Mar Biotechnol (2014) 16:739–747 DOI 10.1007/s10126-014-9587-7
ORIGINAL ARTICLE
Gene Replacement by Zinc Finger Nucleases in Medaka Embryos Guijun Guan & Xi Zhang & Kiyoshi Naruse & Yoshitaka Nagahama & Yunhan Hong
Received: 19 February 2014 / Accepted: 12 July 2014 / Published online: 6 August 2014 # Springer Science+Business Media New York 2014
Abstract Gene replacement (GR) via homologous recombination is a powerful tool for genome editing. Recently, direct GR is achieved successfully by coinjection of mRNAs for engineered endonucleases such as zinc finger nucleases (ZFNs) and donor DNA in developing embryos of diverse organisms. Here, we report the procedures and efficiency for direct GR by using ZFNs in the fish medaka. Upon zygotic coinjection of mRNAs encoding ZFNs that target the gonadspecifically expressed gsdf locus, linear DNA of GR vector pGRgsdf containing the red fluorescent protein (rfp) gene flanked by two homology arms of ~1-kb each underwent GR via homologous recombination. Specifically, 15 of 231 adults from manipulated embryos contained a GR allele in the caudal fin, producing an efficiency of ~7 % for somatic GR. Progeny test revealed that two out of nine fertile fish Electronic supplementary material The online version of this article (doi:10.1007/s10126-014-9587-7) contains supplementary material, which is available to authorized users. G. Guan : X. Zhang : Y. Hong Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore G. Guan : K. Naruse Laboratory of BioResource, National Institute for Basic Biology, Okazaki, Aichi 444-8585, Japan Y. Nagahama South Ehime Fisheries Research Center, Ehime University, Matsuyama 790-8577, Japan G. Guan : Y. Hong (*) College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201306, China e-mail: [email protected] Y. Nagahama (*) Institution for Collaborative Relations, Ehime University, 3 Bunkyo-cho, Matsuyama 790-8577, Japan e-mail: [email protected]
containing the GR allele in the fin were capable of transmitting the GR allele to ~6 % of F1 generation at adulthood, generating an efficiency of ~22 % for germline transmission. Sequencing and Southern blotting validated precise GR. We show that the GR allele expressed a chimeric gsdf:rfp RNA between gsdf and cointegrated rfp specifically in the gonad, demonstrating recapitulation of endogenous RNA expression as predicted for the defined GR allele. Most importantly, RFP expression coincides faithfully with the gonad-specific gsdf expression in developing embryos and adults. These results demonstrate, for the first time, the feasibility and efficiency of ZFN-mediated precise GR directly in the developing embryo of medaka as a lower vertebrate model. Keywords Gene targeting . Gene replacement . gsdf . Homologous recombination . Zinc finger nuclease Abbreviations GFP Green fluorescent protein gfp Gene for GFP gsdf Gene for gonadal soma-derived factor RFP Red fluorescent protein rfp Gene for RFP
Introduction Classical gene targeting (GT) involves the replacement of an endogenous DNA fragment with an exogenously introduced DNA copy via homologous recombination (HR), which was first reported in yeast 30 years ago (Rothstein 1983). GT in embryonic stem (ES) cells followed by germline chimera formation is a routine in mice and has produced many mouse models available for the study of gene functions and genetic diseases (Capecchi 2005, Wang et al. 2008). A low HR
740
frequency of ~10−6 and dependence on ES cells have, however, limited this classic GT strategy to mouse (Capecchi 2005) and rat (Tong et al. 2010). Engineered zinc finger nucleases (ZFNs) have demonstrated a high efficiency for GT, allowing for targeted gene mutagenesis and gene replacement (GR) directly in embryos (Carroll 2011). ZFNs were first designed as chimeric restriction endonucleases in vitro (Kim et al. 1996). In Xenopus oocytes, ZFNs of known specificity exhibit high efficiency of cleavage and recombination (Bibikova et al. 2001). ZFNs have ultimately proven efficient for editing the yellow locus in the Drosophila genome, leading to targeted mutations (Bibikova et al. 2002) and GR (Bibikova et al. 2003). Upon embryo coinjection with a donor DNA, ZFNs mediate high efficiencies of targeted mutations and GR in Drosophila (Beumer et al. 2008). Embryo injection of ZFN mRNAs has been successfully described in several organisms including zebrafish (Doyon et al. 2008, Meng et al. 2008, Foley et al. 2009, Ben et al. 2011), rat (Geurts et al. 2009, Mashimo et al. 2010), mouse (Carbery et al. 2010, Meyer et al. 2010), sea urchin (Ochiai et al. 2010), silkworm embryos (Takasu et al. 2010), and frog (Young et al. 2011). ZFN-induced mutagenesis has also been successful in cell cultures of four mammalian species, in four plant species, four invertebrate species, and five vertebrate species (Carroll 2011, Flisikowska et al. 2011). Since the first success in Drosophila (Beumer et al. 2008), ZFN-targeted GR via cointroduction of a donor vector has been reported also in mammalian cell cultures (Carroll 2011) and embryos (Geurts et al. 2009, Mashimo et al. 2010, Flisikowska et al. 2011, Cui et al. 2011, Meyer et al. 2010). The production of animals with defined GR was recently achieved by using transcription activator-like effector nuclease (TALEN) in zebrafish (Zu et al. 2013). We and others use the fish medaka (Oryziaslatipes) as a lower vertebrate model organism to develop procedures for genome editing. We have established stable lines of diploid embryonic stem (ES) cells (Hong et al. 1996, Hong et al. 1998), haploid ES cells (Yi et al. 2009, Yi et al. 2010) and adult germ stem cells (Hong et al. 2004), and classical GT in medaka ES cells at p53 (Yan et al. 2013) and nanos3 loci (Guan et al. 2013). In this organism, ZFN-induced gene disruption (GD) has recently been demonstrated on an introduced reporter gene (Ansai et al. 2012). We chose the chromosomal gsdf as a model gene to develop ZFNinduced GT in medaka. This gene encodes gonadal somaderived factor and is expressed in gonadal somatic cells (Shibata et al. 2010). Most recently, we have established procedures and parameters for ZFN-mediated gsdf disruption by mRNA injection in medaka embryos (Chen et al. 2012, Zhang et al. 2013). This study was aimed to develop procedures for direct GR in medaka embryos. We show that embryo coinjection of ZFN mRNAs and a donor plasmid leads to somatic and germline
Mar Biotechnol (2014) 16:739–747
GR at high efficiencies in adult animals. We reveal that a GRintroduced reporter transgene is able to form a chimeric mRNA with part of gsdf and more importantly, to express red fluorescent protein (RFP) reporter in the somatic cells of embryonic and adult gonads, thus recapitulates endogenous gsdf expression. Our results demonstrate that, for the first time to our knowledge, precise GR is possible by ZFNs directly in the embryo of medaka as a model of lower vertebrates.
Materials and Methods Fish Maintenance Work with fish followed the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Advisory Committee for Laboratory Animal Research in Singapore and approved by this committee (Permit Number: 27/09). Medaka strains af, HdrR and orange were maintained at 26~28 °C as described (Yi et al. 2009, Hong et al. 2010).
Plasmids Vectors pZF1gsdf and pZF2gsdf were supplied commercially (Toolgen, South Korea). They encode a pair of ZFNs that target a 27-bp sequence (12-bp and 9-bp recognition sequences plus a 6-bp spacer) within exon 1 of the medaka gsdf gene (Chen et al. 2012). The GR vector pGRgsdf was derived by inserting rfp plus CVpf between two gsdf genomic fragments of ~1 kb each as the homology arms (Fig. S1). Upon HR-mediated GR, rfp will become fused in frame with the 5′ part of gsdf exon 1 encoding the first 18 aa and is thus under the transcriptional control of gsdf regulatory elements, whereas the cointegrated CVpf will express PF, a fusion protein containing puromycin acetyltransferase and green fluorescent protein (GFP) under the transcriptional control of the human cytomegalovirus early enhancer/ promoter (CV) (Zhao et al. 2012). To construct GR vector pGRgsdf, a 899-bp XhoI-NdeI fragment from the 5′ region and a 944-bp BamHI-XbaI fragment from the 3′ region were PCR-amplified from genomic DNA of strain af. The rfp open reading frame was amplified as a 687-bp NdeI-ApaI fragment from pDsRed2-N1 (Clontech). The ordered ligation product of the three fragments was inserted between XhoI and XbaI sites in pCVpf (Li et al. 2012). To prevent re-cleavage of the GR allele by the ZFNs, a HindIII site was introduced immediately upstream of the NdeI site in the target sequence. This introduced HindIII site facilitates distinction between the wildtype gsdf allele and GR allele on Southern blots of genomic HindIII-digests.
Mar Biotechnol (2014) 16:739–747
RNA Synthesis After linearization with ApaI, both pZN1gsdf and pZN2gsdf were used for mRNA synthesis with the T7 RNA polymerase (Invitrogen) as described (Li et al. 2009). The mRNAs encoding the pZN1gsdf and pZN2gsdf were quantitated on a Nanodrop (Thermo Scientific), mixed at 1:1 molar ratio and stored at −80 °C until use. Donor vector pGRgsdf was prepared by using the plasmid Midi-kit (Qiagen), linearized with XhoI/XbaI double digestion and recovered by gel purification.
741
Signals were captured by Kodak Image Station 2000 (Carestream Health, Inc). Progeny Test Female and male adult fish containing a PCR-detectable GR allele in the caudal fin clips were out-crossed with normal HdrR fish of opposite sexes. The resultant F1 progeny adults were subjected to PCR genotyping of fin clips and/or to Southern analysis by using DNA isolated from the muscle.
Embryo Microinjection
Sequence Analysis
Embryo microinjection at the one-cell stage was performed as described (Li et al. 2012). Briefly, the synthetic ZFN mRNAs at 10~20 ng/μl were mixed with the linearized pGRgsdf DNA at 25 ng/μl and microinjected with approximately 20~30 pl/ per injection into embryos of medaka strain af, which corresponds to 0.5~0.75 pg of donor DNA per embryo. The injected embryos were analyzed by PCR genotyping or fluorescence microscopy, and subsequently allowed to develop into adulthood.
PCR products were cloned into pGEM-T easy vector (Promega) and sequenced on the ABI3100 automatic sequencer (Applied Biosystems). Sequence analyses were performed by the Vector NTI package (Invitrogen).
PCR Total RNA was isolated by using the Trizol Reagent (Invitrogen). Synthesis of cDNA templates was primed with oligo(dT)18 by using M-MLV transcriptase (Invitrogen). The cDNA reaction was diluted with water to 10 ng/μL. Genomic DNA was extracted from embryos and adult fin clips as described (Chen et al. 2012). The GR fragment was PCRamplified by using primers spanning the unique junction between the 5′ gsdf sequence and rfp (Fig. 1). PCR was run in a 20-μl volume for 35 cycles (94 °C for 10 s, 58 °C for 10 s and 72 °C for 2 min) as described (Yi et al. 2009, Hong et al. 2004). PCR products were separated by agarose gel electrophoresis and documented on a GeneGenius Imaging System (Syngene, England). PCR primers are listed in Table S1. Southern Blot Analysis A 1,142-bp fragment of the medaka 5′-gsdf was PCRamplified, cloned into pGEM-T and used as probe Ext. Probe rfpCV was released from pGRgsdf as a 937-bp insert to detect the predicted 1.8-kb HindIII fragment from GR allele. Probe labeling and Southern analysis were done as described (Yan et al. 2013). Briefly, Hybond N+ nylon membrane blots of genomic DNA HindIII digests from adult fin clips were hybridized with digoxigenin-labeled probes, incubated with an anti-DIG-AP antibody (Roche; 1:5,000 dilution) overnight at 4 °C and subjected to chemiluminescent detection by using the CDP-Star detection reagent (GE Life Sciences).
Microscopy Observation and photography were conducted on a Leica MZFIII stereo microscope, a Zeiss Axiovertinvert, and Axiovert upright microscopes as described (Hong et al. 2012, Yi et al. 2009).
Results Experimental Design for ZFN-Mediated GR The medaka gsdf gene was chosen for ZFN-mediated GR. This gene consists of 5 exons and encodes a protein of 216 amino acids (aa), with the first 19 aa residues encoded by the first exon serving as the signal peptide (Shibata et al. 2010). A pair of engineered ZFNs has proven their efficacy and specificity for gene disruption at the gsdf locus (Chen et al. 2012, Zhang et al. 2013). This pair of ZFNs specifically targets a 27bp DNA sequence within exon 1 (Fig. 1). A donor GR vector designated as pGRgsdf was constructed to contain two homology arms of ~1 kb in length each, which flank rfp as a reporter and CVpf as a cassette (Fig. 1). Dose-Dependent Survival Rate and GR Efficiency We have previously shown the feasibility of targeted gene disruption at the gsdf locus by microinjection of ZFN mRNAs (Chen et al. 2012). We have also revealed that the survival rate and GT efficiency are dependent on the dosage of ZFN mRNAs injected per embryo, with injection of ZFN mRNAs at 10~40 ng/μl giving rise to a satisfactory rate of fry production and gene disruption efficiency (Zhang et al. 2013). To optimize the procedure and efficiency for ZFN-
742
Mar Biotechnol (2014) 16:739–747
Fig. 1 Vector and analysis of ZFN-mediated GR. a Gene structure and the GR vector. The ZFN target site resides within exon 1 of the gsdf locus and consists of two recognition sequences and a spacer. One ZFN has four zinc fingers (Z1~Z4) and the other has three (Z1~Z3). Amino acids are shown with their positions above the codon triplets (underlined). The GR vector pGRgsdf has two homology arms of ~1 kb each (blue line) and two reporters (rfp and pf). Upon GR, the rfp will be knocked in and express RFP under the control of gsdf regulatory sequences, whereas the cointegrated pf will express PF under the human cytomegalovirus early enhancer/promoter (CV). Arrowheads depict the positions and direction
of PCR primers. Filled box translated exon; open box untranslated region; line between boxes intron; cross regions of HR. b PCR genotyping of embryos at 1~6 days post fertilization (dpf). Groups of 10 coinjected (coinjection) and noninjected embryos (Ctr) were analyzed. c PCR genotyping of adult fin clips from injected embryos. d PCR genotyping of adult fin clips. Primer pairs with the sizes of their PCR products are shown to the left. Asterisk GR-specific product; Ctr control fish; F0 founders derived from injected embryos; F1 progeny derived from crossing between a founder and a control fish; H2O negative control without DNA template
mediated GR, we first examined the effect of injection dosages on the survival rate of injected embryos. To this end, medaka embryos at the one-cell stage were injected with 25 ng/μl of linearized HR plasmid DNA plus 10 ng/μl (series I) and 25 ng/μl (series II) of ZFN mRNAs and monitored for the survival rate at the fry stages. Control embryos without injection displayed a 95 % survival rate of fry development. This value decreased to 65 and 59% in series I and series II, respectively (Table 1). These results indicate that injection with 25 ng/μl of linearized HR plasmid DNA plus 10~20 ng/μl of ZFN mRNAs produces a satisfactory survival rate of fry development. We then focused on the possibility of GR. In a pilot experiment, groups of embryos after coinjection with 20 ng/μl of ZFN mRNAs plus 25 ng/μl of linearized pGRgsdf DNA (series II) were PCR-genotyped by using the primer pairs, F9-DR5 and pF2-KOr2. F9 and KOr2 anneals the sequence upstream and downstream of the homology arms in pGRgsdf, whereas DR5 and pF2 anneals the rfp/gfp sequence flanked by the two homology arms (Fig. 1a). This primer pair is expected to define a 1.9-kb product specific to the GR allele. Indeed, this PCR product was easily visible in coinjected embryos at days
1, 3, and 6 post fertilization (dpf) but absent in noninjected wildtype control (Fig. 1b). These results demonstrate the feasibility of GR by coinjection of ZFN mRNAs with a linearized GR plasmid DNA in medaka embryos. We wanted to examine the dosage-dependent effect on the GR efficiency in adult fish from injected embryos. Processes and fate of donor DNA’s integration into host genome via microinjection of foreign DNA into the developing embryo were elucidated in medaka and zebrafish (Stuart et al. 1988; Lu et al. 1992). Like other transgenic animals reviewed by Wall 1996, genetic mosaicism including somatic and germline mosaicisms is observed in our gsdf GD medaka (Zhang et al. 2013). Methods for the screening of founder animals for germline mosaicism prior to mating would reduce labor costing and improve the efficiency of transgenic livestock production. Two approaches of GFP monitoring and PCR prescreening are designed in our experiment to distinguish individuals carrying transgenic fragment from nontransgenic one. GFP expression in operated embryos was evaluated and described in the next section. However, GFP signal was not sensitive to be detected in fry and adults. Therefore, we performed PCR prescreening for candidate F0 founder fish
Mar Biotechnol (2014) 16:739–747
743
Table 1 Survival and GR efficiency in medaka embryos Series of injection
1 2 Sum
ZFN mRNAs (ng/μl)
10 20
Embryo injected
298 274 572
Fry, n (%)a
194 (65 %) 161 (59 %) 355 (62 %)
Adults, n (%)
Germline transmitter, n (%)
Fish
GRb
Fertile fishc
Founder
164 67 231
9 (6 %) 6 (9 %) 15 (7 %)
5 (56 %) 4 (67 %) 9 (60 %)
1 (20 %) 1 (25 %) 2 (22 %)
Embryos at the one-cell stage were injected with 25 ng/μl of linear pGRgsdf DNA and indicated dosages of ZFN mRNAs. The resultant F0 adults were PCR-genotyped by using DNA from the caudal fin clips a
Percentage values were derived by comparison to the number of embryos injected
b
Fish contained the GR allele in the fin and were tested for fertility by outcrossing with normal fish. Percentage values were derived by comparing the number of fish containing a PCR-detectable GR in the fin clips to the number of adult fish c
Fish that were able to produce F1 progeny upon outcrossing with normal fish
using caudal fin clips as template DNA, presuming the HR events occurred in both somatic cells and/or germline. Based on this PCR screening, fry of the two series mentioned above were grown to adulthood, and DNA was extracted from the fin clips and PCR-genotyped by using multiple primer pairs specific to the WT and GR alleles (Fig.1a). This revealed that 9 out of 164 adults in series I contained a GR allele in the caudal fin (Fig. 1c), producing a 6 % efficiency for GR in series-I adults. This efficiency increased to 9 % in series II (n= 67) (Table 1). Therefore, coinjection of ZFN mRNAs with a linear donor DNA can mediate GR in vivo, and a higher injection dose of ZFN mRNAs generates a higher efficiency of GR in medaka embryos, which are capable of developing into adulthood. Germline Transmission The experiments described so far demonstrate the feasibility and dose-dependent GR efficiency in the embryos and adult fin as a somatic organ easily accessible for detection. The question whether the coinjection could generate germline GR remains to be answered. To this end, 9 out of 15 adults from both series of coinjection were fertile and subjected to progeny test. Two of the nine fertile fish turned out to be founders as they were able to transmit a GR allele into their F1 progeny, producing a 20~25 % efficiency for germline GR (Table 1). One founder fish was a male coming from series I, and the other was a female from series II. This demonstrates that a linear GR plasmid DNA in combination with ZFNs is able to generate germline GR in both sexes. The male founder transmitted the GR allele to 3 out of 49 F1 progeny, producing a germline transmission efficiency of 6.1 %. The female founder produced 17 F1 progeny, one of them inherited the GR allele, producing a germline transmission efficiency of 5.9 % (Table 1). The germline transmission of both founders is illustrated in Fig. 1d, where the GR allele was detected as a lighter band compared to the WT band in F0 founder fish. However, the GR band shows equal abundance with the
wildtype band in F1 fish, which indicates the mosaicism in the founder and germline transmission of the GR allele into the F1 generation. Taken together, a combination of ZFNs with GR plasmid DNA is proficient for conducting somatic and germline GR in medaka. Precision of the GR Event In medaka, ZFNs usually generate a wide variety of subtle allelic alterations at particular loci such as gsdf (Chen et al. 2012, Zhang et al. 2013). It provoked us to examine whether ZFN-mediated GR could occur in a precise manner as conventional GR in medaka ES cells (Guan et al. 2013, Yan et al. 2013). PCR products of the GR allele were specifically amplified, cloned and sequenced. All the three clones from injected embryos shared one and the same sequence as the predicted precise GR allele. The GR allele was also observed in six clones from the adult fin of a founder fish and six clones from the adult fin of a F1 progeny (Fig. 2a). This result demonstrates that the gsdf locus was precisely replaced by the sequence of GR vector pGRgsdf within the homologous arms-flanked region in the embryo, adult soma and germline of medaka. Therefore, ZFNs can generate precise GR in medaka. We furthered our examination into Southern blot analysis. For vector construction, two HindIII sites were introduced into pGRgsdf, one in front of rfp to destroy the ZFN target sequence for preventing the GR allele from ZFN cleavage, and the other between CVand pf (Fig. 2a). The introduction of HindIII sites facilitates the distinction between the WT gsdf allele and GR allele on the Southern blot. Upon HindIII digestion, the WT allele will produce a single band of 7.5 kb, whereas the GR allele will produce three bands of 7.5, 3.7, and 1.8 kb, respectively (Fig. 2a). The 7.5- and 3.7-kb bands are detectable by external probe Ext, whereas the 1.8-kb band is specific to internal probe rfpCV. On Southern blots of genomic HindIII digests, probe Ext indeed detected the 7.5-kb band in the control fish and the 3.7-kb band besides the 7.5-kb
744
Mar Biotechnol (2014) 16:739–747
Fig. 2 Precise GR. a Sequence analyses of WT and GR alleles. (Top) Schematic structures of WT and GR gsdf alleles. Shown are the positions and extension directions of PCR primers (arrowheads), two HindIII sites and two probes (Ext and rfpCV) used for Southern analysis. (Bottom) Sequences of the WT allele and cloned PCR products of the GR allele. DNA was from injected embryos, caudal fin clips of adults from operated embryos and F1 progeny. Numbers of sequenced clones are given in parenthesis. Start codon and stop codon are in red. Introduced HindIII and BamHI sites are underlined. b Southern analysis. HindIII digests of genomic DNA (10 μg) from a control fish (+/+) and F1 fish heterozygous
for GR were hybridized to external probe Ext and internal probe rfpCV. Notably, probe rfpCV detects a single band of predicted size for defined GR. c RT-PCR detection of chimeric gsdf:rfp RNA by using indicated primers, highlighting gonad-specific expression of endogenous gsdf and chimeric cgsdf:rfp exclusively in the ovary of a F1 female heterozygous for GR. β-actin was used as a loading control. neg negative control with H2O as a template. d Chimeric gsdf:rfp cDNA (top) and predicted protein (bottom) sequences. underlined primers; ATG start codon; black letter gsdf sequence; red letter rfp sequence
band in F1 progeny, whereas probe rfpCV detected a single band of 1.8 kb diagnostic of GR allele (Fig. 2b). Notably, probe rfpCV was unable to detect additional bands, suggesting the absence of randomly integrated pGRgsdf vector DNA in the genome of this F1 fish. Thus, the ZFN-mediated GR occurs without random integration.
chimeric mRNA expression was indeed limited to the gonad, like the endogenous gsdf (Fig. 2c). Sequencing validated the existence of chimeric mRNA gsdf:rfp (Fig. 2d). This result demonstrates a precise GR event and recapitulation of endogenous gsdf RNA expression by the rfp reporter within the GR allele. As previously described, the GR allele contains rfp under the control of endogenous gsdf regulatory elements and cassette CVpf expressing puromycin acetyltransferase and GFP, under the control of CV promoter (Zhao et al. 2011). After injection of pGRgsdf DNA, PF expression was observed in many cells of the developing embryo, whereas the RFP signal was restricted to a few cells in the region of the developing gonad (Fig. 3a–f). In the adult ovary, The PF signal was found in many somatic cells surrounding oocytes, and the RFP was visible in a subpopulation of these somatic cells (Fig. 3g–i).
Recapitulation of gsdf Expression by a GR-Cointegrated Reporter According to the experimental design, the rfp with the GR allele will be under the transcriptional control of gsdf regulatory sequences, and 5′ part of the gsdf RNA will be linked to rfp RNA, forming a chimeric mRNA fusion gsdf:rfp in the gonad. RT-PCR analyses revealed that this chimeric mRNA was indeed generated in the ovary of a F1 female. This
Mar Biotechnol (2014) 16:739–747
On the squash of this ovary, female germ cells were clearly seen as oocytes that were transparent and much larger than surrounding somatic cells (Fig. 3j). Fluorescence microscopy revealed that RFP and GFP were present in certain somatic cells but absent in the oocytes (Fig. 3k, l). Therefore, RFP expression by the knockin rfp transgene within the GR allele faithfully recapitulates the endogenous gsdf RNA expression in the gonadal soma.
Discussion Medaka and zebrafish are excellent twin model organisms for analyzing gene functions in vertebrate development (Wittbrodt et al. 2002). In both species, efforts have been Fig. 3 Visualization of reporter expression. a–f Embryonic expression. Medaka embryos at the one-cell stage were microinjected with ZFN mRNAs and pGRgsdf DNA and analyzed by fluorescence microscopy at 3 dpf (a–c) and 9 dpf (d–f). The GFP signal (green) is seen in many compartments, whereas the RFP signal (red) is limited largely to the brain (arrows). The insets in c and f are merged fluorescent micrographs of the gonad region at larger magnification. od oil droplet. g–i Adult expression in the ovary of F1 medaka heterozygous for GR allele. j–l Ovarian squash showing the GFP and RFP signal present in somatic cells but absent in oocytes (oc). Scale bars, 100 μm
745
made to introduce defined alterations into specific chromosomal sites. These include conventional GR in ES cell cultures of zebrafish (Fan et al. 2006) and medaka (Guan et al. 2013, Yan et al. 2013). Recently, ZFN and TALEN have been used for GD with a great efficiency in both medaka (Ansai et al. 2012, 2013, Chen et al. 2012, Zhang et al. 2013) and zebrafish (Doyon et al. 2008, Meng et al. 2008, Foley et al. 2009, Ben et al. 2011, Sander et al. 2011, Huang et al. 2011). In zebrafish, TALEN and CRISPR/Cas9 have been used for HR-mediated GR (Zu et al. 2013) and chromosomal deletions and inversions (Xiao et al. 2013). In this study, we have demonstrated—for the first time to our knowledge—the feasibility of ZFN-mediated GR at a chromosomal locus in medaka embryos. We show that embryo coinjection with 10~20 ng/μl of ZFN mRNAs plus 25 ng/μl of linear DNA generates a satisfactory efficiency for somatic GR in the adult fin, and up to
746
25 % of these are capable of germline transmission to the next generation. Two out of nine fertile GR positive fish were GR positive founders with a germline transmission rate at about 6 %. Although PCR prescreening could not increase the frequency of positive founders, it guaranteed us to estimate the approximate proportion of germline bearing GR allele among total operated eggs (2/572=3.5‰). Those injected individuals with only germline GR but no GR allele in its caudal fin clips were escaped by this PCR pre-selection, but should be a minor portion according to the germline specification and formation during medaka embryogenesis (Li et al. 2014, Herpin et al. 2007, Kurokawa et al. 2006, Tanaka et al. 2001). Four lines of evidence demonstrate the precision of ZFNmediated GR in medaka. First, sequencing of the GR allele validates exact junction sequences between the endogenous gsdf and the knock-in region flanked by the two homolog arms. Second, Southern blot analyses reveal a band pattern that is in accordance with a single cointegrated allele at the gsdf locus. Third, we show that the gsdf and the cointegrated rfp generate a chimeric mRNA in a defined fashion. Finally, the gonad-specific expression pattern at both RNA and protein levels from the knock-in GR allele recapitulates the endogenous gsdf expression, which is consistent with the RNA in situ hybridization result reported previously (Shibata et al. 2010). Although targetable nucleases such as ZFN, TALEN, or CRISPR/Cas RNA-guided nucleases are powerful tools available for gene targeting, the successful genome editing requires combination of those meganucleases and efficient knock-in of targeting fragment via homologous recombination. Our direct gene replacement in medaka embryos provided the adequate procedures and parameters for this approach applied in medaka as a lower vertebrate model. Acknowledgments We thank Jiaorong Deng for breeding fish, Veronica Wong and Choy Mei Foong for laboratory management. This work was supported by grant from the National Research Foundation Singapore (NRF-CRP7-2010-03) to YH, by Young Scientists research fellowship from the Japan Society for Promotion of Science (No. 0773) to GG and research scholarship from the National University of Singapore to XZ. Conflict of Interest The authors declare that they have no conflict of interest. Supporting Information Additional information noted in text includes supplementary figures.
References Ansai S, Ochiai H, Kanie Y, Kamei Y, Gou Y, Kitano T, Yamamoto T, Kinoshita M (2012) Targeted disruption of exogenous EGFP gene in medaka using zinc-finger nucleases. Dev Growth Differ 54:546– 556. doi:10.1111/j.1440-169X.2012.01357.x
Mar Biotechnol (2014) 16:739–747 Ansai S, Sakuma T, Yamamoto T, Ariga H, Uemura N, Takahashi R, Kinoshita M (2013) Efficient targeted mutagenesis in medaka using custom-designed transcription activator-like effector nucleases. Genetics 193:739–49. doi:10.1534/genetics.112.147645 Ben J, Elworthy S, Ng AS, Van Eeden F, Ingham PW (2011) Targeted mutation of the talpid3 gene in zebrafish reveals its conserved requirement for ciliogenesis and Hedgehog signalling across the vertebrates. Development 138:4969–78. doi:10.1242/dev.070862 Beumer KJ, Trautman JK, Bozas A, Liu JL, Rutter J, Gall JG, Carroll D (2008) Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases. Proc Natl Acad Sci U S A 105:19821–6. doi:10.1073/pnas.0810475105 0810475105 Bibikova M, Carroll D, Segal DJ, Trautman JK, Smith J, Kim YG, Chandrasegaran S (2001) Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol Cell Biol 21:289–297. doi:10.1128/Mcb.21.1.289-297.2001 Bibikova M, Golic M, Golic KG, Carroll D (2002) Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161:1169–75 Bibikova M, Beumer K, Trautman JK, Carroll D (2003) Enhancing gene targeting with designed zinc finger nucleases. Science 300:764–764. doi:10.1126/science.1079512 Capecchi MR (2005) Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet 6: 507–12. doi:10.1038/nrg1619 Carbery ID, Ji D, Harrington A, Brown V, Weinstein EJ, Liaw L, Cui XX (2010) Targeted genome modification in mice using zinc-finger nucleases. Genetics 186:451–U37. doi:10.1534/genetics.110. 117002 Carroll D (2011) Genome engineering with zinc-finger nucleases. Genetics 188:773–82. doi:10.1534/genetics.111.131433 Chen JB, Zhang X, Wang TS, Li ZD, Guan GJ, Hong YH (2012) Efficient detection, quantification and enrichment of subtle allelic alterations. DNA Res 19:423–433. doi:10.1093/dnares/dss023 Cui X, Ji D, Fisher DA, Wu Y, Briner DM, Weinstein EJ (2011) Targeted integration in rat and mouse embryos with zinc-finger nucleases. Nat Biotechnol 29:64–7. doi:10.1038/nbt.1731 nbt.1731 Doyon Y, Mccammon JM, Miller JC, Faraji F, Ngo C, Katibah GE, Amora R, Hocking TD, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Amacher SL (2008) Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat Biotechnol 26: 702–8. doi:10.1038/nbt1409 Fan L, Moon J, Crodian J, Collodi P (2006) Homologous recombination in zebrafish ES cells. Transgenic Res 15:21–30. doi:10.1007/ s11248-005-3225-0 Flisikowska T, Thorey IS, Offner S, Ros F, Lifke V, Zeitler B, Rottmann O, Vincent A, Zhang L, Jenkins S, Niersbach H, Kind AJ, Gregory PD, Schnieke AE, Platzer J (2011) Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases. Plos One 6:e21045. doi:10.1371/journal.pone.0021045 PONE-D-11-02571 Foley, J.E., Yeh, J.R.J., Maeder, M.L., Reyon, D., Sander, J.D., Peterson, R.T. & Joung, J.K. (2009). Rapid mutation of endogenous zebrafish genes using zinc finger nucleases made by Oligomerized Pool ENgineering (OPEN). Plos One, 4. DOI Artn E4348 Doi 10.1371/ Journal.Pone.0004348. Geurts AM, Cost GJ, Freyvert Y, Zeitler B, Miller JC, Choi VM, Jenkins SS, Wood A, Cui XX, Meng XD, Vincent A, Lam S, Michalkiewicz M, Schilling R, Foeckler J, Kalloway S, Weiler H, Menoret S, Anegon I, Davis GD, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jacob HJ, Buelow R (2009) Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325:433–433. doi:10.1126/ science.1172447 Guan G, Yan Y, Chen T, Yi M, Ni H, Naruse K, Nagahama Y, Hong Y (2013) Nanos3 gene targeting in medaka ES cells. Int J Biol Sci 9: 444–54. doi:10.7150/ijbs.6507
Mar Biotechnol (2014) 16:739–747 Herpin A, Rohr S, Riedel D, Kluever N, Raz E, Schartl M (2007) Specification of primordial germ cells in medaka (Oryzias latipes). BMC Dev Biol 7:3. doi:10.1186/1471-213X-7-3 Hong Y, Winkler C, Schartl M (1996) Pluripotency and differentiation of embryonic stem cell lines from the medakafish (Oryzias latipes). Mech Dev 60:33–44 Hong Y, Winkler C, Schartl M (1998) Production of medakafish chimeras from a stable embryonic stem cell line. Proc Natl Acad Sci U S A 95: 3679–84 Hong Y, Liu T, Zhao H, Xu H, Wang W, Liu R, Chen T, Deng J, Gui J (2004) Establishment of a normal medakafish spermatogonial cell line capable of sperm production in vitro. Proc Natl Acad Sci U S A 101:8011–6. doi:10.1073/pnas.0308668101 0308668101 Hong N, Li M, Zeng Z, Yi M, Deng J, Gui J, Winkler C, Schartl M, Hong Y (2010) Accessibility of host cell lineages to medaka stem cells depends on genetic background and irradiation of recipient embryos. Cell Mol Life Sci 67:1189–1202 Hong N, He BP, Schartl M, Hong Y (2012) Medaka embryonic stem cells are capable of generating entire organs and embryo-like miniatures. Stem Cells Dev. doi:10.1089/scd.2012.0144 Huang P, Xiao A, Zhou M, Zhu Z, Lin S, Zhang B (2011) Heritable gene targeting in zebrafish using customized TALENs. Nat Biotechnol 29:699–700. doi:10.1038/nbt.1939 Kim YG, Cha J, Chandrasegaran S (1996) Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci U S A 93:1156–60 Kurokawa H, Aoki Y, Nakamura S, Ebe Y, Kobayashi D, Tanaka M (2006) Time-lapse analysis reveals different modes of primordial germ cell migration in the medaka Oryzias latipes. Dev Growth Differ 48:209–21. doi:10.1111/j.1440-169X.2006.00858.x Li M, Hong N, Xu H, Yi M, Li C, Gui J, Hong Y (2009) Medaka vasa is required for migration but not survival of primordial germ cells. Mech Dev 126:366–81. doi:10.1016/j.mod.2009.02.004 Li M, Guan G, Hong N, Hong Y (2012) Multiple regulatory regions control the transcription of medaka germ gene vasa. Biochimie. doi: 10.1016/j.biochi.2012.12.003 Li Z, Li M, Hong N, Yi M, Hong Y (2014) Formation and cultivation of medaka primordial germ cells. Cell Tissue Res. doi:10.1007/ s00441-014-1867-z Lu, J.K., Chen, T.T., Chrisman, C.L., Andrisani, O.M. & Dixon, J.E. (1992). Integration, expression and germ-line transmission of foreign growth hormone genes in medaka (Oryzias latipes). Mol Mar Biol Biotechnol 1:366–75 Mashimo, T., Takizawa, A., Voigt, B., Yoshimi, K., Hiai, H., Kuramoto, T. & Serikawa, T (2010) Generation of knockout rats with X-linked severe combined immunodeficiency (X-SCID) using zinc-finger nucleases. Plos One, 5. DOI Artn E8870 Doi 10.1371/Journal.Pone.0008870. Meng XD, Noyes MB, Zhu LHJ, Lawson ND, Wolfe SA (2008) Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat Biotechnol 26:695–701. doi:10.1038/Nbt1398 Meyer M, De Angelis MH, Wurst W, Kuhn R (2010) Gene targeting by homologous recombination in mouse zygotes mediated by zincfinger nucleases. Proc Natl Acad Sci U S A 107:15022–6. doi:10. 1073/pnas.1009424107 Ochiai H, Fujita K, Suzuki K, Nishikawa M, Shibata T, Sakamoto N, Yamamoto T (2010) Targeted mutagenesis in the sea urchin embryo using zinc-finger nucleases. Genes Cells 15:875–885. doi:10.1111/j. 1365-2443.2010.01425.x Rothstein RJ (1983) One-step gene disruption in yeast. Methods Enzymol 101:202–11 Sander JD, Cade L, Khayter C, Reyon D, Peterson RT, Joung JK, Yeh JR (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nat Biotechnol 29:697–8. doi:10.1038/nbt.1934 Shibata Y, Paul-Prasanth B, Suzuki A, Usami T, Nakamoto M, Matsuda M, Nagahama Y (2010) Expression of gonadal soma derived factor (GSDF) is spatially and temporally correlated with early testicular
747 differentiation in medaka. Gene Expr Patterns 10:283–289. doi:10. 1016/j.gep.2010.06.005 Stuart, G.W., McMurray, J.V. & Westerfield, M. (1988). Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos. Dev 103:403–12 Takasu Y, Kobayashi I, Beumer K, Uchino K, Sezutsu H, Sajwan S, Carroll D, Tamura T, Zurovec M (2010) Targeted mutagenesis in the silkworm Bombyx mori using zinc finger nuclease mRNA injection. Insect Biochem Mol Biol 40:759–765. doi:10.1016/j.ibmb.2010.07. 012 Tanaka M, Kinoshita M, Kobayashi D, Nagahama Y (2001) Establishment of medaka (Oryzias latipes) transgenic lines with the expression of green fluorescent protein fluorescence exclusively in germ cells: a useful model to monitor germ cells in a live vertebrate. Proc Natl Acad Sci U S A 98:2544–9. doi:10.1073/ pnas.041315498 Tong C, Li P, Wu NL, Yan Y, Ying QL (2010) Production of p53 gene knockout rats by homologous recombination in embryonic stem cells. Nature 467:211–3. doi:10.1038/nature09368 Wall R.J. (1996). Transgenic livestock: progress and prospects for the future. Theriologenology 45:57–68 Wang RH, Sengupta K, Li C, Kim HS, Cao L, Xiao C, Kim S, Xu X, Zheng Y, Chilton B, Jia R, Zheng ZM, Appella E, Wang XW, Ried T, Deng CX (2008) Impaired DNA damage response, genome instability, and tumorigenesis in SIRT1 mutant mice. Cancer Cell 14:312–23. doi:10.1016/j.ccr.2008.09.001 Wittbrodt J, Shima A, Schartl M (2002) Medaka—a model organism from the far East. Nat Rev Genet 3:53–64 Xiao A, Wang Z, Hu Y, Wu Y, Luo Z, Yang Z, Zu Y, Li W, Huang P, Tong X, Zhu Z, Lin S, Zhang B (2013) Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish. Nucleic Acids Res 41:e141. doi:10.1093/nar/gkt464 Yan Y, Hong N, Chen T, Li M, Wang T, Guan G, Qiao Y, Chen S, Schartl M, Li CM, Hong Y (2013) p53 gene targeting by homologous recombination in fish ES cells. Plos One 8:e59400. doi:10.1371/ journal.pone.0059400 Yi M, Hong N, Hong Y (2009) Generation of medaka fish haploid embryonic stem cells. Science 326:430–3. doi:10.1126/science. 1175151 Yi M, Hong N, Hong Y (2010) Derivation and characterization of haploid embryonic stem cell cultures in medaka fish. Nat Protoc 5:1418–30. doi:10.1038/nprot.2010.104 Young JJ, Cherone JM, Doyon Y, Ankoudinova I, Faraji FM, Lee AH, Ngo C, Guschin DY, Paschon DE, Miller JC, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Harland RM, Zeitler B (2011) Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases. Proc Natl Acad Sci U S A 108:7052–7057. doi:10.1073/pnas. 1102030108 Zhang X, Guan G, Chen J, Naruse K, Hong Y (2013) Parameters and efficiency of direct gene disruption by zinc finger nucleases in Medaka embryos. Marine Biotechnol. doi:10.1007/s10126-0139556-6 Zhao H, Hong N, Lu W, Zeng H, Song J, Hong Y (2011) Fusion gene vectors allowing for simultaneous drug selection, cell labeling and reporter assay in vitro and in vivo. Anal Chem. doi:10.1021/ ac202541t Zhao H, Hong N, Lu W, Zeng H, Song J, Hong Y (2012) Fusion gene vectors allowing for simultaneous drug selection, cell labeling, and reporter assay in vitro and in vivo. Anal Chem 84:987–93. doi:10. 1021/ac202541t Zu Y, Tong X, Wang Z, Liu D, Pan R, Li Z, Hu Y, Luo Z, Huang P, Wu Q, Zhu Z, Zhang B, Lin S (2013) TALEN-mediated precise genome modification by homologous recombination in zebrafish. Nat Methods 10:329–31. doi:10.1038/nmeth. 2374
ORIGINAL ARTICLE
Gene Replacement by Zinc Finger Nucleases in Medaka Embryos Guijun Guan & Xi Zhang & Kiyoshi Naruse & Yoshitaka Nagahama & Yunhan Hong
Received: 19 February 2014 / Accepted: 12 July 2014 / Published online: 6 August 2014 # Springer Science+Business Media New York 2014
Abstract Gene replacement (GR) via homologous recombination is a powerful tool for genome editing. Recently, direct GR is achieved successfully by coinjection of mRNAs for engineered endonucleases such as zinc finger nucleases (ZFNs) and donor DNA in developing embryos of diverse organisms. Here, we report the procedures and efficiency for direct GR by using ZFNs in the fish medaka. Upon zygotic coinjection of mRNAs encoding ZFNs that target the gonadspecifically expressed gsdf locus, linear DNA of GR vector pGRgsdf containing the red fluorescent protein (rfp) gene flanked by two homology arms of ~1-kb each underwent GR via homologous recombination. Specifically, 15 of 231 adults from manipulated embryos contained a GR allele in the caudal fin, producing an efficiency of ~7 % for somatic GR. Progeny test revealed that two out of nine fertile fish Electronic supplementary material The online version of this article (doi:10.1007/s10126-014-9587-7) contains supplementary material, which is available to authorized users. G. Guan : X. Zhang : Y. Hong Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore G. Guan : K. Naruse Laboratory of BioResource, National Institute for Basic Biology, Okazaki, Aichi 444-8585, Japan Y. Nagahama South Ehime Fisheries Research Center, Ehime University, Matsuyama 790-8577, Japan G. Guan : Y. Hong (*) College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201306, China e-mail: [email protected] Y. Nagahama (*) Institution for Collaborative Relations, Ehime University, 3 Bunkyo-cho, Matsuyama 790-8577, Japan e-mail: [email protected]
containing the GR allele in the fin were capable of transmitting the GR allele to ~6 % of F1 generation at adulthood, generating an efficiency of ~22 % for germline transmission. Sequencing and Southern blotting validated precise GR. We show that the GR allele expressed a chimeric gsdf:rfp RNA between gsdf and cointegrated rfp specifically in the gonad, demonstrating recapitulation of endogenous RNA expression as predicted for the defined GR allele. Most importantly, RFP expression coincides faithfully with the gonad-specific gsdf expression in developing embryos and adults. These results demonstrate, for the first time, the feasibility and efficiency of ZFN-mediated precise GR directly in the developing embryo of medaka as a lower vertebrate model. Keywords Gene targeting . Gene replacement . gsdf . Homologous recombination . Zinc finger nuclease Abbreviations GFP Green fluorescent protein gfp Gene for GFP gsdf Gene for gonadal soma-derived factor RFP Red fluorescent protein rfp Gene for RFP
Introduction Classical gene targeting (GT) involves the replacement of an endogenous DNA fragment with an exogenously introduced DNA copy via homologous recombination (HR), which was first reported in yeast 30 years ago (Rothstein 1983). GT in embryonic stem (ES) cells followed by germline chimera formation is a routine in mice and has produced many mouse models available for the study of gene functions and genetic diseases (Capecchi 2005, Wang et al. 2008). A low HR
740
frequency of ~10−6 and dependence on ES cells have, however, limited this classic GT strategy to mouse (Capecchi 2005) and rat (Tong et al. 2010). Engineered zinc finger nucleases (ZFNs) have demonstrated a high efficiency for GT, allowing for targeted gene mutagenesis and gene replacement (GR) directly in embryos (Carroll 2011). ZFNs were first designed as chimeric restriction endonucleases in vitro (Kim et al. 1996). In Xenopus oocytes, ZFNs of known specificity exhibit high efficiency of cleavage and recombination (Bibikova et al. 2001). ZFNs have ultimately proven efficient for editing the yellow locus in the Drosophila genome, leading to targeted mutations (Bibikova et al. 2002) and GR (Bibikova et al. 2003). Upon embryo coinjection with a donor DNA, ZFNs mediate high efficiencies of targeted mutations and GR in Drosophila (Beumer et al. 2008). Embryo injection of ZFN mRNAs has been successfully described in several organisms including zebrafish (Doyon et al. 2008, Meng et al. 2008, Foley et al. 2009, Ben et al. 2011), rat (Geurts et al. 2009, Mashimo et al. 2010), mouse (Carbery et al. 2010, Meyer et al. 2010), sea urchin (Ochiai et al. 2010), silkworm embryos (Takasu et al. 2010), and frog (Young et al. 2011). ZFN-induced mutagenesis has also been successful in cell cultures of four mammalian species, in four plant species, four invertebrate species, and five vertebrate species (Carroll 2011, Flisikowska et al. 2011). Since the first success in Drosophila (Beumer et al. 2008), ZFN-targeted GR via cointroduction of a donor vector has been reported also in mammalian cell cultures (Carroll 2011) and embryos (Geurts et al. 2009, Mashimo et al. 2010, Flisikowska et al. 2011, Cui et al. 2011, Meyer et al. 2010). The production of animals with defined GR was recently achieved by using transcription activator-like effector nuclease (TALEN) in zebrafish (Zu et al. 2013). We and others use the fish medaka (Oryziaslatipes) as a lower vertebrate model organism to develop procedures for genome editing. We have established stable lines of diploid embryonic stem (ES) cells (Hong et al. 1996, Hong et al. 1998), haploid ES cells (Yi et al. 2009, Yi et al. 2010) and adult germ stem cells (Hong et al. 2004), and classical GT in medaka ES cells at p53 (Yan et al. 2013) and nanos3 loci (Guan et al. 2013). In this organism, ZFN-induced gene disruption (GD) has recently been demonstrated on an introduced reporter gene (Ansai et al. 2012). We chose the chromosomal gsdf as a model gene to develop ZFNinduced GT in medaka. This gene encodes gonadal somaderived factor and is expressed in gonadal somatic cells (Shibata et al. 2010). Most recently, we have established procedures and parameters for ZFN-mediated gsdf disruption by mRNA injection in medaka embryos (Chen et al. 2012, Zhang et al. 2013). This study was aimed to develop procedures for direct GR in medaka embryos. We show that embryo coinjection of ZFN mRNAs and a donor plasmid leads to somatic and germline
Mar Biotechnol (2014) 16:739–747
GR at high efficiencies in adult animals. We reveal that a GRintroduced reporter transgene is able to form a chimeric mRNA with part of gsdf and more importantly, to express red fluorescent protein (RFP) reporter in the somatic cells of embryonic and adult gonads, thus recapitulates endogenous gsdf expression. Our results demonstrate that, for the first time to our knowledge, precise GR is possible by ZFNs directly in the embryo of medaka as a model of lower vertebrates.
Materials and Methods Fish Maintenance Work with fish followed the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Advisory Committee for Laboratory Animal Research in Singapore and approved by this committee (Permit Number: 27/09). Medaka strains af, HdrR and orange were maintained at 26~28 °C as described (Yi et al. 2009, Hong et al. 2010).
Plasmids Vectors pZF1gsdf and pZF2gsdf were supplied commercially (Toolgen, South Korea). They encode a pair of ZFNs that target a 27-bp sequence (12-bp and 9-bp recognition sequences plus a 6-bp spacer) within exon 1 of the medaka gsdf gene (Chen et al. 2012). The GR vector pGRgsdf was derived by inserting rfp plus CVpf between two gsdf genomic fragments of ~1 kb each as the homology arms (Fig. S1). Upon HR-mediated GR, rfp will become fused in frame with the 5′ part of gsdf exon 1 encoding the first 18 aa and is thus under the transcriptional control of gsdf regulatory elements, whereas the cointegrated CVpf will express PF, a fusion protein containing puromycin acetyltransferase and green fluorescent protein (GFP) under the transcriptional control of the human cytomegalovirus early enhancer/ promoter (CV) (Zhao et al. 2012). To construct GR vector pGRgsdf, a 899-bp XhoI-NdeI fragment from the 5′ region and a 944-bp BamHI-XbaI fragment from the 3′ region were PCR-amplified from genomic DNA of strain af. The rfp open reading frame was amplified as a 687-bp NdeI-ApaI fragment from pDsRed2-N1 (Clontech). The ordered ligation product of the three fragments was inserted between XhoI and XbaI sites in pCVpf (Li et al. 2012). To prevent re-cleavage of the GR allele by the ZFNs, a HindIII site was introduced immediately upstream of the NdeI site in the target sequence. This introduced HindIII site facilitates distinction between the wildtype gsdf allele and GR allele on Southern blots of genomic HindIII-digests.
Mar Biotechnol (2014) 16:739–747
RNA Synthesis After linearization with ApaI, both pZN1gsdf and pZN2gsdf were used for mRNA synthesis with the T7 RNA polymerase (Invitrogen) as described (Li et al. 2009). The mRNAs encoding the pZN1gsdf and pZN2gsdf were quantitated on a Nanodrop (Thermo Scientific), mixed at 1:1 molar ratio and stored at −80 °C until use. Donor vector pGRgsdf was prepared by using the plasmid Midi-kit (Qiagen), linearized with XhoI/XbaI double digestion and recovered by gel purification.
741
Signals were captured by Kodak Image Station 2000 (Carestream Health, Inc). Progeny Test Female and male adult fish containing a PCR-detectable GR allele in the caudal fin clips were out-crossed with normal HdrR fish of opposite sexes. The resultant F1 progeny adults were subjected to PCR genotyping of fin clips and/or to Southern analysis by using DNA isolated from the muscle.
Embryo Microinjection
Sequence Analysis
Embryo microinjection at the one-cell stage was performed as described (Li et al. 2012). Briefly, the synthetic ZFN mRNAs at 10~20 ng/μl were mixed with the linearized pGRgsdf DNA at 25 ng/μl and microinjected with approximately 20~30 pl/ per injection into embryos of medaka strain af, which corresponds to 0.5~0.75 pg of donor DNA per embryo. The injected embryos were analyzed by PCR genotyping or fluorescence microscopy, and subsequently allowed to develop into adulthood.
PCR products were cloned into pGEM-T easy vector (Promega) and sequenced on the ABI3100 automatic sequencer (Applied Biosystems). Sequence analyses were performed by the Vector NTI package (Invitrogen).
PCR Total RNA was isolated by using the Trizol Reagent (Invitrogen). Synthesis of cDNA templates was primed with oligo(dT)18 by using M-MLV transcriptase (Invitrogen). The cDNA reaction was diluted with water to 10 ng/μL. Genomic DNA was extracted from embryos and adult fin clips as described (Chen et al. 2012). The GR fragment was PCRamplified by using primers spanning the unique junction between the 5′ gsdf sequence and rfp (Fig. 1). PCR was run in a 20-μl volume for 35 cycles (94 °C for 10 s, 58 °C for 10 s and 72 °C for 2 min) as described (Yi et al. 2009, Hong et al. 2004). PCR products were separated by agarose gel electrophoresis and documented on a GeneGenius Imaging System (Syngene, England). PCR primers are listed in Table S1. Southern Blot Analysis A 1,142-bp fragment of the medaka 5′-gsdf was PCRamplified, cloned into pGEM-T and used as probe Ext. Probe rfpCV was released from pGRgsdf as a 937-bp insert to detect the predicted 1.8-kb HindIII fragment from GR allele. Probe labeling and Southern analysis were done as described (Yan et al. 2013). Briefly, Hybond N+ nylon membrane blots of genomic DNA HindIII digests from adult fin clips were hybridized with digoxigenin-labeled probes, incubated with an anti-DIG-AP antibody (Roche; 1:5,000 dilution) overnight at 4 °C and subjected to chemiluminescent detection by using the CDP-Star detection reagent (GE Life Sciences).
Microscopy Observation and photography were conducted on a Leica MZFIII stereo microscope, a Zeiss Axiovertinvert, and Axiovert upright microscopes as described (Hong et al. 2012, Yi et al. 2009).
Results Experimental Design for ZFN-Mediated GR The medaka gsdf gene was chosen for ZFN-mediated GR. This gene consists of 5 exons and encodes a protein of 216 amino acids (aa), with the first 19 aa residues encoded by the first exon serving as the signal peptide (Shibata et al. 2010). A pair of engineered ZFNs has proven their efficacy and specificity for gene disruption at the gsdf locus (Chen et al. 2012, Zhang et al. 2013). This pair of ZFNs specifically targets a 27bp DNA sequence within exon 1 (Fig. 1). A donor GR vector designated as pGRgsdf was constructed to contain two homology arms of ~1 kb in length each, which flank rfp as a reporter and CVpf as a cassette (Fig. 1). Dose-Dependent Survival Rate and GR Efficiency We have previously shown the feasibility of targeted gene disruption at the gsdf locus by microinjection of ZFN mRNAs (Chen et al. 2012). We have also revealed that the survival rate and GT efficiency are dependent on the dosage of ZFN mRNAs injected per embryo, with injection of ZFN mRNAs at 10~40 ng/μl giving rise to a satisfactory rate of fry production and gene disruption efficiency (Zhang et al. 2013). To optimize the procedure and efficiency for ZFN-
742
Mar Biotechnol (2014) 16:739–747
Fig. 1 Vector and analysis of ZFN-mediated GR. a Gene structure and the GR vector. The ZFN target site resides within exon 1 of the gsdf locus and consists of two recognition sequences and a spacer. One ZFN has four zinc fingers (Z1~Z4) and the other has three (Z1~Z3). Amino acids are shown with their positions above the codon triplets (underlined). The GR vector pGRgsdf has two homology arms of ~1 kb each (blue line) and two reporters (rfp and pf). Upon GR, the rfp will be knocked in and express RFP under the control of gsdf regulatory sequences, whereas the cointegrated pf will express PF under the human cytomegalovirus early enhancer/promoter (CV). Arrowheads depict the positions and direction
of PCR primers. Filled box translated exon; open box untranslated region; line between boxes intron; cross regions of HR. b PCR genotyping of embryos at 1~6 days post fertilization (dpf). Groups of 10 coinjected (coinjection) and noninjected embryos (Ctr) were analyzed. c PCR genotyping of adult fin clips from injected embryos. d PCR genotyping of adult fin clips. Primer pairs with the sizes of their PCR products are shown to the left. Asterisk GR-specific product; Ctr control fish; F0 founders derived from injected embryos; F1 progeny derived from crossing between a founder and a control fish; H2O negative control without DNA template
mediated GR, we first examined the effect of injection dosages on the survival rate of injected embryos. To this end, medaka embryos at the one-cell stage were injected with 25 ng/μl of linearized HR plasmid DNA plus 10 ng/μl (series I) and 25 ng/μl (series II) of ZFN mRNAs and monitored for the survival rate at the fry stages. Control embryos without injection displayed a 95 % survival rate of fry development. This value decreased to 65 and 59% in series I and series II, respectively (Table 1). These results indicate that injection with 25 ng/μl of linearized HR plasmid DNA plus 10~20 ng/μl of ZFN mRNAs produces a satisfactory survival rate of fry development. We then focused on the possibility of GR. In a pilot experiment, groups of embryos after coinjection with 20 ng/μl of ZFN mRNAs plus 25 ng/μl of linearized pGRgsdf DNA (series II) were PCR-genotyped by using the primer pairs, F9-DR5 and pF2-KOr2. F9 and KOr2 anneals the sequence upstream and downstream of the homology arms in pGRgsdf, whereas DR5 and pF2 anneals the rfp/gfp sequence flanked by the two homology arms (Fig. 1a). This primer pair is expected to define a 1.9-kb product specific to the GR allele. Indeed, this PCR product was easily visible in coinjected embryos at days
1, 3, and 6 post fertilization (dpf) but absent in noninjected wildtype control (Fig. 1b). These results demonstrate the feasibility of GR by coinjection of ZFN mRNAs with a linearized GR plasmid DNA in medaka embryos. We wanted to examine the dosage-dependent effect on the GR efficiency in adult fish from injected embryos. Processes and fate of donor DNA’s integration into host genome via microinjection of foreign DNA into the developing embryo were elucidated in medaka and zebrafish (Stuart et al. 1988; Lu et al. 1992). Like other transgenic animals reviewed by Wall 1996, genetic mosaicism including somatic and germline mosaicisms is observed in our gsdf GD medaka (Zhang et al. 2013). Methods for the screening of founder animals for germline mosaicism prior to mating would reduce labor costing and improve the efficiency of transgenic livestock production. Two approaches of GFP monitoring and PCR prescreening are designed in our experiment to distinguish individuals carrying transgenic fragment from nontransgenic one. GFP expression in operated embryos was evaluated and described in the next section. However, GFP signal was not sensitive to be detected in fry and adults. Therefore, we performed PCR prescreening for candidate F0 founder fish
Mar Biotechnol (2014) 16:739–747
743
Table 1 Survival and GR efficiency in medaka embryos Series of injection
1 2 Sum
ZFN mRNAs (ng/μl)
10 20
Embryo injected
298 274 572
Fry, n (%)a
194 (65 %) 161 (59 %) 355 (62 %)
Adults, n (%)
Germline transmitter, n (%)
Fish
GRb
Fertile fishc
Founder
164 67 231
9 (6 %) 6 (9 %) 15 (7 %)
5 (56 %) 4 (67 %) 9 (60 %)
1 (20 %) 1 (25 %) 2 (22 %)
Embryos at the one-cell stage were injected with 25 ng/μl of linear pGRgsdf DNA and indicated dosages of ZFN mRNAs. The resultant F0 adults were PCR-genotyped by using DNA from the caudal fin clips a
Percentage values were derived by comparison to the number of embryos injected
b
Fish contained the GR allele in the fin and were tested for fertility by outcrossing with normal fish. Percentage values were derived by comparing the number of fish containing a PCR-detectable GR in the fin clips to the number of adult fish c
Fish that were able to produce F1 progeny upon outcrossing with normal fish
using caudal fin clips as template DNA, presuming the HR events occurred in both somatic cells and/or germline. Based on this PCR screening, fry of the two series mentioned above were grown to adulthood, and DNA was extracted from the fin clips and PCR-genotyped by using multiple primer pairs specific to the WT and GR alleles (Fig.1a). This revealed that 9 out of 164 adults in series I contained a GR allele in the caudal fin (Fig. 1c), producing a 6 % efficiency for GR in series-I adults. This efficiency increased to 9 % in series II (n= 67) (Table 1). Therefore, coinjection of ZFN mRNAs with a linear donor DNA can mediate GR in vivo, and a higher injection dose of ZFN mRNAs generates a higher efficiency of GR in medaka embryos, which are capable of developing into adulthood. Germline Transmission The experiments described so far demonstrate the feasibility and dose-dependent GR efficiency in the embryos and adult fin as a somatic organ easily accessible for detection. The question whether the coinjection could generate germline GR remains to be answered. To this end, 9 out of 15 adults from both series of coinjection were fertile and subjected to progeny test. Two of the nine fertile fish turned out to be founders as they were able to transmit a GR allele into their F1 progeny, producing a 20~25 % efficiency for germline GR (Table 1). One founder fish was a male coming from series I, and the other was a female from series II. This demonstrates that a linear GR plasmid DNA in combination with ZFNs is able to generate germline GR in both sexes. The male founder transmitted the GR allele to 3 out of 49 F1 progeny, producing a germline transmission efficiency of 6.1 %. The female founder produced 17 F1 progeny, one of them inherited the GR allele, producing a germline transmission efficiency of 5.9 % (Table 1). The germline transmission of both founders is illustrated in Fig. 1d, where the GR allele was detected as a lighter band compared to the WT band in F0 founder fish. However, the GR band shows equal abundance with the
wildtype band in F1 fish, which indicates the mosaicism in the founder and germline transmission of the GR allele into the F1 generation. Taken together, a combination of ZFNs with GR plasmid DNA is proficient for conducting somatic and germline GR in medaka. Precision of the GR Event In medaka, ZFNs usually generate a wide variety of subtle allelic alterations at particular loci such as gsdf (Chen et al. 2012, Zhang et al. 2013). It provoked us to examine whether ZFN-mediated GR could occur in a precise manner as conventional GR in medaka ES cells (Guan et al. 2013, Yan et al. 2013). PCR products of the GR allele were specifically amplified, cloned and sequenced. All the three clones from injected embryos shared one and the same sequence as the predicted precise GR allele. The GR allele was also observed in six clones from the adult fin of a founder fish and six clones from the adult fin of a F1 progeny (Fig. 2a). This result demonstrates that the gsdf locus was precisely replaced by the sequence of GR vector pGRgsdf within the homologous arms-flanked region in the embryo, adult soma and germline of medaka. Therefore, ZFNs can generate precise GR in medaka. We furthered our examination into Southern blot analysis. For vector construction, two HindIII sites were introduced into pGRgsdf, one in front of rfp to destroy the ZFN target sequence for preventing the GR allele from ZFN cleavage, and the other between CVand pf (Fig. 2a). The introduction of HindIII sites facilitates the distinction between the WT gsdf allele and GR allele on the Southern blot. Upon HindIII digestion, the WT allele will produce a single band of 7.5 kb, whereas the GR allele will produce three bands of 7.5, 3.7, and 1.8 kb, respectively (Fig. 2a). The 7.5- and 3.7-kb bands are detectable by external probe Ext, whereas the 1.8-kb band is specific to internal probe rfpCV. On Southern blots of genomic HindIII digests, probe Ext indeed detected the 7.5-kb band in the control fish and the 3.7-kb band besides the 7.5-kb
744
Mar Biotechnol (2014) 16:739–747
Fig. 2 Precise GR. a Sequence analyses of WT and GR alleles. (Top) Schematic structures of WT and GR gsdf alleles. Shown are the positions and extension directions of PCR primers (arrowheads), two HindIII sites and two probes (Ext and rfpCV) used for Southern analysis. (Bottom) Sequences of the WT allele and cloned PCR products of the GR allele. DNA was from injected embryos, caudal fin clips of adults from operated embryos and F1 progeny. Numbers of sequenced clones are given in parenthesis. Start codon and stop codon are in red. Introduced HindIII and BamHI sites are underlined. b Southern analysis. HindIII digests of genomic DNA (10 μg) from a control fish (+/+) and F1 fish heterozygous
for GR were hybridized to external probe Ext and internal probe rfpCV. Notably, probe rfpCV detects a single band of predicted size for defined GR. c RT-PCR detection of chimeric gsdf:rfp RNA by using indicated primers, highlighting gonad-specific expression of endogenous gsdf and chimeric cgsdf:rfp exclusively in the ovary of a F1 female heterozygous for GR. β-actin was used as a loading control. neg negative control with H2O as a template. d Chimeric gsdf:rfp cDNA (top) and predicted protein (bottom) sequences. underlined primers; ATG start codon; black letter gsdf sequence; red letter rfp sequence
band in F1 progeny, whereas probe rfpCV detected a single band of 1.8 kb diagnostic of GR allele (Fig. 2b). Notably, probe rfpCV was unable to detect additional bands, suggesting the absence of randomly integrated pGRgsdf vector DNA in the genome of this F1 fish. Thus, the ZFN-mediated GR occurs without random integration.
chimeric mRNA expression was indeed limited to the gonad, like the endogenous gsdf (Fig. 2c). Sequencing validated the existence of chimeric mRNA gsdf:rfp (Fig. 2d). This result demonstrates a precise GR event and recapitulation of endogenous gsdf RNA expression by the rfp reporter within the GR allele. As previously described, the GR allele contains rfp under the control of endogenous gsdf regulatory elements and cassette CVpf expressing puromycin acetyltransferase and GFP, under the control of CV promoter (Zhao et al. 2011). After injection of pGRgsdf DNA, PF expression was observed in many cells of the developing embryo, whereas the RFP signal was restricted to a few cells in the region of the developing gonad (Fig. 3a–f). In the adult ovary, The PF signal was found in many somatic cells surrounding oocytes, and the RFP was visible in a subpopulation of these somatic cells (Fig. 3g–i).
Recapitulation of gsdf Expression by a GR-Cointegrated Reporter According to the experimental design, the rfp with the GR allele will be under the transcriptional control of gsdf regulatory sequences, and 5′ part of the gsdf RNA will be linked to rfp RNA, forming a chimeric mRNA fusion gsdf:rfp in the gonad. RT-PCR analyses revealed that this chimeric mRNA was indeed generated in the ovary of a F1 female. This
Mar Biotechnol (2014) 16:739–747
On the squash of this ovary, female germ cells were clearly seen as oocytes that were transparent and much larger than surrounding somatic cells (Fig. 3j). Fluorescence microscopy revealed that RFP and GFP were present in certain somatic cells but absent in the oocytes (Fig. 3k, l). Therefore, RFP expression by the knockin rfp transgene within the GR allele faithfully recapitulates the endogenous gsdf RNA expression in the gonadal soma.
Discussion Medaka and zebrafish are excellent twin model organisms for analyzing gene functions in vertebrate development (Wittbrodt et al. 2002). In both species, efforts have been Fig. 3 Visualization of reporter expression. a–f Embryonic expression. Medaka embryos at the one-cell stage were microinjected with ZFN mRNAs and pGRgsdf DNA and analyzed by fluorescence microscopy at 3 dpf (a–c) and 9 dpf (d–f). The GFP signal (green) is seen in many compartments, whereas the RFP signal (red) is limited largely to the brain (arrows). The insets in c and f are merged fluorescent micrographs of the gonad region at larger magnification. od oil droplet. g–i Adult expression in the ovary of F1 medaka heterozygous for GR allele. j–l Ovarian squash showing the GFP and RFP signal present in somatic cells but absent in oocytes (oc). Scale bars, 100 μm
745
made to introduce defined alterations into specific chromosomal sites. These include conventional GR in ES cell cultures of zebrafish (Fan et al. 2006) and medaka (Guan et al. 2013, Yan et al. 2013). Recently, ZFN and TALEN have been used for GD with a great efficiency in both medaka (Ansai et al. 2012, 2013, Chen et al. 2012, Zhang et al. 2013) and zebrafish (Doyon et al. 2008, Meng et al. 2008, Foley et al. 2009, Ben et al. 2011, Sander et al. 2011, Huang et al. 2011). In zebrafish, TALEN and CRISPR/Cas9 have been used for HR-mediated GR (Zu et al. 2013) and chromosomal deletions and inversions (Xiao et al. 2013). In this study, we have demonstrated—for the first time to our knowledge—the feasibility of ZFN-mediated GR at a chromosomal locus in medaka embryos. We show that embryo coinjection with 10~20 ng/μl of ZFN mRNAs plus 25 ng/μl of linear DNA generates a satisfactory efficiency for somatic GR in the adult fin, and up to
746
25 % of these are capable of germline transmission to the next generation. Two out of nine fertile GR positive fish were GR positive founders with a germline transmission rate at about 6 %. Although PCR prescreening could not increase the frequency of positive founders, it guaranteed us to estimate the approximate proportion of germline bearing GR allele among total operated eggs (2/572=3.5‰). Those injected individuals with only germline GR but no GR allele in its caudal fin clips were escaped by this PCR pre-selection, but should be a minor portion according to the germline specification and formation during medaka embryogenesis (Li et al. 2014, Herpin et al. 2007, Kurokawa et al. 2006, Tanaka et al. 2001). Four lines of evidence demonstrate the precision of ZFNmediated GR in medaka. First, sequencing of the GR allele validates exact junction sequences between the endogenous gsdf and the knock-in region flanked by the two homolog arms. Second, Southern blot analyses reveal a band pattern that is in accordance with a single cointegrated allele at the gsdf locus. Third, we show that the gsdf and the cointegrated rfp generate a chimeric mRNA in a defined fashion. Finally, the gonad-specific expression pattern at both RNA and protein levels from the knock-in GR allele recapitulates the endogenous gsdf expression, which is consistent with the RNA in situ hybridization result reported previously (Shibata et al. 2010). Although targetable nucleases such as ZFN, TALEN, or CRISPR/Cas RNA-guided nucleases are powerful tools available for gene targeting, the successful genome editing requires combination of those meganucleases and efficient knock-in of targeting fragment via homologous recombination. Our direct gene replacement in medaka embryos provided the adequate procedures and parameters for this approach applied in medaka as a lower vertebrate model. Acknowledgments We thank Jiaorong Deng for breeding fish, Veronica Wong and Choy Mei Foong for laboratory management. This work was supported by grant from the National Research Foundation Singapore (NRF-CRP7-2010-03) to YH, by Young Scientists research fellowship from the Japan Society for Promotion of Science (No. 0773) to GG and research scholarship from the National University of Singapore to XZ. Conflict of Interest The authors declare that they have no conflict of interest. Supporting Information Additional information noted in text includes supplementary figures.
References Ansai S, Ochiai H, Kanie Y, Kamei Y, Gou Y, Kitano T, Yamamoto T, Kinoshita M (2012) Targeted disruption of exogenous EGFP gene in medaka using zinc-finger nucleases. Dev Growth Differ 54:546– 556. doi:10.1111/j.1440-169X.2012.01357.x
Mar Biotechnol (2014) 16:739–747 Ansai S, Sakuma T, Yamamoto T, Ariga H, Uemura N, Takahashi R, Kinoshita M (2013) Efficient targeted mutagenesis in medaka using custom-designed transcription activator-like effector nucleases. Genetics 193:739–49. doi:10.1534/genetics.112.147645 Ben J, Elworthy S, Ng AS, Van Eeden F, Ingham PW (2011) Targeted mutation of the talpid3 gene in zebrafish reveals its conserved requirement for ciliogenesis and Hedgehog signalling across the vertebrates. Development 138:4969–78. doi:10.1242/dev.070862 Beumer KJ, Trautman JK, Bozas A, Liu JL, Rutter J, Gall JG, Carroll D (2008) Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases. Proc Natl Acad Sci U S A 105:19821–6. doi:10.1073/pnas.0810475105 0810475105 Bibikova M, Carroll D, Segal DJ, Trautman JK, Smith J, Kim YG, Chandrasegaran S (2001) Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol Cell Biol 21:289–297. doi:10.1128/Mcb.21.1.289-297.2001 Bibikova M, Golic M, Golic KG, Carroll D (2002) Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161:1169–75 Bibikova M, Beumer K, Trautman JK, Carroll D (2003) Enhancing gene targeting with designed zinc finger nucleases. Science 300:764–764. doi:10.1126/science.1079512 Capecchi MR (2005) Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet 6: 507–12. doi:10.1038/nrg1619 Carbery ID, Ji D, Harrington A, Brown V, Weinstein EJ, Liaw L, Cui XX (2010) Targeted genome modification in mice using zinc-finger nucleases. Genetics 186:451–U37. doi:10.1534/genetics.110. 117002 Carroll D (2011) Genome engineering with zinc-finger nucleases. Genetics 188:773–82. doi:10.1534/genetics.111.131433 Chen JB, Zhang X, Wang TS, Li ZD, Guan GJ, Hong YH (2012) Efficient detection, quantification and enrichment of subtle allelic alterations. DNA Res 19:423–433. doi:10.1093/dnares/dss023 Cui X, Ji D, Fisher DA, Wu Y, Briner DM, Weinstein EJ (2011) Targeted integration in rat and mouse embryos with zinc-finger nucleases. Nat Biotechnol 29:64–7. doi:10.1038/nbt.1731 nbt.1731 Doyon Y, Mccammon JM, Miller JC, Faraji F, Ngo C, Katibah GE, Amora R, Hocking TD, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Amacher SL (2008) Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat Biotechnol 26: 702–8. doi:10.1038/nbt1409 Fan L, Moon J, Crodian J, Collodi P (2006) Homologous recombination in zebrafish ES cells. Transgenic Res 15:21–30. doi:10.1007/ s11248-005-3225-0 Flisikowska T, Thorey IS, Offner S, Ros F, Lifke V, Zeitler B, Rottmann O, Vincent A, Zhang L, Jenkins S, Niersbach H, Kind AJ, Gregory PD, Schnieke AE, Platzer J (2011) Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases. Plos One 6:e21045. doi:10.1371/journal.pone.0021045 PONE-D-11-02571 Foley, J.E., Yeh, J.R.J., Maeder, M.L., Reyon, D., Sander, J.D., Peterson, R.T. & Joung, J.K. (2009). Rapid mutation of endogenous zebrafish genes using zinc finger nucleases made by Oligomerized Pool ENgineering (OPEN). Plos One, 4. DOI Artn E4348 Doi 10.1371/ Journal.Pone.0004348. Geurts AM, Cost GJ, Freyvert Y, Zeitler B, Miller JC, Choi VM, Jenkins SS, Wood A, Cui XX, Meng XD, Vincent A, Lam S, Michalkiewicz M, Schilling R, Foeckler J, Kalloway S, Weiler H, Menoret S, Anegon I, Davis GD, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jacob HJ, Buelow R (2009) Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325:433–433. doi:10.1126/ science.1172447 Guan G, Yan Y, Chen T, Yi M, Ni H, Naruse K, Nagahama Y, Hong Y (2013) Nanos3 gene targeting in medaka ES cells. Int J Biol Sci 9: 444–54. doi:10.7150/ijbs.6507
Mar Biotechnol (2014) 16:739–747 Herpin A, Rohr S, Riedel D, Kluever N, Raz E, Schartl M (2007) Specification of primordial germ cells in medaka (Oryzias latipes). BMC Dev Biol 7:3. doi:10.1186/1471-213X-7-3 Hong Y, Winkler C, Schartl M (1996) Pluripotency and differentiation of embryonic stem cell lines from the medakafish (Oryzias latipes). Mech Dev 60:33–44 Hong Y, Winkler C, Schartl M (1998) Production of medakafish chimeras from a stable embryonic stem cell line. Proc Natl Acad Sci U S A 95: 3679–84 Hong Y, Liu T, Zhao H, Xu H, Wang W, Liu R, Chen T, Deng J, Gui J (2004) Establishment of a normal medakafish spermatogonial cell line capable of sperm production in vitro. Proc Natl Acad Sci U S A 101:8011–6. doi:10.1073/pnas.0308668101 0308668101 Hong N, Li M, Zeng Z, Yi M, Deng J, Gui J, Winkler C, Schartl M, Hong Y (2010) Accessibility of host cell lineages to medaka stem cells depends on genetic background and irradiation of recipient embryos. Cell Mol Life Sci 67:1189–1202 Hong N, He BP, Schartl M, Hong Y (2012) Medaka embryonic stem cells are capable of generating entire organs and embryo-like miniatures. Stem Cells Dev. doi:10.1089/scd.2012.0144 Huang P, Xiao A, Zhou M, Zhu Z, Lin S, Zhang B (2011) Heritable gene targeting in zebrafish using customized TALENs. Nat Biotechnol 29:699–700. doi:10.1038/nbt.1939 Kim YG, Cha J, Chandrasegaran S (1996) Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci U S A 93:1156–60 Kurokawa H, Aoki Y, Nakamura S, Ebe Y, Kobayashi D, Tanaka M (2006) Time-lapse analysis reveals different modes of primordial germ cell migration in the medaka Oryzias latipes. Dev Growth Differ 48:209–21. doi:10.1111/j.1440-169X.2006.00858.x Li M, Hong N, Xu H, Yi M, Li C, Gui J, Hong Y (2009) Medaka vasa is required for migration but not survival of primordial germ cells. Mech Dev 126:366–81. doi:10.1016/j.mod.2009.02.004 Li M, Guan G, Hong N, Hong Y (2012) Multiple regulatory regions control the transcription of medaka germ gene vasa. Biochimie. doi: 10.1016/j.biochi.2012.12.003 Li Z, Li M, Hong N, Yi M, Hong Y (2014) Formation and cultivation of medaka primordial germ cells. Cell Tissue Res. doi:10.1007/ s00441-014-1867-z Lu, J.K., Chen, T.T., Chrisman, C.L., Andrisani, O.M. & Dixon, J.E. (1992). Integration, expression and germ-line transmission of foreign growth hormone genes in medaka (Oryzias latipes). Mol Mar Biol Biotechnol 1:366–75 Mashimo, T., Takizawa, A., Voigt, B., Yoshimi, K., Hiai, H., Kuramoto, T. & Serikawa, T (2010) Generation of knockout rats with X-linked severe combined immunodeficiency (X-SCID) using zinc-finger nucleases. Plos One, 5. DOI Artn E8870 Doi 10.1371/Journal.Pone.0008870. Meng XD, Noyes MB, Zhu LHJ, Lawson ND, Wolfe SA (2008) Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat Biotechnol 26:695–701. doi:10.1038/Nbt1398 Meyer M, De Angelis MH, Wurst W, Kuhn R (2010) Gene targeting by homologous recombination in mouse zygotes mediated by zincfinger nucleases. Proc Natl Acad Sci U S A 107:15022–6. doi:10. 1073/pnas.1009424107 Ochiai H, Fujita K, Suzuki K, Nishikawa M, Shibata T, Sakamoto N, Yamamoto T (2010) Targeted mutagenesis in the sea urchin embryo using zinc-finger nucleases. Genes Cells 15:875–885. doi:10.1111/j. 1365-2443.2010.01425.x Rothstein RJ (1983) One-step gene disruption in yeast. Methods Enzymol 101:202–11 Sander JD, Cade L, Khayter C, Reyon D, Peterson RT, Joung JK, Yeh JR (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nat Biotechnol 29:697–8. doi:10.1038/nbt.1934 Shibata Y, Paul-Prasanth B, Suzuki A, Usami T, Nakamoto M, Matsuda M, Nagahama Y (2010) Expression of gonadal soma derived factor (GSDF) is spatially and temporally correlated with early testicular
747 differentiation in medaka. Gene Expr Patterns 10:283–289. doi:10. 1016/j.gep.2010.06.005 Stuart, G.W., McMurray, J.V. & Westerfield, M. (1988). Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos. Dev 103:403–12 Takasu Y, Kobayashi I, Beumer K, Uchino K, Sezutsu H, Sajwan S, Carroll D, Tamura T, Zurovec M (2010) Targeted mutagenesis in the silkworm Bombyx mori using zinc finger nuclease mRNA injection. Insect Biochem Mol Biol 40:759–765. doi:10.1016/j.ibmb.2010.07. 012 Tanaka M, Kinoshita M, Kobayashi D, Nagahama Y (2001) Establishment of medaka (Oryzias latipes) transgenic lines with the expression of green fluorescent protein fluorescence exclusively in germ cells: a useful model to monitor germ cells in a live vertebrate. Proc Natl Acad Sci U S A 98:2544–9. doi:10.1073/ pnas.041315498 Tong C, Li P, Wu NL, Yan Y, Ying QL (2010) Production of p53 gene knockout rats by homologous recombination in embryonic stem cells. Nature 467:211–3. doi:10.1038/nature09368 Wall R.J. (1996). Transgenic livestock: progress and prospects for the future. Theriologenology 45:57–68 Wang RH, Sengupta K, Li C, Kim HS, Cao L, Xiao C, Kim S, Xu X, Zheng Y, Chilton B, Jia R, Zheng ZM, Appella E, Wang XW, Ried T, Deng CX (2008) Impaired DNA damage response, genome instability, and tumorigenesis in SIRT1 mutant mice. Cancer Cell 14:312–23. doi:10.1016/j.ccr.2008.09.001 Wittbrodt J, Shima A, Schartl M (2002) Medaka—a model organism from the far East. Nat Rev Genet 3:53–64 Xiao A, Wang Z, Hu Y, Wu Y, Luo Z, Yang Z, Zu Y, Li W, Huang P, Tong X, Zhu Z, Lin S, Zhang B (2013) Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish. Nucleic Acids Res 41:e141. doi:10.1093/nar/gkt464 Yan Y, Hong N, Chen T, Li M, Wang T, Guan G, Qiao Y, Chen S, Schartl M, Li CM, Hong Y (2013) p53 gene targeting by homologous recombination in fish ES cells. Plos One 8:e59400. doi:10.1371/ journal.pone.0059400 Yi M, Hong N, Hong Y (2009) Generation of medaka fish haploid embryonic stem cells. Science 326:430–3. doi:10.1126/science. 1175151 Yi M, Hong N, Hong Y (2010) Derivation and characterization of haploid embryonic stem cell cultures in medaka fish. Nat Protoc 5:1418–30. doi:10.1038/nprot.2010.104 Young JJ, Cherone JM, Doyon Y, Ankoudinova I, Faraji FM, Lee AH, Ngo C, Guschin DY, Paschon DE, Miller JC, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Harland RM, Zeitler B (2011) Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases. Proc Natl Acad Sci U S A 108:7052–7057. doi:10.1073/pnas. 1102030108 Zhang X, Guan G, Chen J, Naruse K, Hong Y (2013) Parameters and efficiency of direct gene disruption by zinc finger nucleases in Medaka embryos. Marine Biotechnol. doi:10.1007/s10126-0139556-6 Zhao H, Hong N, Lu W, Zeng H, Song J, Hong Y (2011) Fusion gene vectors allowing for simultaneous drug selection, cell labeling and reporter assay in vitro and in vivo. Anal Chem. doi:10.1021/ ac202541t Zhao H, Hong N, Lu W, Zeng H, Song J, Hong Y (2012) Fusion gene vectors allowing for simultaneous drug selection, cell labeling, and reporter assay in vitro and in vivo. Anal Chem 84:987–93. doi:10. 1021/ac202541t Zu Y, Tong X, Wang Z, Liu D, Pan R, Li Z, Hu Y, Luo Z, Huang P, Wu Q, Zhu Z, Zhang B, Lin S (2013) TALEN-mediated precise genome modification by homologous recombination in zebrafish. Nat Methods 10:329–31. doi:10.1038/nmeth. 2374
