Protective efficacy and immune responses of largemouth bass (Micropterus salmoides) immunized with an inactivated vaccine against the viral hemorrhagic septicemia virus genotype IVa.

Viral hemorrhagic septicemia virus (VHSV) poses a significant threat to the aquaculture industry, prompting the need for effective preventive measures. Here, we developed an inactivated VHSV and revealed the molecular mechanisms underlying the host's protective response against VHSV. The vaccine was created by treating VHSV with 0.05% formalin at 16°C for 48 h, which was determined to be the most effective inactivation method. Compared with nonvaccinated fish, vaccinated fish exhibited a remarkable increase in survival rate (99%) and elevated levels of serum neutralizing antibodies, indicating strong immunization. To investigate the gene changes induced by vaccination, RNA sequencing was performed on spleen samples from control and vaccinated fish 14 days after vaccination. The analysis revealed 893 differentially expressed genes (DEGs), with notable upregulation of immune-related genes such as annexin A1a, coxsackievirus and adenovirus receptor homolog, V-set domain-containing T-cell activation inhibitor 1-like, and heat shock protein 90 alpha class A member 1 tandem duplicate 2, indicating a vigorous innate immune response. Furthermore, KEGG enrichment analysis highlighted significant enrichment of DEGs in processes related to antigen processing and presentation, necroptosis, and viral carcinogenesis. GO enrichment analysis further revealed enrichment of DEGs related to the regulation of type I interferon (IFN) production, type I IFN production, and negative regulation of viral processes. Moreover, protein-protein interaction network analysis identified central hub genes, including IRF3 and HSP90AA1.2, suggesting their crucial roles in coordinating the immune response elicited by the vaccine. These findings not only confirm the effectiveness of our vaccine formulation but also offer valuable insights into the underlying immunological mechanisms, which can be valuable for future vaccine development and disease management in the aquaculture industry.

Sea perch (Lateolabrax japonicus) UBC9 augments RGNNV infection by hindering RLRs-interferon response.

Small ubiquitin-like modifier (SUMO) is a reversible post-translational modification that regulates various biological processes in eukaryotes. Ubiquitin-conjugating enzyme 9 (UBC9) is the sole E2-conjugating enzyme responsible for SUMOylation and plays an important role in essential cellular functions. Here, we cloned the UBC9 gene from sea perch (Lateolabrax japonicus) (LjUBC9) and investigated its role in regulating the IFN response during red-spotted grouper nervous necrosis virus (RGNNV) infection. The LjUBC9 gene consisted of 477 base pairs and encoded a polypeptide of 158 amino acids with an active site cysteine residue and a UBCc domain. Phylogenetic analysis showed that LjUBC9 shared the closest evolutionary relationship with UBC9 from Paralichthys olivaceus. Tissue expression profile analysis demonstrated that LjUBC9 was significantly increased in multiple tissues of sea perch following RGNNV infection. Further experiments showed that overexpression of LjUBC9 significantly increased the mRNA and protein levels of RGNNV capsid protein in LJB cells infected with RGNNV, nevertheless knockdown of LjUBC9 had the opposite effect, suggesting that LjUBC9 exerted a pro-viral effect during RGNNV infection. More importantly, we found that the 93rd cysteine is crucial for its pro-viral effect. Additionally, dual luciferase assays revealed that LjUBC9 prominently attenuated the promoter activities of sea perch type Ⅰ interferon (IFN) in RGNNV-infected cells, and overexpression of LjUBC9 markedly suppressed the transcription of key genes associated with RLRs-IFN pathway. In summary, these findings elucidate that LjUBC9 impairs the RLRs-IFN response, resulting in enhanced RGNNV infection.

Methyltransferase-like 3 suppresses red spotted grouper nervous necrosis virus and viral hemorrhagic septicemia virus infection by enhancing type I interferon responses in sea perch.

Methylation at the N6 position of adenosine (m6A) is the most abundant internal mRNA modification in eukaryotes, tightly associating with regulation of viral life circles and immune responses. Here, a methyltransferase-like 3 homolog gene from sea perch (Lateolabrax japonicus), designated LjMETTL3, was cloned and characterized, and its negative role in fish virus pathogenesis was uncovered. The cDNA of LjMETTL3 encoded a 601-amino acid protein with a MT-A70 domain, which shared the closest genetic relationship with Echeneis naucrates METTL3. Spatial expression analysis revealed that LjMETTL3 was more abundant in the immune tissues of sea perch post red spotted grouper nervous necrosis virus (RGNNV) or viral hemorrhagic septicemia virus (VHSV) infection. LjMETTL3 expression was significantly upregulated at 12 and 24 h post RGNNV and VHSV infection in vitro. In addition, ectopic expression of LjMETTL3 inhibited RGNNV and VHSV infection in LJB cells at 12 and 24 h post infection, whereas knockdown of LjMETTL3 led to opposite effects. Furthermore, we found that LjMETTL3 may participate in boosting the type I interferon responses by interacting with TANK-binding kinase. Taken together, these results disclosed the antiviral role of fish METTL3 against RGNNV and VHSV and provided evidence for understanding the potential mechanisms of fish METTL3 in antiviral innate immunity.

Molecular characterization, transcriptional regulation of sea perch Moloney leukemia virus 10 and its antiviral function against VHSV.

Moloney leukemia virus 10 (MOV10) is a conserved RNA helicase and has multiple biological functions in mammals, but its role remains poorly understood in bony fish. Here, we cloned a MOV10 homolog from sea perch (Lateolabrax japonicus), which contained 23 exons and 22 introns, with an open reading frame of 3000 bp encoding 1000 amino acids. Tissue distribution analysis showed that MOV10 was high expressed in blood of sea perch. Promoter analysis revealed several putative multiple transcription factors binding sites, including upstream transcription factor 1, GATA-box, transcription initiation factor IIB, activator protein 1 and two interferon (IFN) stimulated response elements. Further analysis found that IFNc, IFNh, and IFNγ could not only activate IFN regulatory factor (IRF) 1 expression which in turn led to the induction of MOV10, but also prompted the expression of IRF10 to hinder excessive MOV10 expression. Moreover, IRF2 also suppressed MOV10 expression that was initiated by IRF1. Viral hemorrhagic septicemia virus (VHSV) infection upregulated MOV10 expression in vivo and in vitro, which in turn, enhanced IFNh expression and exhibited strong antiviral activity against VHSV proliferation. This study provides a basis to investigate the immune escape of VHSV by affecting the biological function of transcription factors in the signaling pathways associated with antiviral molecules.

Sea perch (Lateolabrax japonicus) autophagy related gene 5 promotes RGNNV infection via inhibiting RLRs-interferon signaling pathway.

Autophagy-related gene 5 (Atg5), an essential component of autophagy machinery, is associated with innate immune responses. Here, the Atg5 of sea perch (Lateolabrax japonicus) (LjAtg5) was cloned and its role in regulating autophagy and interferon (IFN) response during red-spotted grouper nervous necrosis virus (RGNNV) infection was investigated. The LjAtg5 cDNA encoded a polypeptide of 275 amino acids with an APG5 domain, and had the closet genetic relationship with Micropterus salmoides Atg5. Autophagic detection showed LjAtg5 was conserved in inducing cell autophagy. Spatial expression analysis revealed LjAtg5 had a higher expression level in liver, brain, and kidney tissues of RGNNV-infected sea perch compared with the control group. In RGNNV-infected LJB cells, overexpression of LjAtg5 significantly increased the mRNA and protein levels of capsid protein, whereas knockdown of LjAtg5 led to the opposite effect, indicating LjAtg5 played a pro-viral role during RGNNV infection. Furthermore, dual luciferase reporter assay revealed LjAtg5 significantly suppressed the activation of sea perch type I IFN promoter in vitro, and overexpression of LjAtg5 strongly weaken the expression of genes related to the RIG-I-like receptors (RLRs) signaling pathway and IFN stimulated genes. These results suggested LjAtg5 promoted RGNNV infection by negatively regulating RLRs-IFN signaling pathway.

Capsid protein from red-spotted grouper nervous necrosis virus induces incomplete autophagy by inactivating the HSP90ab1-AKT-MTOR pathway.

As a highly important fish virus, nervous necrosis virus (NNV) has caused severe economic losses to the aquaculture industry worldwide. Autophagy, an evolutionarily conserved intracellular degradation process, is involved in the pathogenesis of several viruses. Although NNV can induce autophagy to facilitate infection in grouper fish spleen cells, how it initiates and mediates autophagy pathways during the initial stage of infection is still unclear. Here, we found that red-spotted grouper NNV (RGNNV) induced autophagosome formation in two fish cell lines at 1.5 and 3 h post infection, indicating that autophagy is activated upon entry of RGNNV. Moreover, autophagic detection showed that RGNNV entry induced incomplete autophagy by impairing the fusion of autophagosomes with lysosomes. Further investigation revealed that binding of the RGNNV capsid protein (CP) to the Lateolabrax japonicus heat shock protein HSP90ab1 (LjHSP90ab1), a cell surface receptor of RGNNV, contributed to RGNNV invasion-induced autophagy. Finally, we found that CP blocked the interaction of L. japonicus protein kinase B (AKT) with LjHSP90ab1 by competitively binding the NM domain of LjHSP90ab1 to inhibit the AKT-mechanistic target of the rapamycin (MTOR) pathway. This study provides novel insight into the relationship between NNV receptors and autophagy, which may help clarify the pathogenesis of NNV.

Ubiquitin-specific protease 5 was involved in the interferon response to RGNNV in sea perch (Lateolabrax japonicus).

Deubiquitinases are widely involved in the regulation of the virus-triggered type I interferon (IFN) signaling. Here, we found sea perch (Lateolabrax japonicus) ubiquitin-specific protease 5 (LjUSP5) was a negative regulatory factor of the red-spotted grouper nervous necrosis virus (RGNNV)-triggered IFN response. LjUSP5 encoded a polypeptide of 830 amino acids, containing a zinc finger UBP domain (residues 197-270 aa), two ubiquitin-associated domains (residues 593-607 aa; 628-665 aa), and one UBP domain (residues 782-807 aa), and shared the closest genetic relationship with the USP5 of Larimichthys crocea. Quantitative RT-PCR analysis showed that LjUSP5 was ubiquitously expressed and up-regulated significantly in all inspected tissues post RGNNV infection, and its transcripts significantly increased in brain, liver and kidney tissues post RGNNV infection. LjUSP5 was up-regulated in cultured LJB cells after poly I:C and RGNNV treatments. In addition, overexpression of LjUSP5 significantly inhibited the activation of zebrafish IFN 1 promoter and promoted RGNNV replication in vitro. Furthermore, LjUSP5 inhibited the activation of zebrafish IFN 1 promoter induced by key genes of retinoic acid-inducible gene I-like receptors signaling pathway. Our findings provides useful information for further elucidating the mechanism underlying NNV infection.

Whole Genome Sequencing of Chinese White Dolphin (Sousa chinensis) for High-Throughput Screening of Antihypertensive Peptides.

Chinese white dolphin (Sousa chinensis), also known as the Indo-Pacific humpback dolphin, has been classified as "Vulnerable" on the IUCN Red List of Threatened Species. It is a special cetacean species that lives in tropical and subtropical nearshore waters, with significant differences from other cetaceans. Here, we sequenced and assembled a draft genome of the Chinese white dolphin with a total length of 2.3 Gb and annotation of 18,387 protein-coding genes. Genes from certain expanded families are potentially involved in DNA replication and repairing, suggesting that they may be related to adaptation of this marine mammal to nearshore environments. We also discovered that its historical population had undergone a remarkable bottleneck incident before the Mindel glaciation. In addition, a comparative genomic survey on antihypertensive peptides (AHTPs) among five representative mammals with various residential habitats (such as remarkable differences in exogenous ion concentrations and sea depth) revealed that these small bioactive peptides were highly conserved among these examined mammals, and they had the most abundant hits in collagen subunit proteins, especially for two putative AHTP peptides Gly-Leu-Pro (GLP) and Leu-Gly-Pro (LGP). Our genome assembly will be a valuable resource for further genetic researches on adaptive ecology and conservation biology of cetaceans, and for in-depth investigations into bioactive peptides in aquatic and terrestrial mammals for development of peptide-based drugs to treat various human cardiovascular diseases.

Comparative transcriptome analysis reveals the role of p53 signalling pathway during red-spotted grouper nervous necrosis virus infection in Lateolabrax japonicus brain cells.

Nervous necrosis virus (NNV) is one of the fish pathogens that have caused mass mortalities of many marine and freshwater fishes in the world. To better comprehend the molecular immune mechanism of sea perch (Lateolabrax japonicus) against NNV infection, the comparative transcriptome analysis of red-spotted grouper nervous necrosis virus (RGNNV)-infected or mock-infected L. japonicus brain (LJB) cells was performed via RNA sequencing technology. Here, 1,969 up-regulated genes and 9,858 down-regulated genes, which were widely implicated in immune response pathways, were identified. Furthermore, we confirmed that p53 signalling pathway was repressed at 48 hr post-RGNNV infection, as indicated by up-regulation of Mdm2 and down-regulation of p53 and its downstream target genes, including Bax, Casp8 and CytC. Overexpression of L. japonicus p53 (Ljp53) significantly inhibited RGNNV replication and up-regulated the expression of apoptosis-related genes, whereas the down-regulation caused by pifithrin-α led to the opposite effect, suggesting Ljp53 might promote cell apoptosis to repress virus replication. Luciferase assay indicated that Ljp53 could enhance the promoter activities of zebrafish interferon (IFN)1, indicating that Ljp53 could exert its anti-RGNNV activities by enforcing the type I IFN response. This study revealed the potential antiviral role of p53 during NNV infection.

Functional characterization of tumor necrosis factor receptor-associated factor 3 of sea perch (Lateolabrax japonicas) in innate immune.

Tumor necrosis factor receptor-associated factor 3 (TRAF3) is a multifunctional regulator implicated in both bacterial defense and antiviral immunity. Here, a TRAF3 gene from the seawater fish sea perch, designated as LjTRAF3, was characterized. The full-length cDNA of LjTRAF3 was 2972 bp including a 5' untranslated region (UTR) of 243 bp, a 3'UTR of 941 bp and a putative open reading frame of 1608 bp encoding a putative protein of 536 amino acid. The deduced LjTRAF3 protein contained a RING finger, two zinc fingers, a coiled-coil, and a meprin and TRAF-C homology domain. Phylogenetic analysis showed that LjTRAF3 shared the closest genetic relationship with Larimichthys crocea TRAF3. Gene expression analyses suggested that LjTRAF3 mRNA was ubiquitously expressed in all the tissues tested, and was up-regulated post red spotted grouper nervous necrosis virus (RGNNV) infection in vivo and in vitro. Reporter gene assay showed that LjTRAF3 significantly activated zebrafish type I interferon (IFN) promoter in vitro. During RGNNV infection, ectopic expression of LjTRAF3 significantly reduced the RNA dependent RNA polymerase transcription of RGNNV, and enhanced the expression of RIG-I-like receptors (RLR), janus kinase-signal transducers and activators of transcription (JAK-STAT) signaling pathway related genes and IFN stimulated genes (ISGs), including ISG15, PKR, VIG and TRIM39. Taken together, our results suggested that LjTRAF3 might trigger the expression of various ISGs to counter RGNNV infection by regulating the RLR-induced IFN and JAK-STAT signaling pathways.

Identification of sea perch (Lateolabrax japonicus) ribonucleoprotein PTB-Binding 1 involved in antiviral immune response against RGNNV.

RIG-I-like receptors (RLRs) can recognize viral RNA and initiate innate antiviral response. In earlier studies, we demonstrated that RLRs were implicated in the antiviral immunity against RGNNV in the seawater fish sea perch (Lateolabrax japonicus). However, potential regulators of RLRs-mediated signaling pathways involved in RGNNV infection remain unclear. In this study, a novel ribonucleoprotein PTB-binding 1 (Raver1) of sea perch (LjRAVER1) was identified for the first time. The cDNA of LjRAVER1 was 4066 bp in length and encoded a deduced polypeptide of 733 amino acids. Phylogenetic analysis revealed a closer affinity of LjRAVER1 with Larimichthys Crocea Raver1. LjRAVER1 mRNA was constitutively expressed in all 10 sampled tissues, and rapidly and significantly increased in vivo upon RGNNV infection. Time course analysis showed that LjRAVER1 transcripts were significantly increased both in vivo and in vitro after RGNNV infection. Viral infection and poly I:C treatment caused translocation of LjRAVER1 from the nucleus to the cytoplasm. Ectopic expression of LjRAVER1 increased the transcription level of several RLR signaling pathway related genes inducible by poly I:C treatment in vitro. Moreover, the viral gene transcription and virus production of RGNNV were significantly decreased in LjRAVER1 overexpressing cells. Luciferase reporter assays demonstrated that overexpression of LjRAVER1 significantly increased the promoter activity of zebrafish IFN1. Taken together, these findings indicated that LjRAVER1 might be an important component of RLR signaling pathway and involved in RLR pathway-mediated IFN response in sea perch.

Molecular characterization and expression pattern of a germ cell marker gene dnd in gibel carp (Carassius gibelio).

As a germ cell marker gene, Dead end (dnd) has been identified and characterized in many vertebrates. Recently, we created a complete germ cell-depleted gonad model by the dnd-specific morpholino-mediated knockdown approach, and revealed sex-biased gene expression alteration through utilizing unisexual gynogenetic superiority in polyploid gibel carp. However, dnd and its expression pattern are still unclear in the gibel carp. In this study, we further analyzed molecular characterization of gibel carp dnd and its dynamic expression pattern during gametogenesis and embryogenesis. Similar to other homologs in vertebrates, gibel carp dnd contains a conserved RRM motif and five other motifs, and is highly evolutionary conserved in genomic organization and neighborhood gene synteny. RT-PCR and Western blot analyses showed its gonad-specific expression intensively in testis and ovary. Section in situ hybridization (SISH) and immunofluorescence localization revealed its dynamic expression pattern specific to oogenic cells and spermatogenetic cells during oogenesis and spermatogenesis. Moreover, its temporal and spatial distribution specific to PGCs were also demonstrated by RT-PCR and whole mount in situ hybridization (WISH) during embryogenesis. Therefore, gibel carp Dnd is a conserved germ cell marker during gametogenesis, and its maternal transcript is also a useful marker for tracing PGC specification and migration.

Identification and characterization of the melanoma differentiation - associated gene 5 in sea perch, Lateolabrax japonicus.

The RIG-I-like receptors family is a group of cytosolic RNA helicase proteins that can recognize viral RNA via binding to pathogen associated molecular pattern motifs within RNA ligands. A novel vertebrate RLR counterpart named LjMDA5 was firstly identified from the marine fish sea perch Lateolabrax japonicus in this study. The full-length cDNA of LjMDA5 is 3750 bp and encodes a polypeptide of 988 amino acids, containing two N-terminal tandem caspase activation and recruitment domains, a DExH (Asp-Glu-X-His) box domain, an HELICc domain, and a C-terminal domain RIG-I. Phylogenetic analysis showed that LjMDA5 shared the closest genetic relationship with the MDA5 of Larimichthys crocea. Quantitative RT-PCR analysis showed that LjMDA5 was ubiquitously expressed and up-regulated significantly in all selected tissues in vivo post NNV infection. Time course analysis showed that LjMDA5 transcripts significantly increased in spleen and kidney. We found LjMDA5 could be regulated in the sea perch LJB and LJF cell lines after lipopolysaccharide, polyinosinic-polycytidylic acid treatment and NNV challenge. RNA interference experiment indicated that silencing of LjMDA5 significantly increased RGNNV replication and virus production in NNV infected LJF cells. Our results revealed that MDA5 was essential for host defense against NNV, which provided new insights into the function of RLR signaling pathway during NNV infection in fish.

Molecular characterization and expression analysis of mitochondrial antiviral signaling protein gene in sea perch, Lateolabrax japonicus.

The mitochondrial antiviral signaling protein (MAVS) is vital for host defenses against viral infection by inducing expression of type I interferon. Here, the MAVS of sea perch (Lateolabrax japonicus) (LjMAVS) was cloned and analyzed. The complete cDNA sequence of LjMAVS was 3207 bp and encoded a polypeptide of 601 amino acids. LjMAVS contains an N-terminal CARD-like domain, a central proline-rich domain and a C-terminal transmembrane domain. Phylogenetic analysis indicated that LjMAVS exhibited the closest relationship to O. fasciatus MAVS. LjMAVS was ubiquitously expressed in all tested tissues of healthy fish. The expression of LjMAVS was significantly increased post nervous necrosis virus (NNV) infection in vivo in all the selected tissues. Furthermore, time course analysis showed that LjMAVS transcripts significantly increased in the brain, spleen and kidney tissues after NNV infection. LjMAVS mRNA expression was significantly up-regulated in vitro after poly I:C stimulation. The viral gene transcription of RGNNV was significantly decreased in LjMAVS over-expressing LJB cells. These findings provide useful information for further elucidating the function ofLjMAVS in antiviral innate immune against NNV in sea perch.

Characterization and expression analysis of laboratory of genetics and physiology 2 gene in sea perch, Lateolabrax japonicus.

LGP2 (laboratory of genetics and physiology 2) as a key component of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), plays a predominant role in modulating RLRs-mediated cellular antiviral signaling during viral infection. In the present study, we cloned the LGP2 gene from the sea perch (Lateolabrax japonicus) (LjLGP2), an economically important farmed fish. The complete cDNA sequence of LjLGP2 was 2790 nt and encoded a polypeptide of 682 amino acids which contains four main structural domains: one DEAD/DEAH box helicase domain, one conserved restriction domain of bacterial type III restriction enzyme, one helicase superfamily c-terminal domain and one C-terminal domain of RIG-I, similar to most vertebrate LGP2. Subcellular localization analysis showed that LjLGP2 spanned the entire cytosol. The LjLGP2 mRNA was widespread expressed in the tested 10 tissues of healthy fish and significantly up-regulated post NNV infection. Furthermore, time course analysis showed that LjLGP2 transcripts significantly increased in the spleen, kidney and liver tissues after NNV infection. LjLGP2 mRNA expression was rapidly and significantly up-regulated in LJB cells after poly I:C stimulation and NNV infection. The present results suggest that LjLGP2 may be involved in recognization of NNV and play a role in antiviral innate immune against NNV in sea perch.

Isolation and characterization of a novel hydrocarbon-degrading bacterium Achromobacter sp. HZ01 from the crude oil-contaminated seawater at the Daya Bay, southern China.

Microorganisms play an important role in the biodegradation of petroleum contaminants, which have attracted great concern due to their persistent toxicity and difficult biodegradation. In this paper, a novel hydrocarbon-degrading bacterium HZ01 was isolated from the crude oil-contaminated seawater at the Daya Bay, South China Sea, and identified as Achromobacter sp. Under the conditions of pH 7.0, NaCl 3% (w/v), temperature 28 °C and rotary speed 150 rpm, its degradability of the total n-alkanes reached up to 96.6% after 10 days of incubation for the evaporated diesel oil. Furthermore, Achromobacter sp. HZ01 could effectively utilize polycyclic aromatic hydrocarbons (PAHs) as its sole carbon source, and could remove anthracene, phenanthrene and pyrence about 29.8%, 50.6% and 38.4% respectively after 30 days of incubation. Therefore, Achromobacter sp. HZ01 may employed as an excellent degrader to develop one cost-effective and eco-friendly method for the bioremediation of marine environments polluted by crude oil.

Interordinal chimera formation between medaka and zebrafish for analyzing stem cell differentiation.

Chimera formation is a standard test for pluripotency of stem cells in vivo. Interspecific chimera formation between distantly related organisms offers also an attractive approach for propagating endangered species. Parameters influencing interspecies chimera formation have remained poorly elucidated. Here, we report interordinal chimera formation between medaka and zebrafish, which separated ∼320 million years ago and exhibit a more than 2-fold difference in developmental speed. We show that, on transplantation into zebrafish blastulae, both noncultivated blastomeres and long-term cultivated embryonic stem (ES) cells of medaka adopted the zebrafish developmental program and differentiated into physiologically functional cell types including pigment cells, blood cells, and cardiomyocytes. We also show that medaka ES cells express differentiation gene markers during chimeric embryogenesis. Therefore, the evolutionary distance and different embryogenesis speeds do not produce donor-host incompatibility to compromise chimera formation between medaka and zebrafish, and molecular markers are valuable for analyzing lineage commitment and cell differentiation in interspecific chimeric embryos.

Medaka fish stem cells and their applications.

Stem cells are present in developing embryos and adult tissues of multicellular organisms. Owing to their unique features, stem cells provide excellent opportunities for experimental analyses of basic developmental processes such as pluripotency control and cell fate decision and for regenerative medicine by stem cell-based therapy. Stem cell cultures have been best studied in 3 vertebrate organisms. These are the mouse, human and a small laboratory fish called medaka. Specifically, medaka has given rise to the first embryonic stem (ES) cells besides the mouse, the first adult testis-derived male stem cells spermatogonia capable of test-tube sperm production, and most recently, even haploid ES cells capable of producing Holly, a semi-cloned fertile female medaka from a mosaic oocyte created by microinjecting a haploid ES cell nucleus directly into a normal oocyte. These breakthroughs make medaka a favoring vertebrate model for stem cell research, the topic of this review.

Generation of medaka fish haploid embryonic stem cells.

Haploid embryonic stem (ES) cells combine haploidy and pluripotency, enabling direct genetic analyses of recessive phenotypes in vertebrate cells. Haploid cells have been elusive for culture, due to their inferior growth and genomic instability. Here, we generated gynogenetic medaka embryos and obtained three haploid ES cell lines that retained pluripotency and competitive growth. Upon nuclear transfer into unfertilized oocytes, the haploid ES cells, even after genetic engineering, generated viable offspring capable of germline transmission. Hence, haploid medaka ES cells stably maintain normal growth, pluripotency, and genomic integrity. Mosaic oocytes created by combining a mitotic nucleus and a meiotic nucleus can generate fertile fish offspring. Haploid ES cells may offer a yeast-like system for analyzing recessive phenotypes in numerous cell lineages of vertebrates in vitro.

Establishment of medakafish as a model for stem cell-based gene therapy: efficient gene delivery and potential chromosomal integration by baculoviral vectors.

Viral vectors hold promise and challenges in gene therapy. Specifically, we have previously shown that baculoviral (BV) vectors have a high efficiency of gene delivery in human embryonic stem (ES) cells. Here we report the development of a complementary system to further our evaluation by utilizing the laboratory fish medaka that has ES cell lines and tools for experimental analyses in vitro and in vivo. We show that BV vectors can give rise to almost 100% of transient gene delivery in the medaka ES cell line MES1. BV-transduced MES1 cells reproducibly (at approximately 10(-5)) produce GFP-expressing colonies that, upon manual isolation, develop into stable clones during 300 days of culture. Surprisingly, BV transduction can also mediate efficient gene integration in the medaka genome, as fluorescent in situ hybridization revealed the presence of the BV-delivered gfp transgene in multiple locations in nuclei and on various chromosomes of metaphase spreads. We show that BV transduction does not compromise the genome stability and pluripotency of MES1 cells. We conclude that BV can efficiently mediate gene delivery and chromosomal integration in medaka ES cells. Therefore, medaka provides a powerful system for analyzing the potential of BV-mediated gene delivery in stem cells and gene therapy.