanthopterygii of ray-finned fish such as pufferfish(tetraodon and fugu) stickleback and killifish while it isseparated from zebrafish by 150 million years (MY) ofdivergent evolution (Figure 1) A growing number of se-quenced teleost genomes and several established geneticmodel systems (zebrafish stickleback medaka) thus providea unique resource for comparative studies relating to verte-brate evolution Medaka is native to Taiwan Korea Chinaand Japan In Japan it is found in small rivers creeks andrice paddies on all main islands with the exception of Hok-kaido Medaka is a euryhaline species and can also live inbrackish water (Figure 2 A and B) (Inoue and Takei 2002)As a resident of a temperate zone medaka can toleratea wide range of temperatures (4ndash40) both as adult and

embryo (Sampetrean et al 2009) Thus in the laboratorytemperature can be used to control the developmental speedwithout adverse effects

Medaka native to Japan and East Korea have a diploidkaryotype of 48 chromosomes but 46 chromosomes inmedaka from West Korea and China (Uwa and Ojima1981) The different chromosome number is due to a fusionof chromosome 11 and 13 by a Robertsonian translocation(Myosho et al 2012b) The haploid genome size is 800Mb (Kasahara et al 2007) Adults can reach a length of upto 4 cm and the wild-type pigmentation is greyish-brown(Figure 2D) However mutant strains are available wherebody pigmentation is strongly reduced both at embryonicand adult stages (Figure 2C) (Wakamatsu et al 2001 Kelshet al 2004) Interestingly sex-specific pigmentation can beused to distinguish male from female embryos as early as3 days postfertilization (dpf) (organogenesis stages) (Wadaet al 1998) since the leucophore free (lf slc2a15b) locus thatis required for pigmentation by leucophores is located on thesex chromosomes X and Y (Kimura et al 2014) The XXndashXYsex determination system (see below) allows the establish-ment of mutant strains with females (XX) that are homozy-gous for a loss-of-function lf allele and males (XY) that areheterozygous and will therefore exhibit wild-type leucophorepigmentation The strains Qurt and FLF2 have the wild allele

Copyright copy 2015 by the Genetics Society of Americadoi 101534genetics114173849Manuscript received December 17 2014 accepted for publication February 6 2015Available freely online through the author-supported open access option1Corresponding authors Karlsruhe Institute of Technology Institute of Toxicology andGenetics Hermann von Helmholtz Platz 1 D-76344 Eggenstein-LeopoldshafenGermany E-mail felixlooslikitedu and COS Heidelberg Heidelberg University ImNeuenheimer Feld 230 D-69120 Heidelberg GermanyE-mail JochenWittbrodtcosuni-heidelbergde

Genetics Vol 199 905ndash918 April 2015 905

on the Y chromosome and a mutant allele on the X chromo-some thus males have leucophores and female do not

The life span of medaka under constant mating con-ditions (14 hr light10 hr dark at 25ndash28) in the laboratoryis 12 months This can be extended to 2 years underconditions where the fish do not mate in combination witha reduced temperature (10 hr light14 hr dark at 19)Medaka originate from a temperate zone and show a sea-sonal mating behavior requiring long light phases and shortdark phases for reproduction whereas temperature has littleeffect on fecundity (Koger et al 1999) Medaka males andfemales can easily be distinguished due to a distinct sexualdimorphism Most conspicuous is a slit in the male dorsal fin(Figure 2D) Medaka is oviparous with transparent eggs andembryos Embryos hatch after 7ndash8 days at 28 as fullydeveloped juvenile fish The generation time of medaka is8ndash12 weeks depending on strain and husbandry conditions

Medaka Wild Populations and Laboratory Strains

In Japan medaka is divided into two geographicallyseparated populations the northern population of thenorthern region of Honshu Island and the southern pop-ulation of southern Honshu Kyushu Shikoku and Okinawawith a divergence time between these two groups of 4MYA (Figure 3) (Takehana et al 2004ab) Based on mor-phological criteria it has recently been suggested that thenorthern population is an independent species (O sakaizu-mii) (Asai et al 2011) Under laboratory conditions pro-ductive matings between these groups can be obtainedand the resulting F1 and F2 male and female hybrids arefully fertile (Sakaizumi et al 1992) The degree of polymor-phism between northern and southern strains is in the rangeof 1 in coding and 4 in noncoding regions (Naruse et al

2004a) Since the northern and southern populations arehighly polymorphic and isogenic inbred lines have beenestablished from both populations these inbred lines areimportant tools for genetic mapping purposes (Naruseet al 2004b)

Medaka is highly tolerant to inbreeding This has beenexploited decades ago to establish highly inbred strains fromdifferent wild populations (Hyodo-Taguchi 1980) In somecases these strains have been inbred for more than 100generations by successive brotherndashsister crosses and cantherefore be considered as isogenic Highly inbred strainsare predominantly homozygous with heterozygosity levelsof 3 3 1025 whereas in wild catch this level is 100-foldhigher (15 3 1023) The inbred strain Nilan has fewercycles of inbreeding and consequently is 2 3 1024 hetero-zygous (Spivakov et al 2014) Currently 60 wild strainsfrom both the northern and southern populations and 14derived inbred strains are available at the Japanese MedakaStock Center (National BioResource Project Medaka NBRPMedaka httpwwwshigennigacjp) Apart from genomicpolymorphisms these inbred strains also exhibit strain-specificdifferences in behavior body shape brain morphology andsusceptibility to mutagens (Ishikawa et al 1999 Kimuraet al 2007) Heritability of craniofacial traits has been dem-onstrated indicating that the inbreeding of polymorphicpopulations in medaka reveals the genetic contribution tovariability of these traits (Kimura et al 2007) To furtherexploit the tolerance to inbreeding combined with geneticpolymorphism recently a detailed analysis of wild popula-tions has been carried out with the aim to establish a panelof100 inbred lines derived from a single polymorphic wildpopulation (Spivakov et al 2014) Such a panel of inbredlines with sequenced genomes will serve as a genetic sourcefor genome-wide association studies (GWAS)

Figure 1 Evolutionary relationship ofmedaka and other teleost model sys-tems Only teleost model systems withpublicly available genome sequencedata on EnsEMBL are shown The spot-ted gar is an outgroup of the teleostwhich did not undergo a whole-genomeduplication (3R) Medaka belongs to thePercomorphaceae the clade with mostsequenced teleost genomes makingit an ideal organism for comparativegenomics For each species the Latinand the common name as well as thegenome size is shown The species treewas calculated using the EnsEMBLcompara team (httpwwwensemblorginfogenomecompara ) Taxonomy dataare available at EnsEMBL and derivedfrom the National Center for Biotechnol-ogy Information taxonomy browser

906 S Kirchmaier et al

Medaka Genomic Information

The medaka genome sequence project has been success-fully completed (Kasahara et al 2007) and the medakadraft genome is accessible through a number of genomebrowsers (httpwwwensemblorgOryzias_latipesInfoIndex httpgenomeucsceducgi-binhgGatewayhgsid=409674941_iubYP8aF1UOAsctCB3qI2E3t1Kffampclade=vertebrateamporg=Medakaampdb=0 and httpviewershigeninfomedakavwmapview) The medaka reference genome se-quence is based on the Hd-rRII1 inbred line In addition thegenome sequence of the HNI-II Kaga HSOK and Nilan andKiyosu strains (Spivakov et al 2014) are available (httpwwwebiacukbirney-srvmedaka-ref-paneldatahtml) It isalso possible to use blast searches against Hd-rR II and HNI-II scaffolds as well as raw shotgun reads of Hd-rRII (httpdolphinnigacjpmedakaindexphp) The NBRP genomebrowser also enables searches for SNPs in the HdrR HNINilan HSOK and Kaga strains (httpmedakalabnigacjpcgi-bingb2gbrowsemedaka)

Related Species

The genus Oryzias comprises 22 species that are widelydistributed in South-Eastern and Western Asia and occupya wide range of different habitats in tropical and temperatezones This provides a rich source of species with a divergencetime ranging from 4 to 30 MYA to study evolution and adap-tation to different habitats The most closely related speciesare O curvinotus from Southern China and Vietnam and Oluzonensis from the Philippines The divergence time of thesesister species has been estimated to be5 MYA (Tanaka et al2007) A comparison of sex determination revealed an un-expected plasticity in the underlying molecular-genetic mech-anism (Tanaka et al 2007) O latipes O curvinotus andO luzonensis possess an XXndashXY sex determination system

In O latipes and O curvinotus the sex determination genesDMYDMRT1Yb respectively are on orthologous and syn-tenic male Y chromosomes (Matsuda et al 2002 2003Nanda et al 2002) On the other hand O luzonensis has noDMY gene There gsdfY (gonadal soma derived growth factoron the Y chromosome) acts as the sex determination gene(Myosho et al 2012a) Recently a third sex determinationgene was identified in O dancena The sex chromosome ofO dancena is orthologous to the autosomal chromosome 10of O latipes Positional cloning and loss-of-function experi-ments using zinc-finger nucleases (ZFNs) showed that thesex determination gene of O dancena is Sox3 which isthought to be orthologous to the mammalian sex determina-tion gene Sry (Takehana et al 2014) Thus these closelyrelated Oryzias species represent a unique paradigm to studythis fast evolving trait As these species live in different hab-itats it is to be expected that also other traits both physio-logical and behavioral have diverged O latipes crosses witheither O curvinotus or O luzonensis result in sterile hybridsHowever O curvinotus and O luzonensis hybrids are fullyfertile thus a genetic segregation analysis of diverged traitsbetween these sister species is possible (Tanaka et al 2007)

Also external factors can influence sex determination inmedaka It has been shown that high temperatures duringembryonic development can induce female-to-male sexreversal whereas males are not affected (Sato et al 2005Hayashi et al 2010) Also hypoxia can induce female-to-male sex reversal in medaka (Cheung et al 2014)

In the following we provide a detailed description ofgenetic and molecular-genetic tools that are available formedaka Table 1 summarizes the described technologies

Forward Genetics

Medaka has been used extensively as a genetic modelfor radiation biology (Shima and Shimada 1991 2001)

Figure 2 The Japanese medaka Oryziaslatipes (A and B) Medaka live in smallrivers and creeks such as irrigation sys-tems of rice paddies (C) The mutantQuiH strain completely lacks pigmen-tation Internal organs are visible inadults Due to the absence of body pig-mentation QuiH medaka are also freeof autofluorescence (D) The pigmenta-tion of wild medakas is shown Notealso the sexual dimorphism of the dor-sal fin The male dorsal fin has a slit(arrowhead in inset)

Genetic Toolbox Review 907

Furthermore systematic mutagenesis approaches such aschemical mutagenesis have been established for medaka(Shima and Shimada 1991 2001 Ishikawa 2000 Loosliet al 2000) The high fecundity extra-uterine developmentand transparency of eggs and embryos render medakaan excellent genetic system for unbiased forward geneticapproaches using chemical mutagenesis Furthermore asoutlined above available highly inbred strains provide theuniform genetic background that is essential to both reducegenetic variance of a given phenotype and allow subsequentmapping of mutations The mutagen of choice is ethylnitro-sourea (ENU) which efficiently induces point mutations bybase alkylation and thus results in genetically separable sin-gle gene mutations Similar to ENU mutagenesis in zebrafishand mouse (Knapik 2000 Nguyen et al 2011) a gene-specificmutation rate of 1 3 1023 has been reported (Loosli et al2000 Furutani-Seiki et al 2004) A number of small- andlarge-scale ENU mutagenesis screens have been carried outwhere classical F3 crossing schemes have been employed toidentify recessive zygotic mutations affecting various pro-cesses (Loosli et al 2000 Furutani-Seiki et al 2004)Mutants from these screens are available at the NBRP Me-daka Stock Center (httpwwwshigennigacjpmedakastrainstrainTopjsp)

Other forward genetic approaches rely on the mutagenicpotential of transgene integrations A major advantage ofsuch approaches is that in most cases these mutations can beidentified by locating the transgene insertion sites within thegenome In medaka gene trap approaches with severaltransposons have been successfully employed (Sano et al2009 Froschauer et al 2012) The transposase Frog Princehas been used to randomly integrate a GFP-based reporter

transgene with upstream splice acceptor sites (Sano et al2009) Insertion of this gene trap cassette results in GFPexpression if the open reading frame is maintained in thespliced transcript A total of 16 of the injected fish weretransgenic founders that gave rise to GFP-expressing em-bryos In addition to Frog Prince the transposon systemAcDs has been used for gene trap approaches (Froschaueret al 2012) The Sleeping Beauty transposon system wasemployed for enhancer trap screens (Grabher et al 2003)In this case a Xenopus cytoskeletal actin (cska) promoterwas used to detect enhancer activities at the transgene in-sertion site Thus transgenic lines were generated with spe-cific temporal and spatial reporter gene activity It hasrecently been shown in zebrafish that such GFP reportergenes can be exchanged for a transactivator such as Gal4using nonhomologous end joining induced by the CrisprCas9 system (Auer et al 2014) Thus once a line is obtainedwith a reporter expression pattern of interest the enhancertrap can be repurposed to fit experimental needs

Reverse Genetics

In fish model systems antisense morpholinos are widelyused to transiently interfere with gene function (Bill et al2009) These typically 25-bp-long oligos specifically inter-fere with gene function based on their complementarity tothe target sequence either by blocking translation initiationor by interfering with splicing The nonribose-based back-bone renders morpholinos insensitive to enzymatic degrada-tion Recently gene knockdown using peptide nucleic acids(PNAs) has been applied in medaka (Dorn et al 2012)Both morpholinos and PNAs are powerful antisense tools

Figure 3 Geographical distribution of differentOryzias latipes populations in Japan Koreaand China Four main populations are distin-guished based on genomic criteria such as allo-zyme variation and variation of mitochondrialDNA The northern and southern populationsare geographically separated and divergedabout 4 MYA The map was modified from thatpublished in Spivakov et al (2014)

908 S Kirchmaier et al

PNAs have a higher affinity for RNA yet they are less solubleand therefore the in vivo use is limited Dorn et al (2012)changed the chemical composition of the PNA backbone toincrease solubility and showed efficient knockdown of thesix3 gene in medaka In most cases these antisense morpho-linos are injected into fertilized eggs at early cleavage stagesto ensure a ubiquitous distribution to all cells of the devel-oping embryo If thus applied they interfere with gene func-tion during early development To study gene functionduring later stages morpholinos can be activated condition-ally by light-induced uncaging (Shestopalov et al 2007)Alternatively they can be applied to specific tissues by elec-troporation (Thummel et al 2006 2008) Also caging ofmRNA has been used to conditionally express genes in fishembryos (Ando et al 2001) However recent results inzebrafish indicate that morpholino-based gene knockdownoften results in unspecific off-target effects (Kok et al 2014)Thus the use of small antisense molecules to study genefunction also needs to be thoroughly evaluated in medaka

To study the function of endogenous microRNAs (miRNAs)several tools have been applied in medaka (Conte et al2010) First morpholinos directed against pre-miRNAs havebeen successfully used to knockdown mir204 and specificeffects during retinal development have been observed Inthe same study commercially available miRNA mimetics(miRIDIAN microRNA Mimics) have been used to mimicmir204 activity This was verified with dosage sensors iefluorescent reporter genes that contain miRNA binding sitesin the 39 UTR such that binding of miRNAs to the reportermessenger RNA (mRNA) affects expression In this way

Meis2 has been verified as a target gene of mir204 by a targetprotector assay Here small RNAs occupy the putativemiRNA binding site on the mRNA and thereby blockmiRNA-dependent inhibition of translation or mRNA degra-dation (Conte et al 2010) So far no targeted approacheshave been described in medaka in which miRNA mimeticsor knockdown tools were expressed in a tissue-specific man-ner However tissue-specific expression of short hairpinRNAs (shRNAs) has been described in several species in-cluding mouse (Zuber et al 2011) and zebrafish (Dong et al2009) Here artificially constructed miRNAs based on theendogenous miRNA backbone of mir30e are inserted withina transgene and transcribed from a pol II promoter How-ever conflicting results in zebrafish indicate that the use ofshRNAs may not be applicable for all target genes and itsfunctionality needs to be thoroughly tested on a case-by-case basis (Dong et al 2009 Kelly and Hurlstone 2011De Rienzo et al 2012) Nevertheless the use of shRNAsseems a promising approach to down-regulate gene expres-sion levels in a controlled and tissue-specific manner in fishmodel systems (Dong et al 2009)

Other genomic resources have been established to isolatemutations for specific genes in medaka The targetedinduced local lesions in genome (TILLING) method is one ofthe reverse genetic or gene-driven approaches based onrandom mutagenesis by ENU A medaka TILLING library hasbeen established that consists of 5760 ENU mutagenized F1fish (offspring from ENU-treated males crossed to wild-typefemales) and their genomic DNA archives The average mu-tation rate of this medaka TILLING library is one mutation

Table 1 Genetic and molecular-genetic tools established for medaka

Topic Approach Method

Forward genetics Chemical mutagenesis ENU mutagenesis (Loosli et al 2000 Furutani-Seiki et al 2004)Transposon mutagenesis Sleeping Beauty (Grabher et al 2003 Grabher and Wittbrodt 2007)

AcDs (Boon Ng and Gong 2011 Froschauer et al 2012)Reverse genetics Antisense technologies Morpholinos (Thummel et al 2006 Shestopalov et al 2007 Thummel et al 2008)

PNA (Dorn et al 2012)miRNA studies miRNA sponges

miRNA knockdownmiRNA mimics(Conte et al 2010)

Tilling ENU mutagenesis (Taniguchi et al 2006)Genome editing ZNF TALEN CRISPRCas9 (Ansai et al 2012 2013 Ansai and Kinoshita 2014 Ansai et al

2014)Transgenesis Meganuclease ISceI (Grabher and Wittbrodt 2008)

Transposons Sleeping Beauty AcDs Frog Prince Tol2 (Grabher et al 2003 Grabher and Wittbrodt2007 Sano et al 2009 Boon Ng and Gong 2011 Froschauer et al 2012 Kirchmaieret al 2013)

Site-specific recombinases PhiC31 (Kirchmaier et al 2013)Molecular-genetic tools Reporter lines Transgenic reporter cassettes with specific promoters

Cell lineage tools Gaudiacute toolbox (Centanin et al 2014)Transactivation system LexPR Gal4 Tet system (Grabher and Wittbrodt 2004 Emelyanov and Parinov 2008

Knopf et al 2010)Gene expression studies RNA detection In situ hybridization (ISH) (Ason et al 2006 Inoue and Wittbrodt 2011)

RNA seq (Schartl et al 2012)Microarray (Kishi et al 2006)

Protein detection Antibody staining (IHC) (Inoue and Wittbrodt 2011)

This is an overview of the vast toolbox that is available to tackle a wide range of biological questions

Genetic Toolbox Review 909

per 350 kb The resource center NBRP Medaka providesa screening system for the genomic DNA archives withhigh-resolution melting (HRM) Identified mutations canbe recovered by in vitro fertilization of the correspondingsperm with wild-type eggs (see httpwwwshigennigacjpmedakastrainaboutTillingjsp) (Taniguchi et al 2006)

Several tools that allow specific manipulations of thegenome have been developed recently that are applicable tomany organisms including medaka These tools are basedon sequence-specific binding of a nuclease that cleaves thegenomic double-stranded DNA (dsDNA) thereby inducinga double strand break (DSB) (Gaj et al 2013) Subsequentlythe DSB repair by nonhomologous end joining (NHEJ) intro-duces small insertiondeletions (indels) at high frequencyand thus introduces mutations In addition to introducingmutations exogenous DNA can be inserted into the site ofthe DSB via NHEJ (Auer et al 2014) or homology-directedrepair (HDR) (Bedell et al 2012) Two types of sequence-specific nucleases are used for these purposes ZFNs as wellas transcription activator-like effector nucleases (TALENs)have a bipartite structure with a flexible and modular DNAbinding domain fused to the effector FokI nuclease A mod-ular set of ZFNs or TALENs can be designed to bind a specificsequence in the genome and induce a DSB at that site (Ansaiet al 2012 2013) Recently it was shown that sequence-specific binding of a guide RNA coupled to a nuclease canvery efficiently induce DSBs (Cong et al 2013 Mali et al2013) This CRISPR (Clustered Regularly Interspaced ShortPalindromic Repeats)Cas9-based system uses a single guideRNA (sgRNA) that targets the nuclease Cas9 in a sequence-specific manner to generate a DSB Importantly the use ofTALENs and the CRISPRCas9 system has been greatly sim-plified Highly efficient cloning methods for TALENs havebeen developed (Cermak et al 2011 Sanjana et al 2012Ma et al 2013 Sakuma et al 2013 Schmid-Burgk et al2013) For the CRISPRCas9 system it suffices to adjustthe short target site within the guide RNA according tothe genomic target sequence

Initial reports of targeted mutagenesis in medaka dem-onstrated the ZFN- and TALEN-mediated knockout of anEGFP transgene (Ansai et al 2012) as well as endogenousgenes (Ansai et al 2013 2014) Also site-directed mutagen-esis using the CRISPRCas9 system has been successfullyemployed in medaka (Ansai and Kinoshita 2014) Theauthors showed that genome modifications by CRISPRCas9 were transmitted to the F1 with frequencies rangingfrom 429 to 100 Recently a medaka target site predictiontool for sgRNAs has been established (CCTop MedakaCRISPR target predictor httpcrisprcosuni-heidelbergdeindexhtml) This tool assists in the design of sgRNAsfor any target sequence The target site as well as potentialoff-target sites in a genome are predicted and the best targetsite can be chosen based on the likelihood for off-site target-ing Importantly potential target and off-target sites in themedaka genome are matched with gene annotations thatwere refined with RNAseq data (T Thumberger J L Mateo

and J Wittbrodt unpublished data) CRISPR sgRNA evalu-ation and microhomology prediction tools are available atNBRP Medaka (httpviewershigeninfocgi-bincrisprcrisprcgi)

Transgenesis Toolbox

Transgenic medaka fish can be generated using severalestablished protocols To introduce the required DNA RNAand proteins into zygotes microinjection is the method ofchoice albeit electroporation (Inoue et al 1990) intracyto-plasmic sperm injection (Liu et al 2011) as well as the useof a particle gun (Kinoshita et al 2003) has been reported Adetailed protocol for microinjection into fertilized medakaeggs is available (Rembold et al 2006a)

Microinjection of plasmid DNA leads to the integration ofmultiple copies of the entire plasmid Often these insertionsare concatemers with a high-copy number Furthermore thegeneration of transgenic fish using plasmid DNA is in-efficient (1ndash10) due to a low genomic integration rateand highly mosaic distribution in the injected animal(Thermes et al 2002) Meganuclease ISceI-mediated trans-genesis is more efficient (25ndash50) Plasmid DNA containingtwo ISceI recognition sites is co-injected with meganucleaseISceI protein (Grabher and Wittbrodt 2008) The transgen-esis vector is cut by the meganuclease that contains nuclearlocalization signals which are thought to increase nuclearimport of the injected DNA The resulting linear DNA isefficiently integrated into the genome via unknown mecha-nisms Meganuclease-based transgenesis results in the inser-tion of low-copy number concatemers at mutlitple sitesAdditionally single-copy transgene integrations are possibleand can be screened for (Thermes et al 2002)

Transgenes can be integrated into the genome via the useof transposons The Sleeping Beauty (SB) transposon belongsto the Tc1mariner transposon family It has been estab-lished by reconstituting a functional transposon from silenttransposon fragments identified in the genomes of rainbowtrout and Atlantic salmon (Ivics et al 1997) Further opti-mization of the system allowed to generate hyperactive ver-sions of SB most notably SB100x (Maacuteteacutes et al 2009) Theoriginal as well as hyperactive versions have been used forthe efficient generation of transgenic medaka fish (Grabheret al 2003 Grabher and Wittbrodt 2007 Kirchmaier et al2013) SB leads to integrations with single-copy insertionsas well as low-copy tandem arrays (Grabher et al 2003)Other transposases that have been successfully used inmedaka are Frog Prince (Sano et al 2009) another Tc1mariner transposase and AcDs (Boon Ng and Gong 2011Froschauer et al 2012) a transposase belonging to the hATfamily like Tol2 Tol2 is an autonomous transposon that hasbeen identified in the genome of medaka (Transposon ory-zias latipes) (Abe et al 2011) This transposon is widelyused to mediate genomic insertion of exogenous DNAin zebrafish Thus a plethora of functionally establishedTol2-based constructs are available Interestingly Tol2 can

910 S Kirchmaier et al

per 350 kb The resource center NBRP Medaka providesa screening system for the genomic DNA archives withhigh-resolution melting (HRM) Identified mutations canbe recovered by in vitro fertilization of the correspondingsperm with wild-type eggs (see httpwwwshigennigacjpmedakastrainaboutTillingjsp) (Taniguchi et al 2006)

Several tools that allow specific manipulations of thegenome have been developed recently that are applicable tomany organisms including medaka These tools are basedon sequence-specific binding of a nuclease that cleaves thegenomic double-stranded DNA (dsDNA) thereby inducinga double strand break (DSB) (Gaj et al 2013) Subsequentlythe DSB repair by nonhomologous end joining (NHEJ) intro-duces small insertiondeletions (indels) at high frequencyand thus introduces mutations In addition to introducingmutations exogenous DNA can be inserted into the site ofthe DSB via NHEJ (Auer et al 2014) or homology-directedrepair (HDR) (Bedell et al 2012) Two types of sequence-specific nucleases are used for these purposes ZFNs as wellas transcription activator-like effector nucleases (TALENs)have a bipartite structure with a flexible and modular DNAbinding domain fused to the effector FokI nuclease A mod-ular set of ZFNs or TALENs can be designed to bind a specificsequence in the genome and induce a DSB at that site (Ansaiet al 2012 2013) Recently it was shown that sequence-specific binding of a guide RNA coupled to a nuclease canvery efficiently induce DSBs (Cong et al 2013 Mali et al2013) This CRISPR (Clustered Regularly Interspaced ShortPalindromic Repeats)Cas9-based system uses a single guideRNA (sgRNA) that targets the nuclease Cas9 in a sequence-specific manner to generate a DSB Importantly the use ofTALENs and the CRISPRCas9 system has been greatly sim-plified Highly efficient cloning methods for TALENs havebeen developed (Cermak et al 2011 Sanjana et al 2012Ma et al 2013 Sakuma et al 2013 Schmid-Burgk et al2013) For the CRISPRCas9 system it suffices to adjustthe short target site within the guide RNA according tothe genomic target sequence

Initial reports of targeted mutagenesis in medaka dem-onstrated the ZFN- and TALEN-mediated knockout of anEGFP transgene (Ansai et al 2012) as well as endogenousgenes (Ansai et al 2013 2014) Also site-directed mutagen-esis using the CRISPRCas9 system has been successfullyemployed in medaka (Ansai and Kinoshita 2014) Theauthors showed that genome modifications by CRISPRCas9 were transmitted to the F1 with frequencies rangingfrom 429 to 100 Recently a medaka target site predictiontool for sgRNAs has been established (CCTop MedakaCRISPR target predictor httpcrisprcosuni-heidelbergdeindexhtml) This tool assists in the design of sgRNAsfor any target sequence The target site as well as potentialoff-target sites in a genome are predicted and the best targetsite can be chosen based on the likelihood for off-site target-ing Importantly potential target and off-target sites in themedaka genome are matched with gene annotations thatwere refined with RNAseq data (T Thumberger J L Mateo

and J Wittbrodt unpublished data) CRISPR sgRNA evalu-ation and microhomology prediction tools are available atNBRP Medaka (httpviewershigeninfocgi-bincrisprcrisprcgi)

Transgenesis Toolbox

Transgenic medaka fish can be generated using severalestablished protocols To introduce the required DNA RNAand proteins into zygotes microinjection is the method ofchoice albeit electroporation (Inoue et al 1990) intracyto-plasmic sperm injection (Liu et al 2011) as well as the useof a particle gun (Kinoshita et al 2003) has been reported Adetailed protocol for microinjection into fertilized medakaeggs is available (Rembold et al 2006a)

Microinjection of plasmid DNA leads to the integration ofmultiple copies of the entire plasmid Often these insertionsare concatemers with a high-copy number Furthermore thegeneration of transgenic fish using plasmid DNA is in-efficient (1ndash10) due to a low genomic integration rateand highly mosaic distribution in the injected animal(Thermes et al 2002) Meganuclease ISceI-mediated trans-genesis is more efficient (25ndash50) Plasmid DNA containingtwo ISceI recognition sites is co-injected with meganucleaseISceI protein (Grabher and Wittbrodt 2008) The transgen-esis vector is cut by the meganuclease that contains nuclearlocalization signals which are thought to increase nuclearimport of the injected DNA The resulting linear DNA isefficiently integrated into the genome via unknown mecha-nisms Meganuclease-based transgenesis results in the inser-tion of low-copy number concatemers at mutlitple sitesAdditionally single-copy transgene integrations are possibleand can be screened for (Thermes et al 2002)

Transgenes can be integrated into the genome via the useof transposons The Sleeping Beauty (SB) transposon belongsto the Tc1mariner transposon family It has been estab-lished by reconstituting a functional transposon from silenttransposon fragments identified in the genomes of rainbowtrout and Atlantic salmon (Ivics et al 1997) Further opti-mization of the system allowed to generate hyperactive ver-sions of SB most notably SB100x (Maacuteteacutes et al 2009) Theoriginal as well as hyperactive versions have been used forthe efficient generation of transgenic medaka fish (Grabheret al 2003 Grabher and Wittbrodt 2007 Kirchmaier et al2013) SB leads to integrations with single-copy insertionsas well as low-copy tandem arrays (Grabher et al 2003)Other transposases that have been successfully used inmedaka are Frog Prince (Sano et al 2009) another Tc1mariner transposase and AcDs (Boon Ng and Gong 2011Froschauer et al 2012) a transposase belonging to the hATfamily like Tol2 Tol2 is an autonomous transposon that hasbeen identified in the genome of medaka (Transposon ory-zias latipes) (Abe et al 2011) This transposon is widelyused to mediate genomic insertion of exogenous DNAin zebrafish Thus a plethora of functionally establishedTol2-based constructs are available Interestingly Tol2 can

910 S Kirchmaier et al

also be used as a transgenesis vector in medaka since ex-pression of Tol2 transposase has no or very few deleteriouseffects on the medaka genome (Takehana et al 2014) It isthus possible to use established Tol2-based constructs fromzebrafish also in medaka It is of course necessary to test ineach case the promotor activity as species-specific differen-ces may result in different temporal and spatial expressionpatterns

To select potential transgenic founder fish a coselectionmarker is often used For example two transgenesis vectorscan be co-injected and often the transgenes on both vectorsare co-inserted (Rembold et al 2006a) Alternatively a selec-tion reporter cassette is included in the transgene For thatstrong tissue-specific promoters such as the lens crystallinpromoter (Vopalensky et al 2010) or the heart muscle-specificcmlc2 promoter (Auman et al 2007) are routinely used Thisallows assessing the distribution of the injected DNA in theinjected embryos (mosaicism) and thereby the preselectionof potential transgenic founder fish However such linkedreporter cassettes can also interfere with the transgeneproper (Kirchmaier et al 2013)

These protocols discussed above result in transgeneinsertions at random genomic loci Since position effectsmay affect transgene function and insertional mutagenesismay occur several independent transgenic lines of a giventransgene have to be assessed to verify an observed traiteither expression or phenotype Recently a site-specifictransgenesis toolbox for medaka has been established thatrelies on the serine integrase derived from the bacteriophagePhiC31 (Kirchmaier et al 2013) The integrase PhiC31mediates the recombination between an attP site in the ge-nome and an attB site in a targeting vector Transgenic linesharboring single intergenic insertion sites containing theattP sequence in gene deserts were established (Figure4B) These insertion sites serve as neutral integration sitesfor all transgenes of interest containing the attB site Impor-tantly the genomic loci have been characterized using theHsp70 promoter indicating that position effects in the tar-geted loci are negligible Since the same integration site canbe used for different transgenes the resulting transgeniclines are directly comparable The landing site is designedsuch that correct transgene integration results in a specificchange of fluorescence allowing to screen for correct geno-mic integration and therefore to preselect for transgenicfounder fish Additionally a Cre-mediated locus cleanupstep removes most parts of the initial integration site re-ducing interference from linked coselection markers

Molecular-Genetic Toolbox

Reporter transgenes based on the expression of fluorescentproteins are often used to study gene function The trans-parency of embryonic tissue including the brain allows theanalysis of such reporter genes in vivo Furthermore pigmen-tation mutants that are almost completely transparent alsoallow reporter gene analysis in adults (Wakamatsu et al

2001) Thus medaka is ideally suited to analyze regulatoryelements in vivo (Mongin et al 2011 ENCODE Project Con-sortium et al 2012 Kirchmaier et al 2013) In addition re-porter gene expression can be used to label specific tissuesand cell types (Tanaka et al 2001 Rembold et al 2006bMartinez-Morales et al 2009 Willems et al 2012) In Figure4B the result from an enhancer test assay using PhiC31 inte-grase is depicted In this case the integration of regulatoryDNA elements by PhiC31-mediated recombination results inspecific expression of the GFP reporter gene that is present inthe landing site on chromosome 18 Importantly GFP expres-sion originates from the targeted locus only and high somaticactivity of PhiC31 integrase allows the analysis of reportergene expression directly in the injected embryos Centaninet al (2011) showed that all cells in a transgenic line can bepermanently labeled using a ubiquitous enhancerpromotor todrive GFP expression Medaka has also been used to study thehigher order neuronal circuits of specific taste bud cells usingtransgenic reporter lines (Ieki et al 2013) In this case thewheat germ agglutinin (WGA) gene was expressed under themfplc-b2 promoter WGA protein is a trans-synaptic tracer thatis used to study neuronal networks (Yoshihara 2002) Recentlyosteoblasts have been conditionally ablated using a fusion ofCFP with the nitroreductase gene (Willems et al 2012) ThisCFPndashNTR fusion gene was specifically expressed in osteoblastsusing the osterix promoter Prolonged treatment with the drugmetronidazole (Mtz) induced apoptosis specifically in CFPndashNTR-expressing cells Also medaka has been used to studyenvironmental influences using transgenic reporter lines asbioindicators for the hormone estrogen androgen activity toxiccompounds such as 2378-Tetrachlordibenzodioxin and heatstress (Zeng et al 2005 Boon Ng and Gong 2011 Kawaguchiet al 2012 Ng and Gong 2013 Seacutebillot et al 2014)

Several tools have been generated to achieve temporal aswell as spatial control of transgene expression Ubiquitousgene expression for a given time period can be achievedusing heat shock promoters The endogenous hsp70 pro-moter from zebrafish (Blechinger et al 2002) as well asartificially constructed heatshock promoters (Bajoghli et al2004) provide temporal control to transgene expression Forexample a heatshock by elevating the ambient temperaturefrom 28 to 37 for 20 min results in a strong activation ofthe heatshock promoter in medaka embryos (Oda et al2010 Herder et al 2013) Additionally heatshocks can beapplied locally using a hot soldering iron (Hardy et al 2007)or infrared (IR) laser light with the IR-laser-evoked geneoperator (LEGO) system (Kamei et al 2008 Deguchi et al2009 Okuyama et al 2013) Given the transparency of themedaka model the temperature can be increased locallyusing infrared laser light with single-cell resolution therebyactivating a heatshock promoter only in that cell This allowsprecise fate-mapping studies (Shimada et al 2013) In thisstudy the expression of Cre recombinase is initiated via IR-LEGO in cells of the somites These cells are permanentlylabeled by Cre-mediated induction of GFP expression andultimately reveal the mesodermal origin of the trunk

Genetic Toolbox Review 911

exoskeleton Heat-inducible Cre transgenic lines and Cre-mediated GFP expression lines (TG918 and TG891 respec-tively) are available from NBRP (httpwwwshigennigacjpmedaka) Alternatively inducible transactivation sys-tems can be used However the use of such systems inmedaka is still quite limited A heatshock-inducible Gal4upstream activating sequence (UAS) transactivation systemhas been published (Grabher and Wittbrodt 2004) Otherinducible systems that are used in zebrafish such as mife-pristone inducible hybrid transcription factor LexPR systems(Emelyanov and Parinov 2008) or the Tet (tetracycline-controlled transcriptional regulation) OnOff system (Knopfet al 2010) are functional in medaka (J Wittbrodt unpub-lished data) Thus several driver lines for the Tet transacti-vation system are available at the Medaka Stock Center ofNBRP (httpwwwshigennigacjpmedaka)

Temporal control of transgene activation can also beachieved using site-specific recombinases Transgene activa-tion can be achieved by recombination-based removal ofa stop cassette or fluorescence reporter gene that separatesthe gene of interest from a promoter These cassettes areflanked by the recognition sites for recombinases such as loxP(Matsuzaki et al 2013) or FRT (Itou et al 2009) Recombi-nase activity can be induced using specific promoters heat-shock (Shimada et al 2013 Kobayashi et al 2013 Okuyamaet al 2013 Centanin et al 2014) or drug admission(Yoshinari et al 2012) In contrast to the tools describedabove in this case transgene activation is irreversible afterthe activation of recombinase In most cases Cre or Flprecombinase have been used for this purpose These site-specific recombinases recognize specific DNA sequencesthe recognition target sites (RTSs) For Cre and Flp recom-binases from the A1 family specific RTSs exist They have

a defined structure with two inverted repeat regions arounda central core Heterologous sites exist in which the se-quence of the central core differs Recombination can onlyoccur between sites with identical central core sequencesThis allows the combinatorial use of heterologous RTSsDepending on the orientation to each other recombinationbetween two RTSs either leads to deletion or inversion ofthe sequence in between (reviewed in Branda and Dymecki2004)

Site-specific recombinases are also used to mediatestochastic activation of transgene expression A prominentexample that relies on this approach is the brainbow system(Livet et al 2007 Pan et al 2013) Here recombinaseactivity leads to a rearrangement of the transgene in a sto-chastic manner such that different fluorescent proteins areexpressed from the transgene in different cells (Figure 4C)Multiple copies of the cassette in the genome lead to thestochastic combination of expressed fluorescent proteins en-abling color barcoding of cells (Houmlckendorf et al 2012 Panet al 2013) So far the functionality of stochastic cell label-ing using recombinases in teleosts has been demonstrated inzebrafish (Gupta and Poss 2012 Pan et al 2013) and inmedaka (Centanin et al 2014) This medaka toolbox Gaudiacuteallows a lineage analysis of individual cells by stochasticlabeling of cells at embryonic or adult stages The toolkitconsists of several ubiquituously expressing brainbow lineswith heatshock- or tamoxifen-inducible Cre driver lines

In many cases it is important to label cells that express anuntagged transgene To achieve this vectors harboringreporter gene constructs can be co-injected with othertransgene-containing vectors using a meganuclease- ortransposase-based approach (Rembold et al 2006a) Thedifferent vectors mostly cosegregate in the progeny of the

Figure 4 Gene expression and cell line-age analysis in medaka (A) Whole-mount in situ hybridization to specificallydetect retinal homeobox gene Rx2mRNA in the retina Rx2 (red) isexpressed in photoreceptor cells (PR)stem cells (RCS) in the ciliary marginalzone of the retina and Mueller glia cells(MGC) L lens Glutamine synthase pro-tein in Mueller glia cells is codetected byantibody staining (white) indicating Rx2expression in Mueller glia cells (red dotsinside white MGCs) (B) Recombinationscheme the GFP reporter gene is pres-ent in the PhiC31 landing site The reg-ulatory DNA to drive GFP reporter geneexpression is provided with a targetingvector PhiC31 integrase activity leads tothe recombination of the regulatoryDNA into the GFP reporter gene cas-sette in the genome In vivo imagingof neurobal GFP expression from a

brain-specific enhancer within a PhiC31 landing site Note autofluorescent pigment cells (red) (C) In vivo analysis of 8-dpf Gaudiacute fish Stochasticrecombination of a brainbow cassette by heatshock-activatable Cre recombinase results in a differential labeling of cells in the entire body A 3Drepresentation of the somites is shown (D) A whole-mount BrdU incorporation assay in a 10-dpf hatchling reveals proliferating cells (yellow) in the bodyNuclei are counterstained with Dapi (blue)

912 S Kirchmaier et al

exoskeleton Heat-inducible Cre transgenic lines and Cre-mediated GFP expression lines (TG918 and TG891 respec-tively) are available from NBRP (httpwwwshigennigacjpmedaka) Alternatively inducible transactivation sys-tems can be used However the use of such systems inmedaka is still quite limited A heatshock-inducible Gal4upstream activating sequence (UAS) transactivation systemhas been published (Grabher and Wittbrodt 2004) Otherinducible systems that are used in zebrafish such as mife-pristone inducible hybrid transcription factor LexPR systems(Emelyanov and Parinov 2008) or the Tet (tetracycline-controlled transcriptional regulation) OnOff system (Knopfet al 2010) are functional in medaka (J Wittbrodt unpub-lished data) Thus several driver lines for the Tet transacti-vation system are available at the Medaka Stock Center ofNBRP (httpwwwshigennigacjpmedaka)

Temporal control of transgene activation can also beachieved using site-specific recombinases Transgene activa-tion can be achieved by recombination-based removal ofa stop cassette or fluorescence reporter gene that separatesthe gene of interest from a promoter These cassettes areflanked by the recognition sites for recombinases such as loxP(Matsuzaki et al 2013) or FRT (Itou et al 2009) Recombi-nase activity can be induced using specific promoters heat-shock (Shimada et al 2013 Kobayashi et al 2013 Okuyamaet al 2013 Centanin et al 2014) or drug admission(Yoshinari et al 2012) In contrast to the tools describedabove in this case transgene activation is irreversible afterthe activation of recombinase In most cases Cre or Flprecombinase have been used for this purpose These site-specific recombinases recognize specific DNA sequencesthe recognition target sites (RTSs) For Cre and Flp recom-binases from the A1 family specific RTSs exist They have

a defined structure with two inverted repeat regions arounda central core Heterologous sites exist in which the se-quence of the central core differs Recombination can onlyoccur between sites with identical central core sequencesThis allows the combinatorial use of heterologous RTSsDepending on the orientation to each other recombinationbetween two RTSs either leads to deletion or inversion ofthe sequence in between (reviewed in Branda and Dymecki2004)

Site-specific recombinases are also used to mediatestochastic activation of transgene expression A prominentexample that relies on this approach is the brainbow system(Livet et al 2007 Pan et al 2013) Here recombinaseactivity leads to a rearrangement of the transgene in a sto-chastic manner such that different fluorescent proteins areexpressed from the transgene in different cells (Figure 4C)Multiple copies of the cassette in the genome lead to thestochastic combination of expressed fluorescent proteins en-abling color barcoding of cells (Houmlckendorf et al 2012 Panet al 2013) So far the functionality of stochastic cell label-ing using recombinases in teleosts has been demonstrated inzebrafish (Gupta and Poss 2012 Pan et al 2013) and inmedaka (Centanin et al 2014) This medaka toolbox Gaudiacuteallows a lineage analysis of individual cells by stochasticlabeling of cells at embryonic or adult stages The toolkitconsists of several ubiquituously expressing brainbow lineswith heatshock- or tamoxifen-inducible Cre driver lines

In many cases it is important to label cells that express anuntagged transgene To achieve this vectors harboringreporter gene constructs can be co-injected with othertransgene-containing vectors using a meganuclease- ortransposase-based approach (Rembold et al 2006a) Thedifferent vectors mostly cosegregate in the progeny of the

Figure 4 Gene expression and cell line-age analysis in medaka (A) Whole-mount in situ hybridization to specificallydetect retinal homeobox gene Rx2mRNA in the retina Rx2 (red) isexpressed in photoreceptor cells (PR)stem cells (RCS) in the ciliary marginalzone of the retina and Mueller glia cells(MGC) L lens Glutamine synthase pro-tein in Mueller glia cells is codetected byantibody staining (white) indicating Rx2expression in Mueller glia cells (red dotsinside white MGCs) (B) Recombinationscheme the GFP reporter gene is pres-ent in the PhiC31 landing site The reg-ulatory DNA to drive GFP reporter geneexpression is provided with a targetingvector PhiC31 integrase activity leads tothe recombination of the regulatoryDNA into the GFP reporter gene cas-sette in the genome In vivo imagingof neurobal GFP expression from a

brain-specific enhancer within a PhiC31 landing site Note autofluorescent pigment cells (red) (C) In vivo analysis of 8-dpf Gaudiacute fish Stochasticrecombination of a brainbow cassette by heatshock-activatable Cre recombinase results in a differential labeling of cells in the entire body A 3Drepresentation of the somites is shown (D) A whole-mount BrdU incorporation assay in a 10-dpf hatchling reveals proliferating cells (yellow) in the bodyNuclei are counterstained with Dapi (blue)

912 S Kirchmaier et al

injected cell thereby leading to coexpression Alternativelyinternal ribosome entry sites (IRESs) have been used success-fully (Fukamachi et al 2009 Matsuzaki et al 2013) HoweverIRESs appear to have context-dependent limitations (Tanakaand Kinoshita 2001) Pseudopolycistronic open readingframes separated by short viral sequences (such as T2A)allow a more robust coexpression of several proteins froma single transgene in equal amounts (Provost et al 2007)The conformation of these short viral peptides inducea cotranslational staggering of the ribosome and resultinglack of a peptide bond formation in the nascent polypeptidechain This results in a physical separation of the polypep-tide chains The use of T2A constructs circumvents the needto use fusion proteins and thereby precludes potential arti-facts resulting from the fusion Recently this technique hasbeen used to label medaka retinal cells that ectopically ex-press variants of small GTPases (Herder et al 2013) Alsothis technique has been utilized in medaka to visualize Creexpression by mCherry fluorescence (Yoshinari et al 2012)

Gene Expression Studies

Established protocols are available for gene expressionstudies either in whole mount embryos or sections Thesein situ hybridization methods use antisense RNA probes la-beled with either fluorescein or digoxygenin to visualizecomplementary RNA molecules in fixed tissues Detectionrelies on color reactions using either alkaline phosphatase-or horseradish peroxidase-coupled antibodies (Henrich et al2005) To analyze coexpression of genes at high resolutionwith confocal microscopy peroxidase-dependent tyramidesignal amplification (PerkinElmer) linked to fluorescentdyes can be used Antisense locked nucleic acid probes havebeen successfully used to visualize the expression of miRNAsin medaka (Ason et al 2006) Detection of mRNAs by in situhybridization can also be coupled with detection of proteinsby antibodies using immunocytochemistry (Figure 4A)(Inoue and Wittbrodt 2011)

The number of available antibodies that are specific formedaka proteins is still limited Nevertheless a largenumber of antibodies against mouse or human epitopes hasbeen shown to be functional for immunocytochemistry inmedaka Furthermore a heatshock-based epitope retrievalstep has been established (Inoue and Wittbrodt 2011) Thisprotocol improves the immunostaining for several antibod-ies by a simple heatshock-based epitope retrieval stepWhile the aforementioned methods are useful to studya limited number of genes genome-wide analysis of geneexpression has been performed in medaka using microar-rays as well as RNAseq Especially DNA oligonucleotidemicroarrays have been used to study the impact of toxiccompounds on gene expression (Kishi et al 2006) RNAseqhas been used to study the transcriptome of melanoma ina medaka tumor model The authors were able to identifyalternatively spliced transcripts between different mela-noma subtypes (Schartl et al 2012) RNAseq in combination

with the analysis of epigenetic marks (histone modifica-tions) was successfully applied for a comparative study toanalyze gene expression during the phylotypic period ofmedaka and zebrafish development (Tena et al 2014)The authors found that similarities in gene expression cor-relate with the phylotypic period and the existence of keyconstrained nodes in the gene networks governing the ver-tebrate body plan has been postulated

Cell Transplantation

Cell transplantation is used to efficiently generate chimericindividuals (Ho and Kane 1990 Hong et al 1998) Thisapproach is frequently used to study cell lineages and toaddress cell autonomy vs nonautonomy of a given process(Winkler et al 2000) Fish model systems such as medakaand zebrafish are especially well suited for transplantationassays due to the extrauterine development of the relativelylarge eggs To generate chimeric embryos cells are trans-planted at the blastula stage from a donor to a host embryousing glass needles The extent of chimeric tissue can bemanipulated by the number of transplanted cells Further-more the developmental potential and determinationof cells can be tested by heterotopic transplantation wherecells are transplanted to an ectopic position Transplantedcells are labeled by either stable inert compounds such asfluorescein-dextran or by genetic means Frequently ubiqui-tous or specific promotors driving expression of fluorescentproteins are used to label donor- and host-derived cells(Rembold et al 2006b Centanin et al 2011)

By cell transplantation into adult fish a melanoma modelwas established in medaka (Hasegawa et al 2009) HereGFP-labeled melanoma cells were introduced into adult fishand thus tumor development was visualized This allowedthe study of tumor cell behavior such as proliferation ormetastasis in vivo

Furthermore cell transplantation can be used to obtaindonor-derived gonads in sterile hosts For example cells offertile donors can be transplanted into sterile Oryziaslatipes 3 Oryzias curvinotus hybrids The transplanted cellscan then give rise to gonads in the otherwise sterile hostShimada et al (2007) used this approach to obtain maternal-zygotic mutant embryos that allowed the study of maternalcontribution to fgfr1 function

Resources

An excellent source of information is Kinoshita et al 2009Researchers working with medaka can rely on a wide

range of resources that are coordinated and provided by theNBRP Medaka NBRP Medaka which was initiated in 2002is a central repository and archive for medaka resources(Sasado et al 2010) The core facilities of NBRP Medaka arethe National Institute for Basic Biology (NIBB OkazakiJapan) Niigata University Riken and Miyazaki UniversityNBRP Medaka provides three different resources namely

Genetic Toolbox Review 913

medaka strains genomic resources and hatching enzymes(httpwwwshigennigacjpmedaka) As of 2014 NBRPMedaka provides 548 medaka strains including 373mutants 74 transgenic lines 11 inbred lines 6 consomiclines (chromosome replacement between two different in-bred lines) 65 wild stocks and 20 medaka-related species(httpwwwshigennigacjpmedakastrainstrainTopjspjsessionid=A637DE046C1536C0251485E216900ECF)NBRP Medaka also provides medaka complementary DNA(cDNA) clones and transcriptome data (httpwwwshigennigacjpmedakaestestjsp) At this website 730259sequences from 33 cDNA libraries including 11 full-lengthcDNA libraries are available Furthermore BAC and fosmidlibraries of O latipes and BAC libraries of O dancena Oluzonensis O celebenesis O hubbsi and O javanicus areavailable End sequence data of O latipes BAC and fosmidclones allow in silico identification of the clones of interestby blast similarity search (httpwwwshigennigacjpmedakaestblastjsp) as well as using a genome browser (httpviewershigeninfomedakavwmapview) In addition 3DPCR pools of all BAC libraries are available Three-dimensional screening requests for libraries should be sub-mitted to NBRP Medaka (mbrcnibbacjp)

The NBRP Medaka website provides several databasesthe Medaka Book is an online lab-manual with basictechniques for medaka maintenance and husbandry as wellas basic and advanced laboratory protocols the Medaka At-las describes the brain anatomy and blood vascular systemthe Medaka Tree depicts phylogenetic relationships of me-daka wild population and medaka related species based onmitochondrial DNA sequences Furthermore NBRP Medakahosts genome browsers Medaka Map (beta) (httpviewershigeninfomedakavwmapview) is a genome browser withseveral useful tools physical maps alignment view blastsearch in silico PCR sequence cutter pattern match-ZFNpattern match CRISPR and search for CRISPR target sitesTILLING provides the basic information of the medaka TILL-ING library and information for its screening (httpwwwshigennigacjpmedakastrainstrainTillingPlateActiondo)

The medaka chorion is very hard and thus has to beremoved for many experimental manipulations of embryosThe most effective way to remove the chorion is usingmedaka hatching enzyme Medaka hatching enzyme is notcommercially available however NBRP Medaka is providinghatching enzyme as a crude hatching solution (supernatantof hatching embryos)

Cryopreservation of Sperm and Primordial GermCells

Medaka sperm cryopreservation is well established androutinely used for long-term preservation of medaka fishstrains Sperm that has been cryopreserved for 10 yearsshows no decrease of sperm motility and fertilization rate Adetailed protocol for sperm cryopreservation is described inKinoshita et al 2009

Medaka eggs as many other teleost eggs are very largeand yolk rich compared to mammalian eggs Thus cryopres-ervation of mature eggs has not been successful so farHowever primordial germ cells (PGCs) of fish can producesperm and eggs after germ cell transplantation In rainbowtrout PGCs were successfully cryopreserved and transplantedto hosts from which donor-derived offspring were obtained(Kobayashi et al 2007) Recently it was shown that eggs andsperm could be generated from cryopreserved whole testes ofrainbow trout (Lee et al 2013) Seki and colleagues foundthat this protocol is applicable to medaka (S Seki personalcommunication) Thus cryopreservation of whole testes andsubsequent transplantation of these cryopreserved to hostgonads will allow the cryopreservation of medaka strainsThis will allow the long-term storage of live inbred strains

Future and Perspectives

Medaka offers a number of unique features and resources thatin combination with established state-of-the art molecular-genetic tools make it an important vertebrate model A centuryof genetic studies has provided a rich resource of strains andgenomic data that serve as a basis for genetic studies such asinteraction with environment and dissecion of complex traitsA wide range of live and genomic resources as well as datarepositories have been centralized and are available throughNBRP Medaka Newly established tools enable targetedmanipulation of gene function Specific genome manipulationsare now possible with the newly established CRISPRCas9 aswell as ZNF and TALEN systems Importantly these novelapproaches enable homologous recombination as a meansfor precise genomic manipulations in medaka Closelyrelated species from a wide range of habitats provide theresources for comparative studies addressing evolution andspeciation This will be complemented with comparativestudies using other established teleost genetic models tostudy vertebrate evolution Finally a panel of inbred lines isin the making that will serve as a medaka reference panel tostudy multigenic traits by GWAS

Acknowledgments

JW was supported by the ERC Advanced Grant - Manipulatingand Imaging Stem Cells at Work KN acknowledges NBRP(Core Facility Upgrading Program Genome InformationUpgrading Program Project and Fundamental TechnologiesUpgrading Program)

Literature Cited

Abe G M L Suster and K Kawakami 2011 Tol2-mediatedtransgenesis gene trapping enhancer trapping and the Gal4-UAS system Methods Cell Biol 104 23ndash49

Ando H T Furuta R Y Tsien and H Okamoto 2001 Photo-mediated gene activation using caged RNADNA in zebrafishembryos Nat Genet 28 317ndash325

914 S Kirchmaier et al

medaka strains genomic resources and hatching enzymes(httpwwwshigennigacjpmedaka) As of 2014 NBRPMedaka provides 548 medaka strains including 373mutants 74 transgenic lines 11 inbred lines 6 consomiclines (chromosome replacement between two different in-bred lines) 65 wild stocks and 20 medaka-related species(httpwwwshigennigacjpmedakastrainstrainTopjspjsessionid=A637DE046C1536C0251485E216900ECF)NBRP Medaka also provides medaka complementary DNA(cDNA) clones and transcriptome data (httpwwwshigennigacjpmedakaestestjsp) At this website 730259sequences from 33 cDNA libraries including 11 full-lengthcDNA libraries are available Furthermore BAC and fosmidlibraries of O latipes and BAC libraries of O dancena Oluzonensis O celebenesis O hubbsi and O javanicus areavailable End sequence data of O latipes BAC and fosmidclones allow in silico identification of the clones of interestby blast similarity search (httpwwwshigennigacjpmedakaestblastjsp) as well as using a genome browser (httpviewershigeninfomedakavwmapview) In addition 3DPCR pools of all BAC libraries are available Three-dimensional screening requests for libraries should be sub-mitted to NBRP Medaka (mbrcnibbacjp)

The NBRP Medaka website provides several databasesthe Medaka Book is an online lab-manual with basictechniques for medaka maintenance and husbandry as wellas basic and advanced laboratory protocols the Medaka At-las describes the brain anatomy and blood vascular systemthe Medaka Tree depicts phylogenetic relationships of me-daka wild population and medaka related species based onmitochondrial DNA sequences Furthermore NBRP Medakahosts genome browsers Medaka Map (beta) (httpviewershigeninfomedakavwmapview) is a genome browser withseveral useful tools physical maps alignment view blastsearch in silico PCR sequence cutter pattern match-ZFNpattern match CRISPR and search for CRISPR target sitesTILLING provides the basic information of the medaka TILL-ING library and information for its screening (httpwwwshigennigacjpmedakastrainstrainTillingPlateActiondo)

The medaka chorion is very hard and thus has to beremoved for many experimental manipulations of embryosThe most effective way to remove the chorion is usingmedaka hatching enzyme Medaka hatching enzyme is notcommercially available however NBRP Medaka is providinghatching enzyme as a crude hatching solution (supernatantof hatching embryos)

Cryopreservation of Sperm and Primordial GermCells

Medaka sperm cryopreservation is well established androutinely used for long-term preservation of medaka fishstrains Sperm that has been cryopreserved for 10 yearsshows no decrease of sperm motility and fertilization rate Adetailed protocol for sperm cryopreservation is described inKinoshita et al 2009

Medaka eggs as many other teleost eggs are very largeand yolk rich compared to mammalian eggs Thus cryopres-ervation of mature eggs has not been successful so farHowever primordial germ cells (PGCs) of fish can producesperm and eggs after germ cell transplantation In rainbowtrout PGCs were successfully cryopreserved and transplantedto hosts from which donor-derived offspring were obtained(Kobayashi et al 2007) Recently it was shown that eggs andsperm could be generated from cryopreserved whole testes ofrainbow trout (Lee et al 2013) Seki and colleagues foundthat this protocol is applicable to medaka (S Seki personalcommunication) Thus cryopreservation of whole testes andsubsequent transplantation of these cryopreserved to hostgonads will allow the cryopreservation of medaka strainsThis will allow the long-term storage of live inbred strains

Future and Perspectives

Medaka offers a number of unique features and resources thatin combination with established state-of-the art molecular-genetic tools make it an important vertebrate model A centuryof genetic studies has provided a rich resource of strains andgenomic data that serve as a basis for genetic studies such asinteraction with environment and dissecion of complex traitsA wide range of live and genomic resources as well as datarepositories have been centralized and are available throughNBRP Medaka Newly established tools enable targetedmanipulation of gene function Specific genome manipulationsare now possible with the newly established CRISPRCas9 aswell as ZNF and TALEN systems Importantly these novelapproaches enable homologous recombination as a meansfor precise genomic manipulations in medaka Closelyrelated species from a wide range of habitats provide theresources for comparative studies addressing evolution andspeciation This will be complemented with comparativestudies using other established teleost genetic models tostudy vertebrate evolution Finally a panel of inbred lines isin the making that will serve as a medaka reference panel tostudy multigenic traits by GWAS

Acknowledgments

JW was supported by the ERC Advanced Grant - Manipulatingand Imaging Stem Cells at Work KN acknowledges NBRP(Core Facility Upgrading Program Genome InformationUpgrading Program Project and Fundamental TechnologiesUpgrading Program)

Literature Cited

Abe G M L Suster and K Kawakami 2011 Tol2-mediatedtransgenesis gene trapping enhancer trapping and the Gal4-UAS system Methods Cell Biol 104 23ndash49

Ando H T Furuta R Y Tsien and H Okamoto 2001 Photo-mediated gene activation using caged RNADNA in zebrafishembryos Nat Genet 28 317ndash325

914 S Kirchmaier et al

Ansai S and M Kinoshita 2014 Targeted mutagenesis usingCRISPRCas system in medaka Biol Open 3 362ndash371

Asai T H Senou and K Hosoya 2011 Oryzias sakaizumiia new ricefish from northern Japan (Teleostei Adrianichthyi-dae) Ichthyol Explor Freshwat 22 289ndash299

Ansai S H Ochiai Y Kanie Y Kamei Y Gou et al2012 Targeted disruption of exogenous EGFP gene in medakausing zinc-finger nucleases Dev Growth Differ 54 546ndash556

Ansai S T Sakuma T Yamamoto H Ariga N Uemura et al2013 Efficient targeted mutagenesis in medaka usingcustom-designed transcription activator-like effector nucleasesGenetics 193 739ndash749

Ansai S K Inohaya Y Yoshiura M Schartl N Uemura et al2014 Design evaluation and screening methods for efficienttargeted mutagenesis with transcription activator-like effectornucleases in medaka Dev Growth Differ 56 98ndash107

Ason B D K Darnell B Wittbrodt E Berezikov W P Kloostermanet al 2006 Differences in vertebrate microRNA expressionProc Natl Acad Sci USA 103 14385ndash14389

Auer T O K Duroure A De Cian J-P Concordet and F delBene 2014 Highly efficient CRISPRCas9-mediated knock-inin zebrafish by homology-independent DNA repair Genome Res24 142ndash153

Auman H J H Coleman H E Riley F Olale H-J Tsai et al2007 Functional modulation of cardiac form through region-ally confined cell shape changes PLoS Biol 5 e53

Bajoghli B N Aghaallaei T Heimbucher and T Czerny2004 An artificial promoter construct for heat-inducible mis-expression during fish embryogenesis Dev Biol 271 416ndash430

Bedell V M Y Wang J M Campbell T L Poshusta C G Starkeret al 2012 In vivo genome editing using a high-efficiencyTALEN system Nature 491 114ndash118

Bill B R A M Petzold K J Clark L A Schimmenti and S CEkker 2009 A primer for morpholino use in zebrafish Zebra-fish 6 69ndash77

Blechinger S R T G Evans P T Tang J Y Kuwada J T Warrenet al 2002 The heat-inducible zebrafish hsp70 gene is ex-pressed during normal lens development under non-stress con-ditions Mech Dev 112 213ndash215

Boon Ng G H and Z Gong 2011 Maize AcDs transposon sys-tem leads to highly efficient germline transmission of transgenesin medaka (Oryzias latipes) Biochimie 93 1858ndash1864

Branda C S and S M Dymecki 2004 Talking about a revolu-tion the impact of site-specific recombinases on genetic analy-ses in mice Dev Cell 6 7ndash28

Centanin L B Hoeckendorf and J Wittbrodt 2011 Fate restrictionand multipotency in retinal stem cells Cell Stem Cell 9 553ndash562

Centanin L J-J Ander B Hoeckendorf K Lust T Kellner et al2014 Exclusive multipotency and preferential asymmetric di-visions in post-embryonic neural stem cells of the fish retinaDevelopment 141 3472ndash3482

Cermak T E L Doyle M Christian L Wang Y Zhang et al2011 Efficient design and assembly of custom TALEN andother TAL effector-based constructs for DNA targeting NucleicAcids Res 39 e82

Cheung C H Y J M Y Chiu and R S S Wu 2014 Hypoxiaturns genotypic female medaka fish into phenotypic males Eco-toxicology 23 1260ndash1269

Cong L F A Ran D Cox S Lin R Barretto et al2013 Multiplex genome engineering using CRISPRCas sys-tems Science (New York NY) 339 819ndash823

Conte I S Carrella R Avellino M Karali R Marco-Ferreres et al2010 miR-204 is required for lens and retinal development viaMeis2 targeting Proc Natl Acad Sci USA 107 15491ndash15496

De Rienzo G J H Gutzman and H Sive 2012 Efficient shRNA-mediated inhibition of gene expression in zebrafish Zebrafish 997ndash107

Deguchi T M Itoh H Urawa T Matsumoto S Nakayama et al2009 Infrared laser-mediated local gene induction in medaka ze-brafish and Arabidopsis thaliana Dev Growth Differ 51 769ndash775

Dong M Y-F Fu T-T Du C-B Jing C-T Fu et al2009 Heritable and lineage-specific gene knockdown in zebra-fish embryo PLoS ONE 4 e6125

Dorn S N Aghaallaei G Jung B Bajoghli B Werner et al2012 Side chain modified peptide nucleic acids (PNA) forknock-down of six3 in medaka embryos BMC Biotechnol 1250

Emelyanov A and S Parinov 2008 Mifepristone-inducibleLexPR system to drive and control gene expression in transgeniczebrafish Dev Biol 320 113ndash121

ENCODE Project Consortium B E Bernstein E Birney I DunhamE D Green et al 2012 An integrated encyclopedia of DNAelements in the human genome Nature 489 57ndash74

Froschauer A D Sprott F Gerwien Y Henker F Rudolph et al2012 Effective generation of transgenic reporter and gene traplines of the medaka (Oryzias latipes) using the AcDs transpo-son system Transgenic Res 21 149ndash162

Fukamachi S T Yada A Meyer and M Kinoshita 2009 Effectsof constitutive expression of somatolactin alpha on skin pigmen-tation in medaka Gene 442 81ndash87

Furutani-Seiki M T Sasado C Morinaga H Suwa K Niwa et al2004 A systematic genome-wide screen for mutations affect-ing organogenesis in Medaka Oryzias latipes Mech Dev 121647ndash658

Gaj T C A Gersbach and C F Barbas 2013 ZFN TALEN andCRISPRCas-based methods for genome engineering TrendsBiotechnol 31 397ndash405

Grabher C and J Wittbrodt 2004 Efficient activation of geneexpression using a heat-shock inducible Gal4Vp16-UAS systemin medaka BMC Biotechnol 4 26

Grabher C and J Wittbrodt 2007 Meganuclease and transposonmediated transgenesis in medaka Genome Biol 8(Suppl 1) S10

Grabher C and J Wittbrodt 2008 Recent advances in meganu-clease-and transposon-mediated transgenesis of medaka andzebrafish Methods Mol Biol 461 521ndash539

Grabher C T Henrich T Sasado A Arenz J Wittbrodt et al2003 Transposon-mediated enhancer trapping in medakaGene 322 57ndash66

Gupta V and K D Poss 2012 Clonally dominant cardiomyo-cytes direct heart morphogenesis Nature 484 479ndash484

Hardy M E L V Ross and C-B Chien 2007 Focal gene mis-expression in zebrafish embryos induced by local heat shockusing a modified soldering iron Dev Dyn 236 3071ndash3076

Hasegawa S K Maruyama H Takenaka T Furukawa and TSaga 2009 A medaka model of cancer allowing direct obser-vation of transplanted tumor cells in vivo at a cellular-levelresolution Proc Natl Acad Sci USA 106 13832ndash13837

Hayashi Y H Kobira T Yamaguchi E Shiraishi T Yazawa et al2010 High temperature causes masculinization of geneticallyfemale medaka by elevation of cortisol Mol Reprod Dev 77679ndash686

Henrich T M Ramialison B Wittbrodt B Assouline F Bourratet al 2005 MEPD a resource for medaka gene expressionpatterns Bioinformatics 21 3195ndash3197

Herder C J M Swiercz C Muumlller R Peravali R Quiring et al2013 ArhGEF18 regulates RhoA-Rock2 signaling to maintainneuro-epithelial apico-basal polarity and proliferation Develop-ment 140 2787ndash2797

Ho R K and D A Kane 1990 Cell-autonomous action of zebra-fish spt-1 mutation in specific mesodermal precursors Nature348 728ndash730

Hong Y H C Winkler and M Schartl 1998 Production of med-akafish chimeras from a stable embryonic stem cell line ProcNatl Acad Sci USA 95 3679ndash3684

Genetic Toolbox Review 915

Houmlckendorf B T Thumberger and J Wittbrodt 2012 Quantitativeanalysis of embryogenesis a perspective for light sheet micros-copy Dev Cell 23 1111ndash1120

Hyodo-Taguchi Y 1980 Establishment of inbred strains of theteleost Oryzias latipes Zoological Magazine 89 283ndash301

Ieki T S Okada Y Aihara M Ohmoto K Abe et al2013 Transgenic labeling of higher order neuronal circuitslinked to phospholipase C-b2-expressing taste bud cells in me-daka fish J Comp Neurol 521 1781ndash1802

Inoue D and J Wittbrodt 2011 One for all a highly efficientand versatile method for fluorescent immunostaining in fishembryos PLoS ONE 6 e19713

Inoue K and Y Takei 2002 Diverse adaptability in oryzias spe-cies to high environmental salinity Zoolog Sci 19 727ndash734

Inoue K S Yamashita J Hata S Kabeno S Asada et al1990 Electroporation as a new technique for producing trans-genic fish Cell Differ Dev 29 123ndash8

Ishikawa Y 2000 Medakafish as a model system for vertebratedevelopmental genetics BioEssays 22 487ndash495

Ishikawa Y M Yoshimoto N Yamamoto and H Ito1999 Different brain morphologies from different genotypesin a single teleost species the medaka (Oryzias latipes) BrainBehav Evol 53 2ndash9

Itou J M Suyama Y Imamura T Deguchi K Fujimori et al2009 Functional and comparative genomics analyses ofpmp22 in medaka fish BMC Neurosci 10 60

Ivics Z P B Hackett R H Plasterk and Z Izsvaacutek1997 Molecular reconstruction of Sleeping Beauty a Tc1-liketransposon from fish and its transposition in human cells Cell91 501ndash510

Kamei Y M Suzuki K Watanabe K Fujimori T Kawasaki et al2008 Infrared laser-mediated gene induction in targeted sin-gle cells in vivo Nat Methods 6 79ndash81

Kasahara M K Naruse S Sasaki Y Nakatani W Qu et al2007 The medaka draft genome and insights into vertebrategenome evolution Nature 447 714ndash719

Kawaguchi M K Misaki H Sato T Yokokawa T Itai et al2012 Transgenic medaka small fresh water teleost (Oryziaslatipes) is now available for environmental science Interdisci-plinary Studies on Environmental Chemistry EnvironmentalPollution and Ecotoxicology 49ndash54 Volume 6 TERRAPUBTokyo

Kelly A and A F Hurlstone 2011 The use of RNAi technologiesfor gene knockdown in zebrafish Brief Funct Genomics 10189ndash196

Kelsh R N C Inoue A Momoi H Kondoh M Furutani-Seikiet al 2004 The Tomita collection of medaka pigmentationmutants as a resource for understanding neural crest cell de-velopment Mech Dev 121 841ndash859

Kimura T A Shimada N Sakai H Mitani K Naruse et al2007 Genetic analysis of craniofacial traits in the medaka Ge-netics 177 2379ndash2388

Kimura T Y Nagao H Hashimoto Y-I Yamamoto-Shiraishi SYamamoto et al 2014 Leucophores are similar to xantho-phores in their specification and differentiation processes in me-daka Proc Natl Acad Sci USA 111 7343ndash7348

Kinoshita M M Yamauchi M Sasanuma Y Ishikawa T Osadaet al 2003 A transgene and its expression profile are stablytransmitted to offspring in transgenic medaka generated by theparticle gun method Zoolog Sci 20 869ndash875

Kinoshita M K Murata K Naruse and M Tanaka2009 Medaka Biology Management and Experimental Proto-cols Wiley-Blackwell USA

Kirchmaier S B Houmlckendorf E K Moumlller D Bornhorst F Spitzet al 2013 Efficient site-specific transgenesis and enhanceractivity tests in medaka using PhiC31 integrase Development140 4287ndash4295

Kishi K E Kitagawa N Onikura A Nakamura and H Iwahashi2006 Expression analysis of sex-specific and 17beta-estradiol-responsive genes in the Japanese medaka Oryzias latipes usingoligonucleotide microarrays Genomics 88 241ndash251

Knapik E W 2000 ENU mutagenesis in zebrafish from genes tocomplex diseases Mamm Genome 11 511ndash519

Knopf F K Schnabel C Haase K Pfeifer K Anastassiadis et al2010 Dually inducible TetON systems for tissue-specific con-ditional gene expression in zebrafish Proc Natl Acad Sci USA107 19933ndash19938

Kobayashi K Y Kamei M Kinoshita T Czerny and M Tanaka2013 A heat-inducible CRELOXP gene induction system inmedaka Genesis 51 59ndash67

Kobayashi T Y Takeuchi T Takeuchi and G Yoshizaki2007 Generation of viable fish from cryopreserved primordialgerm cells Mol Reprod Dev 74 207ndash213

Koger C S S J Teh and D E Hinton 1999 Variations of lightand temperature regimes and resulting effects on reproductiveparameters in medaka (Oryzias latipes) Biol Reprod 61 1287ndash1293

Kok F O M Shin C-W Ni A Gupta A S Grosse et al2014 Reverse genetic screening reveals poor correlation be-tween morpholino-induced and mutant phenotypes in zebrafishDev Cell 32 97ndash108

Lee S Y Iwasaki S Shikina and G Yoshizaki 2013 Generationof functional eggs and sperm from cryopreserved whole testesProc Natl Acad Sci USA 110 1640ndash1645

Liu T L Liu Q Wei and Y Hong 2011 Sperm nuclear transferand transgenic production in the fish medaka Int J Biol Sci 7469ndash475

Livet J T A Weissman H Kang R W Draft J Lu et al2007 Transgenic strategies for combinatorial expression offluorescent proteins in the nervous system Nature 450 56ndash62

Loosli F R W Koumlster M Carl R Kuumlhnlein T Henrich et al2000 A genetic screen for mutations affecting embryonic de-velopment in medaka fish (Oryzias latipes) Mech Dev 97133ndash139

Ma A C H B Lee K J Clark and S C Ekker 2013 Highefficiency in vivo genome engineering with a simplified 15-RVD GoldyTALEN design PLoS ONE 8 e65259

Mali P L Yang K M Esvelt J Aach M Guell et al 2013 RNA-guided human genome engineering via Cas9 Science (NewYork NY) 339 823ndash826

Martinez-Morales J-R M Rembold K Greger J C Simpson K EBrown et al 2009 ojoplano-mediated basal constriction is es-sential for optic cup morphogenesis Development 136 2165ndash2175

Matsuda M Y Nagahama A Shinomiya T Sato C Matsudaet al 2002 DMY is a Y-specific DM-domain gene requiredfor male development in the medaka fish Nature 417 559ndash563

Matsuda M T Sato Y Toyazaki Y Nagahama S Hamaguchiet al 2003 Oryzias curvinotus has DMY a gene that is re-quired for male development in the medaka O latipes ZoologSci 20 159ndash161

Matsuzaki Y H Hosokai Y Mizuguchi S Fukamachi A Shimizuet al 2013 Establishment of HRAS(G12V) transgenic medakaas a stable tumor model for in vivo screening of anticancerdrugs PLoS ONE 8 e54424

Maacuteteacutes L M K L Chuah E Belay B Jerchow N Manoj et al2009 Molecular evolution of a novel hyperactive SleepingBeauty transposase enables robust stable gene transfer in verte-brates Nat Genet 41 753ndash761

Mongin E T O Auer F Bourrat F Gruhl K Dewar et al2011 Combining computational prediction of cis-regulatory el-ements with a new enhancer assay to efficiently label neuronalstructures in the medaka fish PLoS ONE 6 e19747

916 S Kirchmaier et al

Myosho T H Otake H Masuyama M Matsuda Y Kuroki et al2012a Tracing the emergence of a novel sex-determining genein medaka Oryzias luzonensis Genetics 191 163ndash170

Myosho T Y Takehana T Sato S Hamaguchi and M Sakaizumi2012b The origin of the large metacentric chromosome pair inChinese medaka (Oryzias sinensis) Ichthyological Research 59384ndash388

Nanda I M Kondo U Hornung S Asakawa C Winkler et al2002 A duplicated copy of DMRT1 in the sex-determining re-gion of the Y chromosome of the medaka Oryzias latipes ProcNatl Acad Sci USA 99 11778ndash11783

Naruse K K H H Hori N N Shimizu Y Y Kohara and H HTakeda 2004a Medaka genomics a bridge between mutantphenotype and gene function Mech Dev 121 619ndash628

Naruse K M Tanaka K Mita A Shima J Postlethwait et al2004b A medaka gene map the trace of ancestral vertebrateproto-chromosomes revealed by comparative gene mapping Ge-nome Res 14 820ndash828

Ng G H B and Z Gong 2013 GFP transgenic medaka (Oryziaslatipes) under the inducible cyp1a promoter provide a sensitiveand convenient biological indicator for the presence of TCDDand other persistent organic chemicals PLoS ONE 8 e64334

Nguyen N L M Judd A Kalantzis B Whittle A S Giraud et al2011 Random mutagenesis of the mouse genome a strategyfor discovering gene function and the molecular basis of diseaseAm J Physiol Gastrointest Liver Physiol 300 G1ndashG11

Oda S S Mikami Y Urushihara Y Murata Y Kamei et al2010 Identification of a functional medaka heat shock pro-moter and characterization of its ability to induce exogenousgene expression in medaka in vitro and in vivo Zoolog Sci27 410ndash415

Okuyama T Y Isoe M Hoki Y Suehiro G Yamagishi et al2013 Controlled CreloxP site-specific recombination in thedeveloping brain in medaka fish Oryzias latipes PLoS ONE 8e66597

Pan Y A T Freundlich T A Weissman D Schoppik X C Wanget al 2013 Zebrabow multispectral cell labeling for cell trac-ing and lineage analysis in zebrafish Development 140 2835ndash2846

Provost E J Rhee and S D Leach 2007 Viral 2A peptides allowexpression of multiple proteins from a single ORF in transgeniczebrafish embryos Genesis 45 625ndash629

Rembold M K Lahiri N S Foulkes and J Wittbrodt2006a Transgenesis in fish efficient selection of transgenicfish by co-injection with a fluorescent reporter construct NatProtoc 1 1133ndash1139

Rembold M F Loosli R J Adams and J Wittbrodt2006b Individual cell migration serves as the driving forcefor optic vesicle evagination Science 313 1130ndash1134

Sakaizumi M Y Shimizu and S Hamaguchi 1992 Electrophoreticstudies of meiotic segregation in inter- and intraspecific hybridsamong east asian species of the genus Oryzias (Pisces Oryzia-tidae) J Exp Zool 264 85ndash92

Sakuma T H Ochiai T Kaneko T Mashimo D Tokumasu et al2013 Repeating pattern of non-RVD variations in DNA-bindingmodules enhances TALEN activity Sci Rep 3 3379

Sampetrean O S-I Iida S Makino Y Matsuzaki K Ohno et al2009 Reversible whole-organism cell cycle arrest in a livingvertebrate Cell Cycle 8 620ndash627

Sanjana N E L Cong Y Zhou M M Cunniff G Feng et al2012 A transcription activator-like effector toolbox for genomeengineering Nat Protoc 7 171ndash192

Sano S S Takashima H Niwa H Yokoi A Shimada et al2009 Characterization of teleost Mdga1 using a gene-trap ap-proach in medaka (Oryzias latipes) Genesis 47 505ndash513

Sasado T M Tanaka K Kobayashi T Sato M Sakaizumi et al2010 The National BioResource Project Medaka (NBRP Me-

daka) an integrated bioresource for biological and biomedicalsciences Exp Anim 59 13ndash23

Sato T T Endo K Yamahira S Hamaguchi and M Sakaizumi2005 Induction of female-to-male sex reversal by high temper-ature treatment in medaka Oryzias latipes Zoolog Sci 22985ndash988

Schartl M S Kneitz B Wilde T Wagner C V Henkel et al2012 Conserved expression signatures between medaka andhuman pigment cell tumors PLoS ONE 7 e37880

Schmid-Burgk J L T Schmidt V Kaiser K Houmlning and V Hornung2013 A ligation-independent cloning technique for high-throughput assembly of transcription activator-like effectorgenes Nat Biotechnol 31 76ndash81

Seacutebillot A P Damdimopoulou Y Ogino P Spirhanzlova SMiyagawa et al 2014 Rapid fluorescent detection of (anti)androgens with spiggin-gfp medaka Environ Sci Technol48 10919ndash10928

Shestopalov I A S Sinha and J K Chen 2007 Light-controlledgene silencing in zebrafish embryos Nat Chem Biol 3 650ndash651

Shima A and A Shimada 1991 Development of a possible non-mammalian test system for radiation-induced germ-cell muta-genesis using a fish the Japanese medaka (Oryzias latipes)Proc Natl Acad Sci USA 88 2545ndash2549

Shima A and A Shimada 2001 The medaka as a model forstudying germ-cell mutagenesis and genomic instability MarBiotechnol (NY) 3 S162ndashS167

Shimada A M Yabusaki H Niwa H Yokoi K Hatta et al2007 Maternal-zygotic medaka mutants for fgfr1 reveal its es-sential role in the migration of the axial mesoderm but not thelateral mesoderm Development 135 281ndash290

Shimada A T Kawanishi T Kaneko H Yoshihara T Yano et al2013 Trunk exoskeleton in teleosts is mesodermal in originNat Commun 4 1639

Spivakov M T O Auer R Peravali I Dunham D Dolle et al2014 Genomic and phenotypic characterization of a wild me-daka population towards the establishment of an isogenic pop-ulation genetic resource in fish G3 (Bethesda) 4 433ndash445

Takehana Y S-R Jeon and M Sakaizumi 2004a Genetic struc-ture of Korean wild populations of the Medaka Oryzias latipesinferred from allozymic variation Zoolog Sci 21 977ndash988

Takehana Y S Uchiyama M Matsuda S-R Jeon and M Sakaizumi2004b Geographic variation and diversity of the cytochromeb gene in wild populations of medaka (Oryzias latipes) fromKorea and China Zoolog Sci 21 483ndash491

Takehana Y M Matsuda T Myosho M L Suster K Kawakamiet al 2014 Co-option of Sox3 as the male-determining factoron the Y chromosome in the fish Oryzias dancena Nat Com-mun 5 4157

Tanaka K Y Takehana K Naruse S Hamaguchi and M Sakaizumi2007 Evidence for different origins of sex chromosomes inclosely related Oryzias fishes substitution of the master sex-determining gene Genetics 177 2075ndash2081

Tanaka M and M Kinoshita 2001 Recent progress in the gen-eration of transgenic medaka (Oryzias latipes) Zoolog Sci 18615ndash622

Tanaka M M Kinoshita D Kobayashi and Y Nagahama2001 Establishment of medaka (Oryzias latipes) transgeniclines with the expression of green fluorescent protein fluores-cence exclusively in germ cells a useful model to monitor germcells in a live vertebrate Proc Natl Acad Sci USA 98 2544ndash2549

Taniguchi Y S Takeda M Furutani-Seiki Y Kamei T Todoet al 2006 Generation of medaka gene knockout models bytarget-selected mutagenesis Genome Biol 7 R116

Tena J J C Gonzaacutelez-Aguilera A Fernaacutendez-Mintildeaacuten J Vaacutezquez-Mariacuten H Parra-Acero et al 2014 Comparative epigenomics in

Genetic Toolbox Review 917

distantly related teleost species identifies conserved cis-regula-tory nodes active during the vertebrate phylotypic period Ge-nome Res 24 1075ndash1085

Thermes V C Grabher F Ristoratore F Bourrat A Choulikaet al 2002 I-SceI meganuclease mediates highly efficienttransgenesis in fish Mech Dev 118 91ndash98

Thummel R S Bai M P Sarras P Song J McDermott et al2006 Inhibition of zebrafish fin regeneration using in vivoelectroporation of morpholinos against fgfr1 and msxb DevDyn 235 336ndash346

Thummel R S C Kassen J E Montgomery J M Enright andD R Hyde 2008 Inhibition of Muumlller glial cell division blocksregeneration of the light-damaged zebrafish retina Dev Neuro-biol 68 392ndash408

Uwa H and Y Ojima 1981 Detailed and banding karyotypeanalyses of the medaka Oryzias latipes in cultured cells ProcJpn Acad Ser B Phys Biol Sci 57 39ndash43

Vopalensky P J Ruzickova B Pavlu and Z Kozmik 2010 Alens-specific co-injection marker for medaka transgenesis Bio-techniques 48 235ndash236

Wada H A Shimada S Fukamachi K Naruse and A Shima1998 Sex-linked inheritance of the lf locus in the medaka fish(Oryzias latipes) Zoolog Sci 15 123ndash126

Wakamatsu Y S Pristyazhnyuk M Kinoshita M Tanaka and KOzato 2001 The see-through medaka a fish model that is

transparent throughout life Proc Natl Acad Sci USA 9810046ndash10050

Willems B A Buumlttner A Huysseune J Renn P E Witten et al2012 Conditional ablation of osteoblasts in medaka Dev Biol364 128ndash137

Winkler S F Loosli T Henrich Y Wakamatsu and J Wittbrodt2000 The conditional medaka mutation eyeless uncouples pat-terning and morphogenesis of the eye Development 127 1911ndash1919

Yoshihara Y 2002 Visualizing selective neural pathways withWGA transgene combination of neuroanatomy with gene tech-nology Neurosci Res 44 133ndash140

Yoshinari N K Ando A Kudo M Kinoshita and A Kawakami2012 Colored medaka and zebrafish transgenics with ubiqui-tous and strong transgene expression driven by the medakab-actin promoter Dev Growth Differ 54 818ndash828

Zeng Z T Shan Y Tong S H Lam and Z Gong2005 Development of estrogen-responsive transgenic medakafor environmental monitoring of endocrine disrupters EnvironSci Technol 39 9001ndash9008

Zuber J J Shi E Wang A R Rappaport H Herrmann et al2011 RNAi screen identifies Brd4 as a therapeutic target inacute myeloid leukaemia Nature 478 524ndash528

Communicating editor O Hobert

918 S Kirchmaier et al

Houmlckendorf B T Thumberger and J Wittbrodt 2012 Quantitativeanalysis of embryogenesis a perspective for light sheet micros-copy Dev Cell 23 1111ndash1120

Hyodo-Taguchi Y 1980 Establishment of inbred strains of theteleost Oryzias latipes Zoological Magazine 89 283ndash301

Ieki T S Okada Y Aihara M Ohmoto K Abe et al2013 Transgenic labeling of higher order neuronal circuitslinked to phospholipase C-b2-expressing taste bud cells in me-daka fish J Comp Neurol 521 1781ndash1802

Inoue D and J Wittbrodt 2011 One for all a highly efficientand versatile method for fluorescent immunostaining in fishembryos PLoS ONE 6 e19713

Inoue K and Y Takei 2002 Diverse adaptability in oryzias spe-cies to high environmental salinity Zoolog Sci 19 727ndash734

Inoue K S Yamashita J Hata S Kabeno S Asada et al1990 Electroporation as a new technique for producing trans-genic fish Cell Differ Dev 29 123ndash8

Ishikawa Y 2000 Medakafish as a model system for vertebratedevelopmental genetics BioEssays 22 487ndash495

Ishikawa Y M Yoshimoto N Yamamoto and H Ito1999 Different brain morphologies from different genotypesin a single teleost species the medaka (Oryzias latipes) BrainBehav Evol 53 2ndash9

Itou J M Suyama Y Imamura T Deguchi K Fujimori et al2009 Functional and comparative genomics analyses ofpmp22 in medaka fish BMC Neurosci 10 60

Ivics Z P B Hackett R H Plasterk and Z Izsvaacutek1997 Molecular reconstruction of Sleeping Beauty a Tc1-liketransposon from fish and its transposition in human cells Cell91 501ndash510

Kamei Y M Suzuki K Watanabe K Fujimori T Kawasaki et al2008 Infrared laser-mediated gene induction in targeted sin-gle cells in vivo Nat Methods 6 79ndash81

Kasahara M K Naruse S Sasaki Y Nakatani W Qu et al2007 The medaka draft genome and insights into vertebrategenome evolution Nature 447 714ndash719

Kawaguchi M K Misaki H Sato T Yokokawa T Itai et al2012 Transgenic medaka small fresh water teleost (Oryziaslatipes) is now available for environmental science Interdisci-plinary Studies on Environmental Chemistry EnvironmentalPollution and Ecotoxicology 49ndash54 Volume 6 TERRAPUBTokyo

Kelly A and A F Hurlstone 2011 The use of RNAi technologiesfor gene knockdown in zebrafish Brief Funct Genomics 10189ndash196

Kelsh R N C Inoue A Momoi H Kondoh M Furutani-Seikiet al 2004 The Tomita collection of medaka pigmentationmutants as a resource for understanding neural crest cell de-velopment Mech Dev 121 841ndash859

Kimura T A Shimada N Sakai H Mitani K Naruse et al2007 Genetic analysis of craniofacial traits in the medaka Ge-netics 177 2379ndash2388

Kimura T Y Nagao H Hashimoto Y-I Yamamoto-Shiraishi SYamamoto et al 2014 Leucophores are similar to xantho-phores in their specification and differentiation processes in me-daka Proc Natl Acad Sci USA 111 7343ndash7348

Kinoshita M M Yamauchi M Sasanuma Y Ishikawa T Osadaet al 2003 A transgene and its expression profile are stablytransmitted to offspring in transgenic medaka generated by theparticle gun method Zoolog Sci 20 869ndash875

Kinoshita M K Murata K Naruse and M Tanaka2009 Medaka Biology Management and Experimental Proto-cols Wiley-Blackwell USA

Kirchmaier S B Houmlckendorf E K Moumlller D Bornhorst F Spitzet al 2013 Efficient site-specific transgenesis and enhanceractivity tests in medaka using PhiC31 integrase Development140 4287ndash4295

Kishi K E Kitagawa N Onikura A Nakamura and H Iwahashi2006 Expression analysis of sex-specific and 17beta-estradiol-responsive genes in the Japanese medaka Oryzias latipes usingoligonucleotide microarrays Genomics 88 241ndash251

Knapik E W 2000 ENU mutagenesis in zebrafish from genes tocomplex diseases Mamm Genome 11 511ndash519

Knopf F K Schnabel C Haase K Pfeifer K Anastassiadis et al2010 Dually inducible TetON systems for tissue-specific con-ditional gene expression in zebrafish Proc Natl Acad Sci USA107 19933ndash19938

Kobayashi K Y Kamei M Kinoshita T Czerny and M Tanaka2013 A heat-inducible CRELOXP gene induction system inmedaka Genesis 51 59ndash67

Kobayashi T Y Takeuchi T Takeuchi and G Yoshizaki2007 Generation of viable fish from cryopreserved primordialgerm cells Mol Reprod Dev 74 207ndash213

Koger C S S J Teh and D E Hinton 1999 Variations of lightand temperature regimes and resulting effects on reproductiveparameters in medaka (Oryzias latipes) Biol Reprod 61 1287ndash1293

Kok F O M Shin C-W Ni A Gupta A S Grosse et al2014 Reverse genetic screening reveals poor correlation be-tween morpholino-induced and mutant phenotypes in zebrafishDev Cell 32 97ndash108

Lee S Y Iwasaki S Shikina and G Yoshizaki 2013 Generationof functional eggs and sperm from cryopreserved whole testesProc Natl Acad Sci USA 110 1640ndash1645

Liu T L Liu Q Wei and Y Hong 2011 Sperm nuclear transferand transgenic production in the fish medaka Int J Biol Sci 7469ndash475

Livet J T A Weissman H Kang R W Draft J Lu et al2007 Transgenic strategies for combinatorial expression offluorescent proteins in the nervous system Nature 450 56ndash62

Loosli F R W Koumlster M Carl R Kuumlhnlein T Henrich et al2000 A genetic screen for mutations affecting embryonic de-velopment in medaka fish (Oryzias latipes) Mech Dev 97133ndash139

Ma A C H B Lee K J Clark and S C Ekker 2013 Highefficiency in vivo genome engineering with a simplified 15-RVD GoldyTALEN design PLoS ONE 8 e65259

Mali P L Yang K M Esvelt J Aach M Guell et al 2013 RNA-guided human genome engineering via Cas9 Science (NewYork NY) 339 823ndash826

Martinez-Morales J-R M Rembold K Greger J C Simpson K EBrown et al 2009 ojoplano-mediated basal constriction is es-sential for optic cup morphogenesis Development 136 2165ndash2175

Matsuda M Y Nagahama A Shinomiya T Sato C Matsudaet al 2002 DMY is a Y-specific DM-domain gene requiredfor male development in the medaka fish Nature 417 559ndash563

Matsuda M T Sato Y Toyazaki Y Nagahama S Hamaguchiet al 2003 Oryzias curvinotus has DMY a gene that is re-quired for male development in the medaka O latipes ZoologSci 20 159ndash161

Matsuzaki Y H Hosokai Y Mizuguchi S Fukamachi A Shimizuet al 2013 Establishment of HRAS(G12V) transgenic medakaas a stable tumor model for in vivo screening of anticancerdrugs PLoS ONE 8 e54424

Maacuteteacutes L M K L Chuah E Belay B Jerchow N Manoj et al2009 Molecular evolution of a novel hyperactive SleepingBeauty transposase enables robust stable gene transfer in verte-brates Nat Genet 41 753ndash761

Mongin E T O Auer F Bourrat F Gruhl K Dewar et al2011 Combining computational prediction of cis-regulatory el-ements with a new enhancer assay to efficiently label neuronalstructures in the medaka fish PLoS ONE 6 e19747

916 S Kirchmaier et al

Myosho T H Otake H Masuyama M Matsuda Y Kuroki et al2012a Tracing the emergence of a novel sex-determining genein medaka Oryzias luzonensis Genetics 191 163ndash170

Myosho T Y Takehana T Sato S Hamaguchi and M Sakaizumi2012b The origin of the large metacentric chromosome pair inChinese medaka (Oryzias sinensis) Ichthyological Research 59384ndash388

Nanda I M Kondo U Hornung S Asakawa C Winkler et al2002 A duplicated copy of DMRT1 in the sex-determining re-gion of the Y chromosome of the medaka Oryzias latipes ProcNatl Acad Sci USA 99 11778ndash11783

Naruse K K H H Hori N N Shimizu Y Y Kohara and H HTakeda 2004a Medaka genomics a bridge between mutantphenotype and gene function Mech Dev 121 619ndash628

Naruse K M Tanaka K Mita A Shima J Postlethwait et al2004b A medaka gene map the trace of ancestral vertebrateproto-chromosomes revealed by comparative gene mapping Ge-nome Res 14 820ndash828

Ng G H B and Z Gong 2013 GFP transgenic medaka (Oryziaslatipes) under the inducible cyp1a promoter provide a sensitiveand convenient biological indicator for the presence of TCDDand other persistent organic chemicals PLoS ONE 8 e64334

Nguyen N L M Judd A Kalantzis B Whittle A S Giraud et al2011 Random mutagenesis of the mouse genome a strategyfor discovering gene function and the molecular basis of diseaseAm J Physiol Gastrointest Liver Physiol 300 G1ndashG11

Oda S S Mikami Y Urushihara Y Murata Y Kamei et al2010 Identification of a functional medaka heat shock pro-moter and characterization of its ability to induce exogenousgene expression in medaka in vitro and in vivo Zoolog Sci27 410ndash415

Okuyama T Y Isoe M Hoki Y Suehiro G Yamagishi et al2013 Controlled CreloxP site-specific recombination in thedeveloping brain in medaka fish Oryzias latipes PLoS ONE 8e66597

Pan Y A T Freundlich T A Weissman D Schoppik X C Wanget al 2013 Zebrabow multispectral cell labeling for cell trac-ing and lineage analysis in zebrafish Development 140 2835ndash2846

Provost E J Rhee and S D Leach 2007 Viral 2A peptides allowexpression of multiple proteins from a single ORF in transgeniczebrafish embryos Genesis 45 625ndash629

Rembold M K Lahiri N S Foulkes and J Wittbrodt2006a Transgenesis in fish efficient selection of transgenicfish by co-injection with a fluorescent reporter construct NatProtoc 1 1133ndash1139

Rembold M F Loosli R J Adams and J Wittbrodt2006b Individual cell migration serves as the driving forcefor optic vesicle evagination Science 313 1130ndash1134

Sakaizumi M Y Shimizu and S Hamaguchi 1992 Electrophoreticstudies of meiotic segregation in inter- and intraspecific hybridsamong east asian species of the genus Oryzias (Pisces Oryzia-tidae) J Exp Zool 264 85ndash92

Sakuma T H Ochiai T Kaneko T Mashimo D Tokumasu et al2013 Repeating pattern of non-RVD variations in DNA-bindingmodules enhances TALEN activity Sci Rep 3 3379

Sampetrean O S-I Iida S Makino Y Matsuzaki K Ohno et al2009 Reversible whole-organism cell cycle arrest in a livingvertebrate Cell Cycle 8 620ndash627

Sanjana N E L Cong Y Zhou M M Cunniff G Feng et al2012 A transcription activator-like effector toolbox for genomeengineering Nat Protoc 7 171ndash192

Sano S S Takashima H Niwa H Yokoi A Shimada et al2009 Characterization of teleost Mdga1 using a gene-trap ap-proach in medaka (Oryzias latipes) Genesis 47 505ndash513

Sasado T M Tanaka K Kobayashi T Sato M Sakaizumi et al2010 The National BioResource Project Medaka (NBRP Me-

daka) an integrated bioresource for biological and biomedicalsciences Exp Anim 59 13ndash23

Sato T T Endo K Yamahira S Hamaguchi and M Sakaizumi2005 Induction of female-to-male sex reversal by high temper-ature treatment in medaka Oryzias latipes Zoolog Sci 22985ndash988

Schartl M S Kneitz B Wilde T Wagner C V Henkel et al2012 Conserved expression signatures between medaka andhuman pigment cell tumors PLoS ONE 7 e37880

Schmid-Burgk J L T Schmidt V Kaiser K Houmlning and V Hornung2013 A ligation-independent cloning technique for high-throughput assembly of transcription activator-like effectorgenes Nat Biotechnol 31 76ndash81

Seacutebillot A P Damdimopoulou Y Ogino P Spirhanzlova SMiyagawa et al 2014 Rapid fluorescent detection of (anti)androgens with spiggin-gfp medaka Environ Sci Technol48 10919ndash10928

Shestopalov I A S Sinha and J K Chen 2007 Light-controlledgene silencing in zebrafish embryos Nat Chem Biol 3 650ndash651

Shima A and A Shimada 1991 Development of a possible non-mammalian test system for radiation-induced germ-cell muta-genesis using a fish the Japanese medaka (Oryzias latipes)Proc Natl Acad Sci USA 88 2545ndash2549

Shima A and A Shimada 2001 The medaka as a model forstudying germ-cell mutagenesis and genomic instability MarBiotechnol (NY) 3 S162ndashS167

Shimada A M Yabusaki H Niwa H Yokoi K Hatta et al2007 Maternal-zygotic medaka mutants for fgfr1 reveal its es-sential role in the migration of the axial mesoderm but not thelateral mesoderm Development 135 281ndash290

Shimada A T Kawanishi T Kaneko H Yoshihara T Yano et al2013 Trunk exoskeleton in teleosts is mesodermal in originNat Commun 4 1639

Spivakov M T O Auer R Peravali I Dunham D Dolle et al2014 Genomic and phenotypic characterization of a wild me-daka population towards the establishment of an isogenic pop-ulation genetic resource in fish G3 (Bethesda) 4 433ndash445

Takehana Y S-R Jeon and M Sakaizumi 2004a Genetic struc-ture of Korean wild populations of the Medaka Oryzias latipesinferred from allozymic variation Zoolog Sci 21 977ndash988

Takehana Y S Uchiyama M Matsuda S-R Jeon and M Sakaizumi2004b Geographic variation and diversity of the cytochromeb gene in wild populations of medaka (Oryzias latipes) fromKorea and China Zoolog Sci 21 483ndash491

Takehana Y M Matsuda T Myosho M L Suster K Kawakamiet al 2014 Co-option of Sox3 as the male-determining factoron the Y chromosome in the fish Oryzias dancena Nat Com-mun 5 4157

Tanaka K Y Takehana K Naruse S Hamaguchi and M Sakaizumi2007 Evidence for different origins of sex chromosomes inclosely related Oryzias fishes substitution of the master sex-determining gene Genetics 177 2075ndash2081

Tanaka M and M Kinoshita 2001 Recent progress in the gen-eration of transgenic medaka (Oryzias latipes) Zoolog Sci 18615ndash622

Tanaka M M Kinoshita D Kobayashi and Y Nagahama2001 Establishment of medaka (Oryzias latipes) transgeniclines with the expression of green fluorescent protein fluores-cence exclusively in germ cells a useful model to monitor germcells in a live vertebrate Proc Natl Acad Sci USA 98 2544ndash2549

Taniguchi Y S Takeda M Furutani-Seiki Y Kamei T Todoet al 2006 Generation of medaka gene knockout models bytarget-selected mutagenesis Genome Biol 7 R116

Tena J J C Gonzaacutelez-Aguilera A Fernaacutendez-Mintildeaacuten J Vaacutezquez-Mariacuten H Parra-Acero et al 2014 Comparative epigenomics in

Genetic Toolbox Review 917

distantly related teleost species identifies conserved cis-regula-tory nodes active during the vertebrate phylotypic period Ge-nome Res 24 1075ndash1085

Thermes V C Grabher F Ristoratore F Bourrat A Choulikaet al 2002 I-SceI meganuclease mediates highly efficienttransgenesis in fish Mech Dev 118 91ndash98

Thummel R S Bai M P Sarras P Song J McDermott et al2006 Inhibition of zebrafish fin regeneration using in vivoelectroporation of morpholinos against fgfr1 and msxb DevDyn 235 336ndash346

Thummel R S C Kassen J E Montgomery J M Enright andD R Hyde 2008 Inhibition of Muumlller glial cell division blocksregeneration of the light-damaged zebrafish retina Dev Neuro-biol 68 392ndash408

Uwa H and Y Ojima 1981 Detailed and banding karyotypeanalyses of the medaka Oryzias latipes in cultured cells ProcJpn Acad Ser B Phys Biol Sci 57 39ndash43

Vopalensky P J Ruzickova B Pavlu and Z Kozmik 2010 Alens-specific co-injection marker for medaka transgenesis Bio-techniques 48 235ndash236

Wada H A Shimada S Fukamachi K Naruse and A Shima1998 Sex-linked inheritance of the lf locus in the medaka fish(Oryzias latipes) Zoolog Sci 15 123ndash126

Wakamatsu Y S Pristyazhnyuk M Kinoshita M Tanaka and KOzato 2001 The see-through medaka a fish model that is

transparent throughout life Proc Natl Acad Sci USA 9810046ndash10050

Willems B A Buumlttner A Huysseune J Renn P E Witten et al2012 Conditional ablation of osteoblasts in medaka Dev Biol364 128ndash137

Winkler S F Loosli T Henrich Y Wakamatsu and J Wittbrodt2000 The conditional medaka mutation eyeless uncouples pat-terning and morphogenesis of the eye Development 127 1911ndash1919

Yoshihara Y 2002 Visualizing selective neural pathways withWGA transgene combination of neuroanatomy with gene tech-nology Neurosci Res 44 133ndash140

Yoshinari N K Ando A Kudo M Kinoshita and A Kawakami2012 Colored medaka and zebrafish transgenics with ubiqui-tous and strong transgene expression driven by the medakab-actin promoter Dev Growth Differ 54 818ndash828

Zeng Z T Shan Y Tong S H Lam and Z Gong2005 Development of estrogen-responsive transgenic medakafor environmental monitoring of endocrine disrupters EnvironSci Technol 39 9001ndash9008

Zuber J J Shi E Wang A R Rappaport H Herrmann et al2011 RNAi screen identifies Brd4 as a therapeutic target inacute myeloid leukaemia Nature 478 524ndash528

Communicating editor O Hobert

918 S Kirchmaier et al

Myosho T H Otake H Masuyama M Matsuda Y Kuroki et al2012a Tracing the emergence of a novel sex-determining genein medaka Oryzias luzonensis Genetics 191 163ndash170

Myosho T Y Takehana T Sato S Hamaguchi and M Sakaizumi2012b The origin of the large metacentric chromosome pair inChinese medaka (Oryzias sinensis) Ichthyological Research 59384ndash388

Nanda I M Kondo U Hornung S Asakawa C Winkler et al2002 A duplicated copy of DMRT1 in the sex-determining re-gion of the Y chromosome of the medaka Oryzias latipes ProcNatl Acad Sci USA 99 11778ndash11783

Naruse K K H H Hori N N Shimizu Y Y Kohara and H HTakeda 2004a Medaka genomics a bridge between mutantphenotype and gene function Mech Dev 121 619ndash628

Naruse K M Tanaka K Mita A Shima J Postlethwait et al2004b A medaka gene map the trace of ancestral vertebrateproto-chromosomes revealed by comparative gene mapping Ge-nome Res 14 820ndash828

Ng G H B and Z Gong 2013 GFP transgenic medaka (Oryziaslatipes) under the inducible cyp1a promoter provide a sensitiveand convenient biological indicator for the presence of TCDDand other persistent organic chemicals PLoS ONE 8 e64334

Nguyen N L M Judd A Kalantzis B Whittle A S Giraud et al2011 Random mutagenesis of the mouse genome a strategyfor discovering gene function and the molecular basis of diseaseAm J Physiol Gastrointest Liver Physiol 300 G1ndashG11

Oda S S Mikami Y Urushihara Y Murata Y Kamei et al2010 Identification of a functional medaka heat shock pro-moter and characterization of its ability to induce exogenousgene expression in medaka in vitro and in vivo Zoolog Sci27 410ndash415

Okuyama T Y Isoe M Hoki Y Suehiro G Yamagishi et al2013 Controlled CreloxP site-specific recombination in thedeveloping brain in medaka fish Oryzias latipes PLoS ONE 8e66597

Pan Y A T Freundlich T A Weissman D Schoppik X C Wanget al 2013 Zebrabow multispectral cell labeling for cell trac-ing and lineage analysis in zebrafish Development 140 2835ndash2846

Provost E J Rhee and S D Leach 2007 Viral 2A peptides allowexpression of multiple proteins from a single ORF in transgeniczebrafish embryos Genesis 45 625ndash629

Rembold M K Lahiri N S Foulkes and J Wittbrodt2006a Transgenesis in fish efficient selection of transgenicfish by co-injection with a fluorescent reporter construct NatProtoc 1 1133ndash1139

Rembold M F Loosli R J Adams and J Wittbrodt2006b Individual cell migration serves as the driving forcefor optic vesicle evagination Science 313 1130ndash1134

Sakaizumi M Y Shimizu and S Hamaguchi 1992 Electrophoreticstudies of meiotic segregation in inter- and intraspecific hybridsamong east asian species of the genus Oryzias (Pisces Oryzia-tidae) J Exp Zool 264 85ndash92

Sakuma T H Ochiai T Kaneko T Mashimo D Tokumasu et al2013 Repeating pattern of non-RVD variations in DNA-bindingmodules enhances TALEN activity Sci Rep 3 3379

Sampetrean O S-I Iida S Makino Y Matsuzaki K Ohno et al2009 Reversible whole-organism cell cycle arrest in a livingvertebrate Cell Cycle 8 620ndash627

Sanjana N E L Cong Y Zhou M M Cunniff G Feng et al2012 A transcription activator-like effector toolbox for genomeengineering Nat Protoc 7 171ndash192

Sano S S Takashima H Niwa H Yokoi A Shimada et al2009 Characterization of teleost Mdga1 using a gene-trap ap-proach in medaka (Oryzias latipes) Genesis 47 505ndash513

Sasado T M Tanaka K Kobayashi T Sato M Sakaizumi et al2010 The National BioResource Project Medaka (NBRP Me-

daka) an integrated bioresource for biological and biomedicalsciences Exp Anim 59 13ndash23

Sato T T Endo K Yamahira S Hamaguchi and M Sakaizumi2005 Induction of female-to-male sex reversal by high temper-ature treatment in medaka Oryzias latipes Zoolog Sci 22985ndash988

Schartl M S Kneitz B Wilde T Wagner C V Henkel et al2012 Conserved expression signatures between medaka andhuman pigment cell tumors PLoS ONE 7 e37880

Schmid-Burgk J L T Schmidt V Kaiser K Houmlning and V Hornung2013 A ligation-independent cloning technique for high-throughput assembly of transcription activator-like effectorgenes Nat Biotechnol 31 76ndash81

Seacutebillot A P Damdimopoulou Y Ogino P Spirhanzlova SMiyagawa et al 2014 Rapid fluorescent detection of (anti)androgens with spiggin-gfp medaka Environ Sci Technol48 10919ndash10928

Shestopalov I A S Sinha and J K Chen 2007 Light-controlledgene silencing in zebrafish embryos Nat Chem Biol 3 650ndash651

Shima A and A Shimada 1991 Development of a possible non-mammalian test system for radiation-induced germ-cell muta-genesis using a fish the Japanese medaka (Oryzias latipes)Proc Natl Acad Sci USA 88 2545ndash2549

Shima A and A Shimada 2001 The medaka as a model forstudying germ-cell mutagenesis and genomic instability MarBiotechnol (NY) 3 S162ndashS167

Shimada A M Yabusaki H Niwa H Yokoi K Hatta et al2007 Maternal-zygotic medaka mutants for fgfr1 reveal its es-sential role in the migration of the axial mesoderm but not thelateral mesoderm Development 135 281ndash290

Shimada A T Kawanishi T Kaneko H Yoshihara T Yano et al2013 Trunk exoskeleton in teleosts is mesodermal in originNat Commun 4 1639

Spivakov M T O Auer R Peravali I Dunham D Dolle et al2014 Genomic and phenotypic characterization of a wild me-daka population towards the establishment of an isogenic pop-ulation genetic resource in fish G3 (Bethesda) 4 433ndash445

Takehana Y S-R Jeon and M Sakaizumi 2004a Genetic struc-ture of Korean wild populations of the Medaka Oryzias latipesinferred from allozymic variation Zoolog Sci 21 977ndash988

Takehana Y S Uchiyama M Matsuda S-R Jeon and M Sakaizumi2004b Geographic variation and diversity of the cytochromeb gene in wild populations of medaka (Oryzias latipes) fromKorea and China Zoolog Sci 21 483ndash491

Takehana Y M Matsuda T Myosho M L Suster K Kawakamiet al 2014 Co-option of Sox3 as the male-determining factoron the Y chromosome in the fish Oryzias dancena Nat Com-mun 5 4157

Tanaka K Y Takehana K Naruse S Hamaguchi and M Sakaizumi2007 Evidence for different origins of sex chromosomes inclosely related Oryzias fishes substitution of the master sex-determining gene Genetics 177 2075ndash2081

Tanaka M and M Kinoshita 2001 Recent progress in the gen-eration of transgenic medaka (Oryzias latipes) Zoolog Sci 18615ndash622

Tanaka M M Kinoshita D Kobayashi and Y Nagahama2001 Establishment of medaka (Oryzias latipes) transgeniclines with the expression of green fluorescent protein fluores-cence exclusively in germ cells a useful model to monitor germcells in a live vertebrate Proc Natl Acad Sci USA 98 2544ndash2549

Taniguchi Y S Takeda M Furutani-Seiki Y Kamei T Todoet al 2006 Generation of medaka gene knockout models bytarget-selected mutagenesis Genome Biol 7 R116

Tena J J C Gonzaacutelez-Aguilera A Fernaacutendez-Mintildeaacuten J Vaacutezquez-Mariacuten H Parra-Acero et al 2014 Comparative epigenomics in

Genetic Toolbox Review 917

distantly related teleost species identifies conserved cis-regula-tory nodes active during the vertebrate phylotypic period Ge-nome Res 24 1075ndash1085

Thermes V C Grabher F Ristoratore F Bourrat A Choulikaet al 2002 I-SceI meganuclease mediates highly efficienttransgenesis in fish Mech Dev 118 91ndash98

Thummel R S Bai M P Sarras P Song J McDermott et al2006 Inhibition of zebrafish fin regeneration using in vivoelectroporation of morpholinos against fgfr1 and msxb DevDyn 235 336ndash346

Thummel R S C Kassen J E Montgomery J M Enright andD R Hyde 2008 Inhibition of Muumlller glial cell division blocksregeneration of the light-damaged zebrafish retina Dev Neuro-biol 68 392ndash408

Uwa H and Y Ojima 1981 Detailed and banding karyotypeanalyses of the medaka Oryzias latipes in cultured cells ProcJpn Acad Ser B Phys Biol Sci 57 39ndash43

Vopalensky P J Ruzickova B Pavlu and Z Kozmik 2010 Alens-specific co-injection marker for medaka transgenesis Bio-techniques 48 235ndash236

Wada H A Shimada S Fukamachi K Naruse and A Shima1998 Sex-linked inheritance of the lf locus in the medaka fish(Oryzias latipes) Zoolog Sci 15 123ndash126

Wakamatsu Y S Pristyazhnyuk M Kinoshita M Tanaka and KOzato 2001 The see-through medaka a fish model that is

transparent throughout life Proc Natl Acad Sci USA 9810046ndash10050

Willems B A Buumlttner A Huysseune J Renn P E Witten et al2012 Conditional ablation of osteoblasts in medaka Dev Biol364 128ndash137

Winkler S F Loosli T Henrich Y Wakamatsu and J Wittbrodt2000 The conditional medaka mutation eyeless uncouples pat-terning and morphogenesis of the eye Development 127 1911ndash1919

Yoshihara Y 2002 Visualizing selective neural pathways withWGA transgene combination of neuroanatomy with gene tech-nology Neurosci Res 44 133ndash140

Yoshinari N K Ando A Kudo M Kinoshita and A Kawakami2012 Colored medaka and zebrafish transgenics with ubiqui-tous and strong transgene expression driven by the medakab-actin promoter Dev Growth Differ 54 818ndash828

Zeng Z T Shan Y Tong S H Lam and Z Gong2005 Development of estrogen-responsive transgenic medakafor environmental monitoring of endocrine disrupters EnvironSci Technol 39 9001ndash9008

Zuber J J Shi E Wang A R Rappaport H Herrmann et al2011 RNAi screen identifies Brd4 as a therapeutic target inacute myeloid leukaemia Nature 478 524ndash528

Communicating editor O Hobert

918 S Kirchmaier et al

distantly related teleost species identifies conserved cis-regula-tory nodes active during the vertebrate phylotypic period Ge-nome Res 24 1075ndash1085

Thermes V C Grabher F Ristoratore F Bourrat A Choulikaet al 2002 I-SceI meganuclease mediates highly efficienttransgenesis in fish Mech Dev 118 91ndash98

Thummel R S Bai M P Sarras P Song J McDermott et al2006 Inhibition of zebrafish fin regeneration using in vivoelectroporation of morpholinos against fgfr1 and msxb DevDyn 235 336ndash346

Thummel R S C Kassen J E Montgomery J M Enright andD R Hyde 2008 Inhibition of Muumlller glial cell division blocksregeneration of the light-damaged zebrafish retina Dev Neuro-biol 68 392ndash408

Uwa H and Y Ojima 1981 Detailed and banding karyotypeanalyses of the medaka Oryzias latipes in cultured cells ProcJpn Acad Ser B Phys Biol Sci 57 39ndash43

Vopalensky P J Ruzickova B Pavlu and Z Kozmik 2010 Alens-specific co-injection marker for medaka transgenesis Bio-techniques 48 235ndash236

Wada H A Shimada S Fukamachi K Naruse and A Shima1998 Sex-linked inheritance of the lf locus in the medaka fish(Oryzias latipes) Zoolog Sci 15 123ndash126

Wakamatsu Y S Pristyazhnyuk M Kinoshita M Tanaka and KOzato 2001 The see-through medaka a fish model that is

transparent throughout life Proc Natl Acad Sci USA 9810046ndash10050

Willems B A Buumlttner A Huysseune J Renn P E Witten et al2012 Conditional ablation of osteoblasts in medaka Dev Biol364 128ndash137

Winkler S F Loosli T Henrich Y Wakamatsu and J Wittbrodt2000 The conditional medaka mutation eyeless uncouples pat-terning and morphogenesis of the eye Development 127 1911ndash1919

Yoshihara Y 2002 Visualizing selective neural pathways withWGA transgene combination of neuroanatomy with gene tech-nology Neurosci Res 44 133ndash140

Yoshinari N K Ando A Kudo M Kinoshita and A Kawakami2012 Colored medaka and zebrafish transgenics with ubiqui-tous and strong transgene expression driven by the medakab-actin promoter Dev Growth Differ 54 818ndash828

Zeng Z T Shan Y Tong S H Lam and Z Gong2005 Development of estrogen-responsive transgenic medakafor environmental monitoring of endocrine disrupters EnvironSci Technol 39 9001ndash9008

Zuber J J Shi E Wang A R Rappaport H Herrmann et al2011 RNAi screen identifies Brd4 as a therapeutic target inacute myeloid leukaemia Nature 478 524ndash528

Communicating editor O Hobert

918 S Kirchmaier et al