Expression and biological activity of two types of interferon genes in medaka (Oryzias latipes).

Type I interferon (IFN) is one of most important cytokines for antiviral responses in fish innate immunity, after the induction pathway following pattern recognition. In this study, 2 types of type I IFN mRNA from a medaka (Japanese rice fish; Oryzias latipes) were identified and classified (phylogenetic analysis) into subgroup-a and -d by (designated olIFNa and olIFNd, respectively). Both olIFNa and olIFNd (encoding 197 and 187 amino acid residues, respectively) contained 2 cysteines. Gene expression pattern of olIFNa, olIFNd and IFN-stimulated genes (ISGs) was assessed (quantitative real-time reverse transcriptase PCR, qRT-PCR) in various organs (i.e., whole kidney, liver and spleen) of medaka stimulated by polyI:C or infected with nervous necrosis virus (NNV). Expression of olIFNa, olIFNd and ISGs, especially the ISG15 gene, were significantly upregulated after NNV-infection. Furthermore, olIFNa, olIFNd and ISGs mRNAs were sufficiently induced in DIT cells (i.e., medaka hepatoma cell line) transfected with polyI:C or infected with NNV. In addition, in vitro biological activities of recombinant olIFNa and olIFNd (rolIFNa and rolIFNd) produced by mammalian cell line HEK293T were also characterized. Expression of GIG1a and ISG15 genes in kidney cells of adult medaka were induced by rolIFNa or rolIFNd. The olIFNs-overexpressing DIT cells had reduced viral titers following NNV infection. Therefore, we inferred that 2 type I IFNs were involved in innate immunity (antiviral response) in medaka fish.

Transient and permanent gene transfer into the brain of the teleost fish medaka (Oryzias latipes) using human adenovirus and the Cre-loxP system.

In this study, we demonstrated that human type-5 adenovirus infected the brain of the teleost fish, medaka (Oryzias latipes), in vivo. Injection of adenoviral vector into the mesencephalic ventricle of medaka larvae induced the expression of reporter genes in some parts of the telencephalon, the periventricular area of the mesencephalon and diencephalon, and the cerebellum. Additionally, the Cre-loxP system works in medaka brains using transgenic medaka carrying a vector containing DsRed2, flanked by loxP sites under control of the beta-actin promoter and downstream promoterless enhanced green fluorescent protein (EGFP). We demonstrated that the presence of green fluorescence depended on injection of adenoviral vector expressing the Cre gene and confirmed that EGFP mRNA was transcribed in the virus-injected larvae.

Pantropic retroviral vector integration, expression, and germline transmission in medaka (Oryzias latipes).

Pantropic retroviral vectors were used to introduce transgenes into Japanese medaka (Oryzias latipes). These vectors contain the long terminal repeat (LTR) sequence of Moloney murine leukemia virus (Mo-MLV) and a reporter gene (neo or lacZ) regulated by the LTR sequence of rous sarcoma virus (RSV). Because these pseudotyped retroviral vectors contain the vesicular stomatitis virus envelope glycoprotein (VSV-G), they have an extremely broad host cell range and can infect many no mammalian species. Newly fertilized medaka eggs (intact or dechorionated) were electroporated at different voltage settings in the presence of 4 x 10(4) cfu of pantropic retroviral vector. The survival rates of the pantropic retroviral vector-treated embryos ranged from 65% to 20% with increasing amplitude of electroporation. Dechorionation did not substantially affect the survival rate of embryos. PCR amplification demonstrated proviral sequences in up to 60% of the 2-month-old fish. The efficiency of gene transfer was enhanced by dechorionation. Furthermore, overnight incubation of dechorionated embryos with pantropic retroviral vectors without electroporation also resulted in proviral integration in 60% of the embryos without compromising survival rate. Southern blot analysis of DNA samples isolated from polymerase chain reaction (PCR) as positive F1 reaction animals confirmed the integration of a single copy of the provirus into the host genome. Three P1 transgenic females transmitted the proviral sequence to 50% of their F1 progeny in a back cross with wild-type males, suggesting that the entire germline of these P1 fish was transformed by the pantropic retroviral vector. Expression of the neomycin phosphotranferase transgene in F1 transgenic individuals was detected by reverse transcription (RT)-PCR amplification of the neo mRNA sequence. Furthermore, expression of a beta-galactosidase transgene was also observed in 4-day-old F1 transgenic individuals. Thus, pantropic retroviral vectors provide a convenient method to stably introduce and express foreign genes in medaka.