RecA-mediated, targeted mutagenesis in zebrafish

We have evaluated the efficacy of RecA, a prokaryotic protein involved with homologous recombination, to direct site-specific mutagenesis in zebrafish embryos. For this we coinjected a vector containing a mutated enhanced green fluorescent protein (EGFP) gene plus 236-nucleotide corrective single-stranded DNAs coated with RecA into I-cell zebrafish embryos. Twenty-hours after fertilization, about 5% to 20% of injected embryos showed EGFP expression in I or more cells when RecA-coated corrective DNAs were used, but not when RecA was omitted. Mutated EGFP genes with 1-bp insertions or deletions were inefficiently activated, whereas those with 7-bp insertions were activated about 4-fold more efficiently. RecA-coated template strand had a higher efficiency than its complementary strand in activation of EGFP expression. Prior irradiation of the embryos with UV light enhanced RecA-mediated restoration of gene activity, suggesting that the effects we observed were augmented by one or more factors of zebrafish DNA repair systems.

Nuclear transplantation in different strains of zebrafish

Single later blastula nuclei from AB strain of zebrafish (Danio rerio) were transplanted into enucleated unfertilized eggs of Long fin strain. Of 1119 cloning embryos, 14 reconstructed embryos developed into fry. DNA fingerprinting systems of the cloned fish were similar to those of the nuclear donor fish, but were distinctly different from those of the unclear recipient fish. It confirmed that the genetic material originated from nuclear donor cell other than from nuclear recipient egg. The research suggested that the basic technique for nuclear transplantation performed with different strains of zebrafish has made a breakthrough. It should be helpful for the study of some important developmental problems such as gene function, the regulation of gene expression during animal development, the developmental potential of a nucleus and the interactions between the donor nucleus and the recipient cytoplasm, etc.

Utilization of several plant proteins by gibel carp (Carassius auratus gibelio)

Six isonitrogenous (gross protein content 35%) and isoenergetic (gross energy content 17 kJ g(-1)) diets were formulated to investigate the effects of inclusion of plant proteins on the gibel carp (Carassius auratus gibelio L.). The plant proteins tested were: soybean cake (SBC), potato protein concentrate (PPC), peanut cake (PNC), cottonseed cake (CSC) and rapeseed cake (RSC). Fish meal (FM) was used as control. In each diet, 27% of the protein was supplied by fish meal, and the rest supplied by the plant protein tested. Each diet was fed to three groups of gibel carp for 8 weeks in a recirculation system. Specific growth rate (SGR) in fish fed the control diet was significantly higher than those in the other groups, and SGR in fish fed the PPC was significantly lower than in fish fed other plant proteins. There was no significant difference in SGR among the other groups. Feeding rates were ranked in the order: RSC > CSC > FM > PNC > SBC > PPC. Conversion efficiency was highest in groups fed FM, SBC and PNC, followed by groups fed CSC and RSC, and was lowest in the group fed PPC. The fish fed PPC showed lower protein retention than those fed FM and SBC. FM showed highest energy retention while PPC showed lowest, There was no significant relationship between SGR and intake of digestible protein (g g(-1) day(-1)), digestible lysine (g g(-1) day(-1)), digestible methionine (g g(-1) day(-1)) or digestible total essential amino acids (g g(-1) day(-1)), suggesting that the differences in SGR could not alone account for any of these variables.

A zebrafish forebrain-specific zinc finger gene can induce ectopic dlx2 and dlx6 expression

Identifcation of the earliest forebrain-specific markers should facilitate the elucidation of molecular events underlying vertebrate forebrain determination and specification. Here we report the sequence and characterization of fez (forebrain embryonic zinc finger), a gene that is specifically expressed in the embryonic forebrain of zebrafish. Fez encodes a putative nuclear zinc finger protein that is highly conserved in Drosophila, zebrafish, Xenopus, mouse, and human. In zebrafish, the expression of fez becomes detectable at the anterior edge of the presumptive neuroectoderm by 70% epiboly. During the segmentation period, its expression is completely restricted to the rostral region of the prospective forebrain. At approximately 24 h postfertilization, fez expression is mostly confined to the telencephalon and the anterior-ventral region of the diencephalon. Although fez expression is present in one-eyed pinhead (oep) and cyclops (cyc) zebrfish mutants, the pattern is altered. Forced expression of fez induces ectopic expression of dlx2 and dlx6, two genes involved in brain development. Knockdown of fez function using a morpholino-based antisense oligo inhibited dlx2 expression in the ventral forebrain. Our studies indicate that fez is one of the earliest markers specific for the anterior neuroectoderm and it may play a role in forebrain development by regulating Dlx gene expression. (C) 2001 Academic Press.

Waterborne exposure to PFOS causes disruption of the hypothalamus-pituitary-thyroid axis in zebrafish larvae

Thyroid hormones (THs) play an important role in the normal development and physiological functions in fish. Environmental chemicals may adversely affect thyroid function by disturbing gene transcription. Perfluorooctane sulfonate (PFOS), a persistent compound, is widely distributed in the aquatic environment and wildlife. In the present study, we investigated whether PFOS could disrupt the hypothalamic– pituitary–thyroid (HPT) axis. Zebrafish embryos were exposed to various concentrations of PFOS (0, 100, 200 and 400 lg L 1) and gene expression patterns were examined 15 d post-fertilization. The expression of several genes in the HPT system, i.e., corticotropin-releasing factor (CRF), thyroid-stimulating hormone (TSH), sodium/iodide symporter (NIS), thyroglobulin (TG), thyroid peroxidase (TPO), transthyretin (TTR), iodothyronine deiodinases (Dio1 and Dio2) and thyroid receptor (TRa and TRb), was quantitatively measured using real-time PCR. The gene expression levels of CRF and TSH were significantly up-regulated and down-regulated, respectively, upon exposure to 200 and 400 lg L 1 PFOS. A significant increase in NIS and Dio1 gene expression was observed at 200 lg L 1 PFOS exposure, while TG gene expression was down-regulated at 200 and 400 lg L 1 PFOS exposure. TTR gene expression was down-regulated in a concentration-dependent manner. Up-regulation and down-regulation of TRa and TRb gene expression, respectively, was observed upon exposure to PFOS. The whole body thyroxine (T4) content remained unchanged, whereas triiodothyronine (T3) levels were significantly increased, which could directly reflect disrupted thyroid hormone status after PFOS exposure. The overall results indicated that PFOS exposure could alter gene expression in the HPT axis and that mechanisms of disruption of thyroid status by PFOS could occur at several steps in the synthesis, regulation, and action of thyroid hormones.

Pressurized electrochromatography coupled with electrospray ionization mass spectrometry for analysis of peptides and proteins

Pressurized capillary electrochromatography (pCEC) was coupled with electrospray ionization mass spectrometry (ESI-MS) using a coaxial sheath liquid interface. It was used for separation and analysis of peptides and proteins. The effects of organic modifier and applied voltage on separation were investigated, and the effects of pH value of the mobile phase and the concentration of the electrolyte on ESI-MS signal were investigated. The resolution and detection sensitivity with different separation methods (pCEC, capillary high-performance liquid chromatography) coupled on-line with mass spectrometry were compared for the separation of a peptide mixture. To evaluate the feasibility and reliability of the experimental setup of the system, tryptic digests of cytochrome c and modified protein as real samples were analyzed by using pCEC-ESI-MS.

Immobilized metal-ion chelating capillary microreactor for peptide mapping analysis of proteins by matrix assisted laser desorption/ionization-time of flight-mass spectrometry

Peptide mass mapping analysis, utilizing a regenerable enzyme microreactor with metal-ion chelated adsorption of enzyme, combined with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was developed. Different procedures from the conventional approaches were adopted to immobilize the chelator onto the silica supports, that is, the metal chelating agent of iminodiacetic acid (IDA) was reacted with glycidoxypropyltrimethoxysilane (GLYMO) before its immobilization onto the inner wall of the fused-silica capillary pretreated with NH4HF2. The metal ion of copper and subsequently enzyme was specifically adsorbed onto the surface to form the immobilized enzyme capillary microreactor, which was combined with MALDI-TOF-MS to apply for the mass mapping analysis of nL amounts of protein samples. The results revealed that the peptide mapping could routinely be generated from 0.5 pmol protein sample in 15 min at 50degreesC, even 20 fmol cytochrome c could be well digested and detected.

Affinity membrane chromatography for the analysis and purification of proteins

Affinity chromatography is unique among separation methods as it is the only technique that permits the purification of proteins based on biological functions rather than individual physical or chemical properties. The high specificity of affinity chromatography is due to the strong interaction between the ligand and the proteins of interest. Membrane separation allows the processing of a large amount of sample in a relatively short time owing to its structure, which provides a system with rapid reaction kinetics. The integration of membrane and affinity chromatography provides a number of advantages over traditional affinity chromatography with porous-bead packed columns, especially with regard to time and recovery of activity. This review gives detailed descriptions of materials used as membrane substrates, preparation of basic membranes, coupling of affinity ligands to membrane supports, and categories of affinity membrane cartridges. It also summarizes the applications of cellulose/glycidyl methacrylate composite membranes for proteins separation developed in our laboratory. (C) 2001 Elsevier Science B.V. All rights reserved.

Early Embryonic Exposure to Polychlorinated Biphenyls Disrupts Heat-Shock Protein 70 Cognate Expression in Zebrafish

Polychlorinated biphenyls (PCBs) are persistent environmental contaminants that have documented neurological effects in children exposed in utero. To better define neuronally linked molecular targets during early development, zebrafish embryos were exposed to Aroclor 1254, a mixture of PCB congeners that are common environmental contaminants. Microarray analysis of the zebrafish genome revealed consistent significant changes in 38 genes. Of these genes, 55% (21) are neuronally related. One gene that showed a consistent 50% reduction in expression in PCB-treated embryos was heat-shock protein 70 cognate (Hsc70). The reduction in Hsc70 expression was confirmed by real-time polymerase chain reaction (PCR), revealing a consistent 30% reduction in expression in PCB-treated embryos. Early embryonic exposure to PCBs also induced structural changes in the ventro-rostral cluster as detected by immunocytochemistry. In addition, there was a significant reduction in dorso-rostral neurite outgrowth emanating from the RoL1 cell cluster following PCB exposure. The serotonergic neurons in the developing diencephalon showed a 34% reduction in fluorescence when labeled with a serotonin antibody following PCB exposure, corresponding to a reduction in serotonin concentration in the neurons. The total size of the labeled neurons was not significantly different between treated and control embryos, indicating that the development of the neurons was not affected, only the production of serotonin within the neurons. The structural and biochemical changes in the developing central nervous system following early embryonic exposure to Aroclor 1254 may lead to alterations in the function of the affected regions.

Quantitative electrochemiluminescence detection of proteins: Avidin-based sensor and tris(2,2 '-bipyridine) ruthenium(II) label

Quantitative electrochemilumineseence (ECL) detection of a model protein, bovine serum albumin (BSA) was achieved via biotin-avidin interaction using an avidin-based sensor and a well-developed ECL system of tris(2,2'-bipyridine) ruthenium(II) derivative as label and tri-n-propylamine (TPA) as coreactant. To detect the protein, avidin was linked to the glassy carbon electrode through passive adsorptions and covalent interaction with carboxylate-terminated carbon nanotubes that was used as binder to immobilize avidin onto the electrode. Then, biotinylated BSA tagged with tris(2,2'-bipyridine) ruthenium(II) label was attached to the prepared avidin surface.

Microchip Micellar Electrokinetic Chromatography Based on One Functionalized Ionic Liquid and Its Excellent Performance on Proteins Separation

Herein, one water-soluble functionalized ionic liquid (IL), 1-butyl-3-methylimidazolium dodecanesulfonate (BAS), was designed, investigated and successfully applied to microchip micellar electrokinetic chromatography (MEKC) construction. It possessed the properties of both IL and surfactant. A fairly stable pH value similar to 7.4, which was fit to pH values of general biological buffers, was nicely placed at the optimum concentration of 20 mM BAS solution. While applying BAS solution as running buffer in poly(dimethylsiloxane) (PDMS) microfluidic systems, significantly enhanced electroosmotic flow (8-fold) and resolutions between analytes were obtained than that using other supporting electrolytes or surfactants.