Characterization of Promoter Activities of Four Different Japanese Flounder Promoters in Transgenic Zebrafish

An important consideration in transgenic research is the choice of promoter for regulating the expression of a foreign gene. In this study several tissue-specific and inducible promoters derived from Japanese flounder Paralichthys olivaceus were identified, and their promoter activity was examined in transgenic zebrafish. The 5′ flanking regions of the Japanese flounder complement component C3, gelatinase B, keratin, and tumor necrosis factor (TNF) genes were linked to green fluorescence protein (GFP) as a reporter gene. The promoter regulatory constructs were introduced into fertilized zebrafish eggs. As a result we obtained several stable transgenic zebrafish that displayed green fluorescence in different tissues. Complement component C3 promoter regulated GFP expression in liver, and gelatinase B promoter regulated it in the pectoral fin and gills. Keratin promoter regulated GFP expression in skin and liver. TNF gene promoter regulated GFP expression in the pharynx and heart. TNF promoter had lipoplysaccharide-inducible activity, such that when transgenic embryos were immersed lipopolysaccharide, GFP expression increased in the epithelial tissues. These 4 promoters regulated the expression of GFP in different patterns in transgenic zebrafish.     

Cloning and characterization of the IkappaBalpha gene from Japanese flounder, Paralichthys olivaceus

We identified and characterized the Japanese flounder (Paralichthys olivaceus) inhibitor kappa B alpha (JFIKBA) cDNA. The JFIKBA cDNA contains an open reading frame of 960bp encoding 320 amino acid residues. JFIKBA contains 6 ankyrin repeats in the central coding region. Expression studies by RT-PCR showed constitutive expression of the JFIKBA gene in several Japanese flounder tissues (brain, muscle, gill, heart, kidney, liver, spleen and intestine). Moreover, expression of JFIKBA mRNA was induced in kidney by LPS stimulation. To investigate the role of JFIKBA, we constructed a recombinant plasmid expressing the JFIKBA coding region under the control of the cytomegalovirus (CMV) promoter. Over-expression of the JFIKBA gene in the Japanese flounder cultured cell line derived from kidney, suppressed the expression of the TNF alpha gene with lipopolysaccharide stimulation. These results indicated that JFIKBA has an important role in the innate immune system, especially in the signaling of the cytokine network.     

Transgenic Zebrafish Expressing Chicken Lysozyme Show Resistance against Bacterial Diseases

We established a transgenic zebrafish strain expressing chicken lysozyme gene under the control of the Japanese flounder keratin gene promoter, and investigated its resistance to a pathogenic bacterial infection. To generate the lysozyme transgenic construct, Japanese flounder keratin promoter was linked to both the hen egg white (HEW) lyoszyme gene and green fluorescence protein (GFP) gene used as a selection marker for the transgenic strains, in a recombinant plasmid. The recombinant plasmid was microinjected into fertilized zebrafish eggs. In F2 transgenic zebrafish, GFP expression was strong in the epithelial tissues, liver and gill from the embryonic stage to the adult stage. The expressions of HEW lysozyme and GFP mRNA were confirmed in the liver and skin by RT-PCR. Western blot analysis showed that both HEW lysozyme and GFP were present in protein extracts from the liver of transgenic zebrafish, but not in protein extracts from the muscle. The lytic activity of protein extracts from the liver (assessed by a lysoplate assay using Micrococcus lysodeikticus as a substrate) was 1.75 times higher in F2 transgenic zebrafish than in the wild type. In a challenge experiment, 65% of the F2 transgenic fish survived an infection of Flavobacterium columnare and 60% survived an infection of Edwardsiella tarda, whereas 100% of the control fish were killed by both pathogens. However, the survival rates of the transgenic fish were not significantly higher when higher concentrations of bacteria were used.     

Molecular cloning, characterization, and expression of TNF cDNA and gene from Japanese flounder Paralychthys olivaceus

We cloned a cDNA and the gene for Japanese flounder TNF. The TNF cDNA consisted of 1217 bp, which encoded 225 amino acid residues. The identities between Japanese flounder TNF and members of the mammalian TNF family were approximately 20-30%. The positions of cysteine residues that are important for disulfide bonds were conserved with respect to those in mammalian TNF-alpha. The Japanese flounder TNF gene has a length of approximately 2 kbp and consists of four exons and three introns. The positions of the exon-intron junction positions of Japanese flounder TNF gene are similar to those of human TNF-alpha. However, the length of the first intron of Japanese flounder is much shorter than that of the human TNF-alpha gene. There are simple CA or AT dinucleotide repeats in the 5'-upstream and 3'-downstream regions of the Japanese flounder TNF gene. Southern blot hybridization indicted that Japanese flounder TNF exists as a single copy. Expression of Japanese flounder TNF mRNA is greatly induced after stimulation of PBLs with LPS, Con A, or PMA. These results indicated that Japanese flounder TNF is more like mammalian TNF-alpha than mammalian lymphotoxin-alpha, with respect to its gene structure, length of amino acid sequence, number and position of cysteine residues, and regulation of gene expression.     

Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters

Although the zebrafish has become a popular model organism for vertebrate developmental and genetic analyses, its use in transgenic studies still suffers from the scarcity of homologous gene promoters. In the present study, three different zebrafish cDNA clones were isolated and sequenced completely, and their expression patterns were characterized by whole‐mount in situ hybridization as well as by Northern blot hybridization. The first clone encodes a type II cytokeratin (CK), which is specifically expressed in skin epithelia in early embryos and prominently expressed in the adult skin tissue. The second clone is muscle specific and encodes a muscle creatine kinase (MCK). The third clone, expressed ubiquitously in all tissues, is derived from an acidic ribosomal phosphoprotein P0 (arp) gene. In order to test the fidelity of zebrafish embryos in transgenic expression, the promoters of the three genes were isolated using a rapid linker‐mediated PCR approach and subsequently ligated to a modified green fluorescent protein (gfp) reporter gene. When the three hybrid GFP constructs were introduced into zebrafish embryos by microinjection, the three promoters were activated faithfully in developing zebrafish embryos. The 2.2‐kb ck promoter was sufficient to direct GFP expression in skin epithelia, although a weak expression in muscle was also observed in a few embryos. This pattern of transgenic expression is consistent with the expression pattern of the endogenous cytokeratin gene. The 1.5‐kb mck promoter/gfp was expressed exclusively in skeletal muscles and not elsewhere. By contrast, the 0.8‐kb ubiquitous promoter plus the first intron of the arp gene were capable of expressing GFP in a variety of tissues, including the skin, muscle, lens, neurons, notochord, and circulating blood cells. Our experiments, therefore, further demonstrated that zebrafish embryos can faithfully express exogenously introduced genes under the control of zebrafish promoters. Dev. Genet. 25:158–167, 1999. © 1999 Wiley‐Liss, Inc.     

Generation of Two-color Transgenic Zebrafish Using the Green and Red Fluorescent Protein Reporter Genes gfp and rfp

Two tissue-specific promoters were used to express both green fluorescent protein (GFP) and red fluorescent protein (RFP) in transgenic zebrafish embryos. One promoter (CK), derived from a cytokeratin gene, is active specifically in skin epithelia in embryos, and the other promoter (MLC) from a muscle-specific gene encodes a myosin light chain 2 polypeptide. When the 2 promoters drove the 2 reporter genes to express in the same embryos, both genes were faithfully expressed in the respective tissues, skin or muscle. When the 2 fluorescent proteins were expressed in the same skin or muscle cells under the same promoter, GFP fluorescence appeared earlier than RFP fluorescence in both skin and muscle tissues, probably owing to a higher detection sensitivity of GFP. However, RFP appeared to be more stable as its fluorescence steadily increased during development. Finally, F₁ transgenic offspring were obtained expressing GFP in skin cells under the CK promoter and RFP in muscle cells under the MLC promoter. Our study demonstrates the feasibility of monitoring expression of multiple genes in different tissues in the same transgenic organism.     

Development of a heat shock inducible gfp transgenic zebrafish line by using the zebrafish hsp27 promoter

In the present study, a zebrafish hsp27 promoter was isolated and used to develop heat shock inducible gfp transgenic zebrafish. The endogenous hsp27 mRNAs were constitutively expressed from 4 hpf and increased in several regions of brain, heart and somites in early embryogenesis until 24 hpf. Subsequently, the expression was reduced significantly but maintained in the heart and ears. Heat shock induced hsp27 mRNAs in the blastoderm from 6 hpf and later in somites, branchial arches and several regions of brain. Similarly in hsp27-gfp transgenic zebrafish, constitutive GFP expression was observed from 11 hpf. GFP expression was mainly in the skin cells and increased to the peak level at 7 dpf, followed by a reduction. The constitutive GFP expression in the heart was initiated from 50 hpf and maintained even in the adult fish. After heat shock, GFP expression was mainly induced in the muscle in addition to a mild increase in the skin and heart. The early stages of the embryos were more sensitive than late stages as the time required for induced GFP expression in the muscle is shorter. Thus, the hsp27-gfp transgenic line generally recapitulates the expression pattern and heat shock inducibility of endogenous hsp27 RNAs. We also tested the potential of using the hsp27-gfp transgenic zebrafish embryos for heavy metal induction and demonstrated the inducibility of GFP expression by arsenic; this pattern of induction was also supported by examination of endogenous hsp27 mRNA.     

High-level expression of human lactoferrin in milk of transgenic mice using genomic lactoferrin sequence.

In our previous study, transgenic mice were generated that expressed human lactoferrin (hLF) in milk using cDNA under control of the 2 kb bovine beta-casein promoter. The expression level of the protein in milk of 7 mice ranged from 1 to 200 microg/ml; 1 to 34 microg/ml in 6 mice and 200 microg/ml in 1 mouse. With the aim of inducing higher expression of the protein, we constructed an expression cassette comprised of 10 kb of the bovine beta-casein gene promoter and the hLF genomic sequence in place of the cDNA. The hLF genomic sequence of about 27 kb, spanning 23 kb of the entire coding region and 4 kb of the 3'-flanking sequence, was placed downstream the bovine beta-casein promoter. In total, 8 transgenic mice were generated from 31 mice (transgenic rate of 25.8%) born from the embryos microinjected with the 40-kb hLF expression cassette. Mammary-specific expression of the transgene was addressed by performing Northern hybridization of the total RNAs from various tissues of transgenic mice. Immunoblot analysis showed that the recombinant protein expressed in milk has the same molecular weight as the native protein. The amount of the protein in milk of 5 mice ranged from 60 to 6,600 microg/ml when judged by ELISA analysis. Three mice expressed the protein at the level higher than 500 microg/ml. These data suggest that the genomic lactoferrin sequence represents a valuable element for the efficient expression of the protein in milk of transgenic animals.     

Characterization of Japanese flounder (Paralichthys olivaceus) NK-lysin, an antimicrobial peptide

The NK-lysin cDNA of Japanese flounder, Paralichthys olivaceus, consists of 657bp, containing an open reading frame (ORF) of 444bp, which encodes 147 amino acid residues. The amino acid sequence of Japanese flounder NK-lysin has 21% identity to porcine NK-lysin and bovine NK-lysin, 23% to equine NK-lysin, and 46% to zebrafish NK-lysin-like protein. Multiple alignments of Japanese flounder NK-lysin and other known saposin-like proteins revealed that the six cysteine residues important for structural folding are completely conserved. The Japanese flounder NK-lysin gene is approximately 2kb and consists of five exons and four introns. Japanese flounder NK-lysin mRNA constitutive expression was mainly detected in gills, heart, head kidney, intestines, peripheral blood leukocytes (PBLs), spleen and trunk kidney, and was detected at low levels in liver, muscle and ovary. However, expression was not detected in brain, skin and stomach of apparently healthy Japanese flounder. Gene expression of Japanese flounder NK-lysin was not inducible by lipopolysaccharide (LPS) treatment. A synthesized NK-lysin peptide, consisting of 27 amino acid residues, showed antimicrobial activity against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Photobacterium damselae subsp. piscicida.     

An Apo-14 promoter-driven transgenic zebrafish that marks liver organogenesis

Several transgenic zebrafish lines for liver development studies had been obtained in the first decade of this century, but not any transgenic GFP zebrafish lines that mark the through liver development and organogenesis were reported. In this study, we analyzed expression pattern of endogenous Apo-14 in zebrafish embryogenesis by whole-mount in situ hybridization, and revealed its expression in liver primordium and in the following liver development. Subsequently, we isolated zebrafish Apo-14 promoter of 1763 bp 5'-flanking sequence, and developed an Apo-14 promoter-driven transgenic zebrafish Tg(Apo14: GFP). And, maternal expression and post-fertilization translocation of Apo-14 promoter-driven GFP were observed in the transgenic zebrafish line. Moreover, we traced onset expression of Apo-14 promoter-driven GFP and developmental behavior of the expressed cells in early heterozygous embryos by out-crossing the Tg(Apo14: GFP) male to the wild type female. Significantly, the Apo-14 promoter-driven GFP is initially expressed around YSL beneath the embryo body at 10 hpf when the embryos develop to tail bud prominence. In about 14-somite embryos at 16-17 hpf, a typical "salt-and-pepper" expression pattern is clearly observed in YSL around the yolk sac. Then, a green fluorescence dot begins to appear between the notochord and the yolk sac adjacent to otic vesicle at about 20 hpf, which is later demonstrated to be liver primordium that gives rise to liver. Furthermore, we investigated dynamic progression of liver organogenesis in the Tg(Apo14: GFP) zebrafish, because the Apo-14 promoter-driven GFP is sustainably expressed from hepatoblasts and liver progenitor cells in liver primordium to hepatocytes in the larval and adult liver. Additionally, we observed similar morphology between the liver progenitor cells and the GFP-positive nuclei on the YSL, suggesting that they might originate from the same progenitor cells in early embryos. Overall, the current study provides a transgenic zebrafish line that marks the through liver organogenesis.     

Pituitary corticotroph ontogeny and regulation in transgenic zebrafish

We characterized zebrafish proopiomelanocortin (POMC) gene promoter, and sequence analysis revealed that the promoter contains regulatory elements conserved among vertebrate species. To monitor the ontogeny of the pituitary POMC lineage in living vertebrates, we generated transgenic zebrafish expressing green fluorescent protein (GFP) driven by the POMC promoter. Zebrafish POMC-GFP is first expressed asymmetrically as two bilateral groups of cells most anterior to the neural ridge midline at 18-20 h post fertilization (hpf). POMC-GFP-positive cells then fuse into a single-cell mass within the pituitary anlage after 24 hpf and subsequently organize as distinct anterior and posterior domains between 48 and 64 hpf. Immunohistochemical studies with ACTH and alphaMSH antisera showed that POMC-GFP was mainly targeted to both anterior and posterior pituitary corticotrophs, whereas posterior pituitary region melanotrophs did not express GFP. To determine in vivo zebrafish corticotroph responses, dexamethasone (10(-5) m) was added to live embryos, which selectively suppressed POMC-GFP expression in the anterior group of corticotrophs, suggesting a distinct domain that is responsive to glucocorticoid feedback. Transgenic zebrafish with specific POMC-GFP expression in pituitary corticotrophs offers a powerful genetic system for in vivo study of vertebrate corticotroph lineage development.     

Role of p52 (NF-kappaB2) in LPS tolerance in a human B cell line

Cells of the weakly CD14 positive human B cell line RPMI 8226, clone 1, will mobilize NF-kappaB (p50/p65 and p50/p50) proteins and produce TNF mRNA when stimulated with lipopolysaccharide (LPS). When such cells are precultured with a low amount of LPS (50-250 ng/ml) for 3 - 4 days followed by a secondary stimulation with a high dose of LPS (1 microg/ml) then the cytokine expression is strongly reduced, i. e. the cells have become tolerant. Western blot analysis of proteins of the NF-kappaB/rel family demonstrates cytoplasmic p50 and p65 for naive B cells plus a low level of p52. While with tolerance induction the pattern of p50 and p65 proteins remains essentially unchanged, the LPS tolerant 8226 cells show a dramatic increase of both p52 protein and its p100 precursor in the cytosol. This p52 is found strongly upregulated in Western blots of extracts from purified nuclei of tolerant cells. Also, gelshift analysis with the -605 kappaB motif of the human TNF 5'-region shows an additional high mobility complex in LPS tolerant cells -a complex that is supershifted with an anti-p52 antibody. Functional analysis with the -1064 TNF 5'-region in front of the luciferase reporter gene demonstrates that transactivation of the TNF promoter is strongly reduced in tolerant cells. Also, overexpression of p52 will suppress activity of TNF promoter reporter gene constructs. Taken together these data show that tolerance to LPS in the human RPMI 8226 B cell line involves upregulation of the p52 (NF-kappaB2) gene, which appears to be instrumental in the blockade of TNF gene expression.     

赤点石斑鱼促甲状腺激素TSHβ启动子的克隆及其在斑马鱼胚胎原始性腺和垂体中定位表达

促甲状腺激素(Thyroid-stimulating hormone,TSH)具有调节生长,发育,代谢和生殖的功能,但是在低等脊椎动物中的功能研究较少。斜带石斑鱼(Epinephelus coioides)促甲状腺激素TSHβ除在垂体中表达外还在性腺中表达,进一步发现TSHβ也在赤点石斑鱼(Epinephelus akaara)性腺中表达。且在人工诱导的赤点石斑鱼中,随着雄性化的转变,TSHβ转录水平升高。为研究石斑鱼TSHβ特殊表达方式的调控机制,采用基因组步移技术扩增获得赤点石斑鱼TSHβ5′端序列共5112bp。将1864bp核心片段顺向克隆到pBK-TOL2载体中,pTSHβ-TOL2重组质粒与转座酶mRNA共注射斑马鱼一细胞期受精卵。Western blot结果显示,在受精后10h的尾芽胚中可检测到GFP的表达,胚体晚期GFP表达量最高。荧光显微镜追踪观察发现,受精后12-30hpf(Hours post fertilization)观察到GFP在原始生殖细胞(PGC)区域表达;36hpf-7dpf观察到GFP除在PGC区域表达外还在垂体中表达。由此可见,赤点石斑鱼TSHβ5′端1864bp的序列具有特异定位的启动子活性。