Development of a zebrafish spleen cell line, ZSSJ, and its growth arrest by gamma irradiation and capacity to act as feeder cells.

A zebrafish spleen cell line, ZSSJ, was developed and its growth arrest by gamma radiation determined and its capacity to stimulate the proliferation of the zebrafish blastula cell line, ZEB2J, measured. ZSSJ was initiated by explant outgrowth, grew adherent with mainly an epithelial-like morphology, and stained strongly for alkaline phosphatase. ZSSJ was not only grown in L-15 with 15% fetal bovine serum at 26 degrees C to 28 degrees degrees C but also grew at room temperature. Cultures of ZSSJ have undergone approximately 40 population doublings, had few cells staining for b-galactosidase activity, which is commonly present in senescent cultures, and many cells with an aneuploid karyotype, which is frequently associated with immortalization. ZSSJ growth was arrested by 30 to 50 Gy of g-irradiation, whereas after 20 Gy, some slight growth was observed. By contrast, growth of the rainbow trout spleen stromal cell line, RTS34st, which has been used as a feeder for zebrafish ES cell cultures, was arrested completely by 20 Gy. In cocultures, nongrowth-arrested ZSSJ stimulated ZEB2J proliferation better than growth-arrested ZSSJ and better than RTS34st. ZSSJ should be useful as a feeder cell line for zebrafish ES cell cultures.

Evaluating the performance and acceptance of teleconference instruction versus traditional teaching methods for undergraduate and graduate students.

Institutions are currently seeking alternative ways to deliver a full-line of course materials without acquiring additional staffing. Hence, faculty is charged with creating alternative ways to deliver or offer course content to students. The purpose of this study was to evaluate undergraduate and graduate performance and perception of teleconferencing versus traditional blackboard lectures. In the undergraduate course, we discovered that students performed equally as well on exams and provided favorable reviews of the course; however, the acceptance of this new format is lacking given the enrollment and number of students dropping, e.g., 30 to 40% reduction in the course before semester's end. On the other hand, students taking the graduate course appear to accept the technology well with consistent enrollments and achievement in course content. In summary, using teleconferencing as a way to teach students may be better suited for graduate students when compared to undergraduates.

Identification and characterization of a novel fibronectin in zebrafish.

Sequence analysis of zebrafish fibronectin (FN) cDNAs indicates that at least two forms of the protein exist in fish. One form (FN1) is very similar to FNs identified in other vertebrates possessing 12 type I, 2 type II, and 17 type III repeats including two alternative splice sites (EIIIA and EIIIB) and a variable region (V). Zebrafish FN1 contains the RGD cell adhesion site in type III(10) and a second cell-binding site (LDV) in the V region. In addition to this conserved form of FN, a novel truncated form of zebrafish FN (FN2) was identified. The predicted structure of FN2 is identical to FN1 at the N-terminal region possessing 9 type I, 2 type II, and the first 3 type III repeats. Following III(3), FN2 contains a unique 20-amino-acid C-terminal tail that is different from the C-terminus of FN1, lacking the two cysteines that are usually involved in the formation of interchain disulfide bonds. Genomic sequence analysis has revealed that FN2 is generated by an alternative RNA splicing pattern that has not been described for FN in other organisms. Reverse transcription-polymerase chain reaction analysis and RNase protection assays reveal that FN2 mRNA is present in the zebrafish embryo throughout development as well as in cultures of an established liver cell line. Experiments conducted with recombinant FN2 synthesized in insect cells demonstrate that the protein promotes the attachment and spreading of fish embryo cells in culture.

Production of zebrafish germ-line chimeras from embryo cell cultures.

Although the zebrafish possesses many characteristics that make it a valuable model for genetic studies of vertebrate development, one deficiency of this model system is the absence of methods for cell-mediated gene transfer and targeted gene inactivation. In mice, embryonic stem cell cultures are routinely used for gene transfer and provide the advantage of in vitro selection for rare events such as homologous recombination and targeted mutation. Transgenic animals possessing a mutated copy of the targeted gene are generated when the selected cells contribute to the germ line of a chimeric embryo. Although zebrafish embryo cell cultures that exhibit characteristics of embryonic stem cells have been described, successful contribution of the cells to the germ-cell lineage of a host embryo has not been reported. In this study, we demonstrate that short-term zebrafish embryo cell cultures maintained in the presence of cells from a rainbow trout spleen cell line (RTS34st) are able to produce germ-line chimeras when introduced into a host embryo. Messenger RNA encoding the primordial germ-cell marker, vasa, was present for more than 30 days in embryo cells cocultured with RTS34st cells or their conditioned medium and disappeared by 5 days in the absence of the spleen cells. The RTS34st cells also inhibited melanocyte and neuronal cell differentiation in the embryo cell cultures. These results suggest that the RTS34st splenic-stromal cell line will be a valuable tool in the development of a cell-based gene transfer approach to targeted gene inactivation in zebrafish.

Expression of the porcine beta2-adrenergic receptor in Chinese hamster ovary cells.

The gene for the porcine beta2-adrenergic receptor (pbeta2AR) was transfected into Chinese hamster ovary (CHO) cells for expression. Fourteen stable cell lines were obtained and exhibited receptor densities ranging from 12 to 2,371 fmol/mg membrane protein. The receptor density was not correlated with estimates of gene copy number obtained by Southern hybridization. The pbeta2AR in CHO cells exhibited saturable binding of [125I]CYP (Kd = 14.5 pM) and stereospecificity for (-)- and (+)-isoproterenol. The relative affinities for (-)-isoproterenol (ISO), (-)-epinephrine (EPI), and (-)-norepinephrine (NEPI) were ISO > EPI > NEPI, which are characteristic of beta2AR. The affinity values for these ligands were similar to those in other species. Binding of ISO, EPI, and NE revealed two affinity states of the betaAR; the high-affinity state was eliminated by adding Gpp(NH)p, a nonhydrolyzable GTP analogue. Binding of the antagonist propranolol modeled to only one affinity state, and Gpp(NH)p did not affect binding. Multiple affinity states are characteristic of agonist-induced coupling of betaAR with G-proteins, and the data suggest that the cloned pbetaAR is functionally competent. Data confirm that the pbeta2AR is the pig version of beta2AR. Stable CHO cell lines will be useful for characterization of pbeta2AR and screening and designing potential drugs that may be used to enhance pig production.

Preparation of primary cell cultures from lamprey.

The lamprey is an important model for studies of evolution and comparative biology. The ability to culture cells from lamprey tissues makes it possible to employ an in vitro approach to address basic questions in these areas. Methods are described for the initiation of cell cultures derived from tissues of adult and larval sea lamprey (Petromyzon marinus). Primary cultures initiated from gill, muscle, gut, brain, ovary, heart and kidney were viable for up to eight months and several of the cultures were propagated for multiple passages. Most cultures were initiated from tissue explants in basal nutrient medium supplemented with fetal bovine and trout sera on a culture surface treated with fibronectin and collagen. Variations of these culture conditions to meet the specific growth requirements of certain cell types are discussed.

Cell cultures derived from early zebrafish embryos differentiate in vitro into neurons and astrocytes.

The zebrafish is a polular nonmammalian model for studies of neural development. We have derived cell cultures, initiated from blastula-stage zebrafish embryos, that differentiate in vitro into neurons and astrocytes. Cultures were initiated in basal nutrient medium supplemented with bovine insulin, trout serum, trout embryo extract and fetal bovine serum. After two weeks in culture the cells exhibited extensive neurite outgrowth and possessed elevated levels of acetylcholinesterase enzyme activity. Ultrastructural analysis revealed that the neurites possessed microtubules, synaptic vessicles and areas exhibiting growth cone morphology. The cultures expressed proteins recognized by antibodies to the neuronal and astrocyte-specific markers, neurofilament and glial fibrillary acidic protein (GFAP). Poly-D-lysine substrate stimulated neurite outgrowth in the cultures and inhibited the growth of nonneuronal cells. Medium conditioned by the buffalo rat liver line, BRL, promoted the growth and survival of the cells in culture. Mitotically active cells were identified in cultures that had undergone extensive differentiation. The embryo cell cultures provide an in vitro system for investigations of biochemical parameters influencing zebrafish neuronal cell growth and differentiation.

Culture of cells from tissues of adult and larval sea lamprey.

Methods were developed for the culture of cells derived from tissues of the sea lamprey (Petromyzon marinus). Cultures were initiated from gill, liver, muscle and gut from larvae and newly transformed individuals and brain, heart, kidney and ovary from sexually mature adults. The lamprey cells were viable for up to six months in culture and cells from ovary, muscle, gut, gill and liver were propagated for multiple passages. For all cultures except liver, optimal cell attachment and spreading was obtained on surfaces coated with fibronectin and collagen. Optimal liver cell attachment was achieved on basement membrane. Cells synthesizing DNA were detected by precursor incorporation in five week-old cultures derived from adult and larval tissues. Metabolic labeling experiments with [(35)S]-methionine demonstrated that cultures initiated from liver and ovary continued to synthesize and release proteins into the medium for several weeks. Ultrastructural examination revealed the presence of ciliated cells in cultures from brain and the accumulation of lipid in epithelial cells derived from liver and gill.

ES-like cell cultures derived from early zebrafish embryos.

Pluripotent embryonic stem (ES) cell cultures provide an efficient method for genome manipulation with many applications in marine biotechnology. To develop this technology we have been working to derive fish ES cell lines for in vitro studies of embryo cell growth and differentiation and for the generation of transgenic fish. Zebrafish embryonal cell cultures were derived from blastula-stage embryos in LDF medium supplemented with fetal bovine serum, trout serum, trout embryo extract, selenium, insulin, and leukemia inhibitory factor. Cultures derived under these conditions on feeder layers of zebrafish embryonic fibroblasts possessed a diploid karyotype and exhibited an ES-like morphology with elevated levels of alkaline phosphatase enzyme activity. Injection of primary cell cultures derived from embryos of transgenic fish carrying neo produced chimeric fish detected by polymerase chain reaction analysis. Embryo cells cultured on poly-D-lysine substrate in the presence of retinoic acid or Buffalo rat liver cell-conditioned medium (BRL-CM) and a reduced serum concentration differentiated into neuronal cell types exhibiting elevated levels of acetylcholinesterase enzyme activity and expression of neurofilament and glial fibrillary acidic protein.

Derivation and characterization of a zebrafish liver cell line.

ZF-L cells were derived from normal adult zebrafish liver, and have been growing in culture for more than 100 generations. The cells were derived in basal nutrient medium supplemented with fetal bovine serum (FBS), trout serum, trout embryo extract, bovine insulin and mouse epidermal growth factor. After 50 generations in culture, optimal growth of the cells was achieved in medium supplemented with FBS (5%) and trout serum (0.5%). ZF-L cells were hypodiploid (modal chromosome number = 46) and exhibited an epithelial morphology. ZF-L cell homogenates exhibited alanine and aspartate aminotransferase, glucose-6-phosphatase and alkaline phosphatase enzyme activities. The cells synthesized and released several proteins into the culture medium, including a 70 kDa protein recognized by anti-bovine serum albumin IgG.

Induction of zebrafish (Brachydanio rerio) P450 in vivo and in cell culture.

1. Induction of zebrafish P450 by 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) was studied in liver tissue, primary liver cell culture and multipassage cell culture derived from zebrafish haploid and diploid embryos and liver. 2. TCDD induced two hepatic proteins (54 and 50 kDa) in vivo which were recognized by anti-trout P4501A1 IgG. The 54-kDa protein was induced by TCDD in primary and multipassage hepatocyte cultures and in haploid and diploid embryo-derived cells. The proteins in liver homogenates were not induced by aqueous exposure of zebrafish to beta-naphthoflavone (BNF). 3. Homogenates of zebrafish liver, cultured hepatocytes and embryo-derived cells also exhibited increased ethoxyresorufin O-deethylase (EROD) and 7-12-dimethyl-benz[a]anthracene (DMBA) hydroxylase activity following TCDD exposure.

Culture of cells from zebrafish (Brachydanio rerio) blastula-stage embryos.

The zebrafish has become a popular model for studies of vertebrate development and toxicology. However, in vitro approaches utilizing this organism have not been fully exploited due to the absence of suitable cell culture systems. Previously, we developed methods for the culture of cells derived from zebrafish blastula-stage embryos. One of these cultures, ZEM-2, was derived in a complex medium containing trout embryo extract, trout serum and medium conditioned by buffalo rat liver cells. In this study we describe a zebrafish embryo cell line, ZEM-2A, derived from ZEM-2 following selection for growth in a simplified medium. Optimal growth of ZEM-2A cells is attained in nutrient medium supplemented with 5% fetal bovine serum.

Culture of cells from juvenile worms of Schistosoma mansoni.

Tissue disruption methods were developed and serum-free cell culture media formulated for the maintenance in vitro of cells from juvenile worms (day 18 after infection) of Schistosoma mansoni. Cultures maintained viability for up to 6 mo when plated on a feeder layer of irradiated rat liver cells and survived primarily as clusters of small (2.5-4 microns diameter) cells with a high nuclear-to-cytoplasmic ratio and relatively few organelles identified by electron microscopy. Cultures synthesized a protein profile similar to that of intact worms, and the cell clusters maintained a time- and concentration-dependent contractile response to serotonin. Cells synthesizing DNA were detected by precursor incorporation and flow cytometry in cultures initially and also after several weeks in vitro, although the percentage of cells synthesizing DNA decreased with time. Efforts to identify peptide growth factor-responsive tyrosine phosphorylation were negative, and the overall amount of S. mansoni phosphotyrosine-containing proteins identified by western blot with anti-phosphotyrosine monoclonal antibody was much less than that found in a peptide growth factor-responsive mouse cell line.

Regulation of glial fibrillary acidic protein in serum-free mouse embryo (SFME) cells by leukemia inhibitory factor and related peptides.

The serum-free mouse embryo (SFME) cell line, derived in serum-free medium from 16-day-old mouse embryos, exhibits unique properties. SFME cells grow indefinitely in culture without senescence, require epidermal growth factor (EGF) or fibroblast growth factor (FGF) for survival and are growth-inhibited by serum. The cell line expresses glial fibrillary acidic protein (GFAP) in response to transforming growth factor beta or serum and cells with similar properties can be isolated directly from brain. Culture of SFME cells with leukemia inhibitory factor (LIF), a peptide implicated in neural tissue development, also resulted in expression of GFAP. Other peptides that share signal transduction mechanisms with LIF--ciliary neurotropic factor, oncostatin M and interleukin-6--also caused expression of GFAP in these cells. These effects were inhibited by concentrations of EGF or FGF that promoted rapid cell growth.

Regulation of cytochrome P450 expression in a novel liver cell line from zebrafish (Brachydanio rerio).

The expression and induction of cytochrome P450 by 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) and beta-naphthoflavone (BNF) in a new liver cell line from adult zebrafish (Brachydanio rerio) were studied. Subcellular fractions from control, BNF- or TCDD-treated cells did not show detectable bands in immunoblots probed with antibodies to the constitutive forms of trout P450 (LMC1, LMC2, LMC3, LMC4, and LMC5), suggesting that either zebrafish liver cells lack P450s closely related to those constitutively expressed in trout or that the concentrations of the orthologous P450s were too low to be detected. However, upon exposure to TCDD, the cells expressed a major immunoreactive 54-kDa protein and a minor 50-kDa protein recognized by antibodies to rainbow trout P4501A1. These immunoreactive proteins were observed in microsomal and mitochondrial fractions of TCDD-treated cells but were not detected in cell cultures treated with dimethyl sulfoxide (DMSO) (vehicle control) or BNF. The activities of ethoxyresorufin O-deethylase (EROD) and 7,12-dimethylbenzanthracene (DMBA) hydroxylase were markedly increased by TCDD but not by BNF in this cell line. EROD activity was more sensitive than DMBA hydroxylase activity of TCDD-treated liver cells to diagnostic inhibitors such as alpha-naphthoflavone and anti-trout P4501A1 IgG. The TCDD-treated cells converted DMBA to various metabolites, one of which is the putative proximate carcinogen, DMBA-3,4-diol. These results suggest that TCDD, but not BNF, induces one or possibly two forms of P450 immunochemically and functionally related to trout P4501A1, in cultured zebrafish liver cells.

Fish embryo cell cultures for derivation of stem cells and transgenic chimeras.

It has been demonstrated in mammalian systems that techniques using embryonal stem cells provide advantages over conventional injection of DNA into embryos for generation of transgenic animals. We employed cell culture approaches in an attempt to develop this technology for fish transgenesis. Using a trout embryo-derived mitogenic preparation in a specialized culture medium, we initiated replication of zebrafish blastula-derived cell cultures and expressed marker genes introduced into the cells by plasmid transfection. Reintroduction of cells from the cultures into blastula-stage embryos indicated that the cultured cells survived and may contribute to the developing organism.

Culture of cells from zebrafish (Brachydanio rerio) embryo and adult tissues.

The zebrafish is a popular model for studies of vertebrate development and toxicology. However, in vitro approaches with this organism have not been fully exploited because cell culture systems have been unavailable. We developed methods for the culture of cells from blastula-stage diploid and haploid zebrafish embryos, as well as cells from the caudal and pelvic fin, gill, liver, and viscera of adult fish. The haploid embryo-derived cells differentiated in culture to a pigmented phenotype and expressed, upon exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin, a protein that was immunologically and functionally similar to rainbow trout cytochrome P450IA1. Zebrafish cultures were grown in a complex basal nutrient medium supplemented with insulin, trout embryo extract, and low concentrations of trout and fetal bovine serum; they could not be maintained in conventional culture medium containing a high concentration of mammalian serum. Using calcium phosphate-mediated transfection, a plasmid constructed for use in mammalian cells was introduced into zebrafish embryo cell cultures and expressed in a stable manner. These results indicated that the transfection procedures utilized in mammalian systems can also be applied to zebrafish cell cultures, providing a means for in vitro alteration of the genotype and phenotype of the cells.

Oncogene transformation frequency of nonsenescent SFME cells is increased by c-myc.

Immortalized, postcrisis mouse embryo cell cultures derived in serum-containing medium display genomic abnormalities and an altered, preneoplastic phenotype. These lines can be transformed with single oncogenes, such as Ha-ras, while efficient transformation of precrisis, genomically unaltered rodent embryo cultures require cooperating oncogenes, such as Ha-ras and the mouse c-myc gene constitutively expressed. Serum-free mouse embryo (SFME) cells, cultured under conditions in which serum is replaced by growth factors and other supplements, are 'immortalized' in the genomically unaltered state. SFME cells do not exhibit growth crisis or gross chromosomal aberration, and are dependent on epidermal growth factor for survival, growth inhibited by serum, and are nontumorigenic. Transformation of SFME cells can be achieved with ras alone, but the introduction of c-myc increased the transfection frequency upon subsequent transfection with ras by as much as twenty fold. Similar results were obtained with mutationally activated neu oncogene and with genomic human tumor DNA. Constitutive expression of c-myc alone did not alter the properties of the SFME cells. These results demonstrate that c-myc alters cellular responses to oncogenes in a culture system in which oncogene-induced immortalization is not a factor, indicating that the effects of myc may extend beyond an 'immortalization' function in these cells.

Human and mouse S-protein mRNA detected in northern blot experiments and evidence for the gene encoding S-protein in mammals by Southern blot analysis.

Human S-protein is a serum glycoprotein that binds and inhibits the activated complement complex, mediates coagulation through interaction with antithrombin III and plasminogen activator inhibitor I, and also functions as a cell adhesion protein through interactions with extracellular matrix and cell plasma membranes. A full length cDNA clone for human S-protein was isolated from a lambda gt11 cDNA library of mRNA from the HepG2 hepatocellular carcinoma cell line using mixed oligonucleotide sequences predicted from the amino-terminal amino acid sequence of human S-protein. The cDNA clone in lambda was subcloned into pUC18 for Southern and Northern blot experiments. Hybridization with radiolabeled human S-protein cDNA revealed a single copy gene encoding S-protein in human and mouse genomic DNA. In addition, the S-protein gene was detected in monkey, rat, dog, cow and rabbit genomic DNA. A 1.7 Kb mRNA for S-protein was detected in RNA from human liver and from the PLC/PRF5 human hepatoma cell line. No S-protein mRNA was detected in mRNA from human lung, placenta, or leukocytes or in total RNA from cultured human embryonal rhabdomyosarcoma (RD cell line) or cultured human fibroblasts from embryonic lung (IMR90 cell line) and neonatal foreskin. A 1.6 Kb mRNA for S-protein was detected in mRNA from mouse liver and brain. No S-protein mRNA was detected in mRNA from mouse skeletal muscle, kidney, heart or testis.