Characterization of isolated ventricular myocytes from adult zebrafish (Danio rerio)

The zebrafish is widely used for human related disease studies. Surprisingly, there is no information about the electrical activity of single myocytes freshly isolated from adult zebrafish ventricle. In this study, we present an enzymatic method to isolate ventricular myocytes from zebrafish heart that yield a large number of calcium tolerant cells. Ventricular myocytes from zebrafish were imaged using light and confocal microscopy. Myocytes were mostly rod shaped and responded by vigorous contraction to field electrical stimulation. Whole cell configuration of the patch clamp technique was used to record electrophysiological characteristics of myocytes. Action potentials present a long duration and a plateau phase and action potential duration decreases when increasing stimulation frequency (as observed in larger mammals). Together these results indicate that zebrafish is a species ideally suited for investigation of ion channels related mutation screening of cardiac alteration important in human.     

Cell electroporation is a highly efficient method for introducing restriction endonucleases into cells

Restriction endonucleases that make either blunt- or cohesive-end DNA double-strand breaks can induce chromosome aberrations. We have used cell electroporation with great success to permeabilize Chinese hamster ovary cells for the introduction of restriction enzymes. The introduction of restriction enzymes by this method resulted in extremely high frequencies (greater than 90%) of aberrant metaphase cells and also a dramatic decrease in cell survival, as measured by subsequent colony formation. Cell electroporation by itself caused no increase in aberrant chromosomes and had only a slight effect on cell survival.     

Performance of an automated system for capillary microinjection into living cells

An automated capillary microinjection system for nuclear and cytoplasmic injections into cells is described. The system has been tested with samples of DNA, RNA and proteins. Movements of the capillary with precise cell positioning and time of injections are controlled by a computer. This first automated microinjection system allows injection of more than 1500 cells per hour with a minimum of practical training, volumes injected are more reproducible and cells are less damaged when compared with the standard manual injection technique. Retrieval of the injected cells is accurate to within 1% without complicated and laborious produced orientation marks on the cell support. The number of successfully injected cells is easily determined with great accuracy and the error of the statistical evaluation of the results is reduced to a minimum. Standardized procedures for pulling, handling and storage of the injection capillaries were developed.     

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.[/ p]Abbreviations TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin - EROD, 7-ethyoxyresorufin - HDPDS, 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - EDTA, ethylanediaminetetraacetic acid - FBS, fetal bovine serum - LDF, limit dilution factor - DMSO, dimethyl sulfoxide - ES, embryonal stem - PAH, polycylic aromatic hydrocarbons - ZG, zebrafish gill - ZBF, zebrafish pelvic fin - ZV, Zebrafish viscera - ZCF, zebrafish caudal fin - ZEM, diploid blastula-derived     

Microporation is a valuable transfection method for efficient gene delivery into human umbilical cord blood-derived mesenchymal stem cells

Background

Mesenchymal stem cells (MSCs) are an attractive source of adult stem cells for therapeutic application in clinical study. Genetic modification of MSCs with beneficial genes makes them more effective for therapeutic use. However, it is difficult to transduce genes into MSCs by common transfection methods, especially nonviral methods. In this study, we applied microporation technology as a novel electroporation technique to introduce enhanced green fluorescent protein (EGFP) and brain-derived neurotropfic factor (BDNF) plasmid DNA into human umbilical cord blood-derived MSCs (hUCB-MSCs) with significant efficiency, and investigated the stem cell potentiality of engineered MSCs through their phenotypes, proliferative capacity, ability to differentiate into multiple lineages, and migration ability towards malignant glioma cells.

Results

Using microporation with EGFP as a reporter gene, hUCB-MSCs were transfected with higher efficiency (83%) and only minimal cell damage than when conventional liposome-based reagent (<20%) or established electroporation methods were used (30-40%). More importantly, microporation did not affect the immunophenotype of hUCB-MSCs, their proliferation activity, ability to differentiate into mesodermal and ectodermal lineages, or migration ability towards cancer cells. In addition, the BDNF gene could be successfully transfected into hUCB-MSCs, and BDNF expression remained fairly constant for the first 2 weeks in vitro and in vivo. Moreover, microporation of BDNF gene into hUCB-MSCs promoted their in vitro differentiation into neural cells.

Conclusion

Taken together, the present data demonstrates the value of microporation as an efficient means of transfection of MSCs without changing their multiple properties. Gene delivery by microporation may enhance the feasibility of transgenic stem cell therapy.     

The laser method for efficient introduction of foreign DNA into cultured cells

A minute hole upon a cultured cell, perforated with a finely focused laser beam, was found to repair itself within a short period of time. The procedure constitutes a new way of introducing exogenous gene materials dissolved in medium into cells. The 'laser-aided' DNA transfection is better than the existing methods because it allows the treatment of a large number of cells in a shorter time, and an improved success rate.     

Conjugate for efficient delivery of short interfering RNA (siRNA) into mammalian cells

Cholesterol enrichment of rat liver mitochondria (CHM) impairs atractyloside-induced mitochondrial permeability transition (MPT) due to decreased membrane fluidity. In this study we addressed the effect of cholesterol enrichment on MPT induced by reactive oxygen species (ROS). Superoxide anion generated by xanthine plus xanthine oxidase triggered mitochondrial swelling and cytochrome c release in CHM, which was prevented by butylated hydroxytoluene, an anti-voltage-dependent anion channel antibody, or cyclosporin A. Furthermore, hydrogen peroxide generated by the combination of ganglioside GD3 and mitochondrial GSH depletion elicited mitochondrial swelling and release of cytochrome c, Smac/Diablo and apoptosis-inducing factor in control mitochondria and CHM. Thus, ROS induce MPT and apoptosome activation regardless of decreased mitochondrial membrane dynamics due to cholesterol enrichment.     

Nucleofection as an efficient nonviral transfection method for human monocytic cells

Despite some progress in the field of gene transfer into hard-to-transfect cells, so far an efficient nonviral method for monocytes has not been available. A comparison of plasmid DNA with capped and polyadenylated mRNA for enhanced green fluorescent protein gene delivery into the commonly used monocytic cell lines U937 and THP-1 suggested that limited DNA trafficking may be the underlying cause of poor transfection results. As Nucleofector technology delivers DNA (or mRNA) straight into the nucleus, we obtained nucleofection efficiencies of up to 80% without significant cell toxicity. Moreover, as the DNA quickly reaches the nucleus, nucleofected cells were ready for analysis after only 2–6 h. The technique is suitable not only for monocytes but also for other hard-to-transfect cells.     

An efficient method for introducing defined lipids into the plasma membrane of mammalian cells

An efficient method has been devised to introduce lipid molecules into the plasma membrane of mammalian cells. This method has been applied to fuse lipid vesicles with the apical plasma membrane of Madin-Darby canine kidney cells. The cells were infected with fowl plague or influenza N virus. 4 h after infection, the hemagglutinin (HA) spike glycoprotein of the virus was present in the apical plasma membrane of the cells. Lipid vesicles containing egg phosphatidylcholine, cholesterol, and an HA receptor (ganglioside) were then bound to the cells at 0 degrees C. More than 85% of the vesicles were released by external neuraminidase at 0 degrees C or by simply warming the cells to 37 degrees C for 10 s, probably because of the action of the viral neuraminidase at the cell surface. However, when the cells were warmed to 37 degrees C in a pH 5.3 medium for 30 s, 50% of the bound vesicles could no longer be released by external neuraminidase. This only occurred when the HA protein had been cleaved into its HA1 and HA2 subunits. When we used influenza N virus, whose HA is not cleaved in Madin-Darby canine kidney cells, cleavage with external trypsin was required. The fact that the HA protein has fusogenic properties at low pH only in its cleaved form suggests that fusion of the vesicles with the plasma membrane had taken place. Further confirmation for fusion was obtained using an assay based on the decrease of energy transfer between two fluorescent phospholipids in a vesicle upon fusion of the vesicle with the plasma membrane (Struck, D. K., D. Hoekstra, and R. E. Pagano. 1981. Biochemistry, 20:4093-4099).     

ReCLIP (reversible cross-link immuno-precipitation): an efficient method for interrogation of labile protein complexes

The difficulty of maintaining intact protein complexes while minimizing non-specific background remains a significant limitation in proteomic studies. Labile interactions, such as the interaction between p120-catenin and the E-cadherin complex, are particularly challenging. Using the cadherin complex as a model-system, we have developed a procedure for efficient recovery of otherwise labile protein-protein interactions. We have named the procedure "ReCLIP" (Reversible Cross-Link Immuno-Precipitation) to reflect the primary elements of the method. Using cell-permeable, thiol-cleavable crosslinkers, normally labile interactions (i.e. p120 and E-cadherin) are stabilized in situ prior to isolation. After immunoprecipitation, crosslinked binding partners are selectively released and all other components of the procedure (i.e. beads, antibody, and p120 itself) are discarded. The end result is extremely efficient recovery with exceptionally low background. ReCLIP therefore appears to provide an excellent alternative to currently available affinity-purification approaches, particularly for studies of labile complexes.     

Base excision repair is efficient in cells lacking poly(ADP-ribose) polymerase 1

Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that is activated by binding to DNA breaks induced by ionizing radiation or through repair of altered bases in DNA by base excision repair. Mice lacking PARP-1 and, in certain cases, the cells derived from these mice exhibit hypersensitivity to ionizing radiation and alkylating agents. In this study we investigated base excision repair in cells lacking PARP-1 in order to elucidate whether their augmented sensitivity to DNA damaging agents is due to an impairment of the base excision repair pathway. Extracts prepared from wild-type cells or cells lacking PARP-1 were similar in their ability to repair plasmid DNA damaged by either X-rays (single-strand DNA breaks) or by N-methyl-N′-nitro-N-nitrosoguanidine (methylated bases). In addition, we demonstrated in vivo that PARP-1-deficient cells treated with N-methyl-N′-nitro-N-nitrosoguanidine repaired their genomic DNA as efficiently as wild-type cells. Therefore, we conclude that cells lacking PARP-1 have a normal capacity to repair single-strand DNA breaks inflicted by X-irradiation or breaks formed during the repair of modified bases. We propose that the hypersensitivity of PARP-1 null mutant cells to γ-irradiation and alkylating agents is not directly due to a defect in DNA repair itself, but rather results from greatly reduced poly(ADP-ribose) formation during base excision repair in these cells.     

Enantioselective carbon-hydrogen insertion is an effective and efficient methodology for the synthesis of (R)-(-)-baclofen

A highly enantioselective methodology for the synthesis of the GABA(B) receptor agonist (R)-(-)-baclofen is described. This synthesis begins with p-chlorophenethyl alcohol and involves a catalytic carbon-hydrogen insertion reaction of a chiral dirhodium(II) carboxamidate with the corresponding diazoacetate (81% yield, 95% ee). Subsequent steps convert the intermediate gamma-lactone to (R)- (-)-baclofen in a 60% overall yield. The amount of catalyst required for the C-H insertion transformation is only 0.5 mol%.     

Organotypic brain slice cultures: an efficient and reliable method for neurotoxicological screening and mechanistic studies

This paper reviews the current state of the use of organotypic brain slice cultures for neurotoxicological and neuropharmacological screening and mechanistic studies, as exemplified by excitotoxin application. At present, no in vitro systems have been approved by the regulatory authorities for neurotoxicity testing. For the evaluation of the slice culture method, organotypic hippocampal slice cultures were exposed to toxic doses of the excitotoxins, glutamate, N-methyl-D-aspartate (NMDA), kainic acid and 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and the glial toxin, DL-alpha-aminoadipic acid (DLAAA). Neuronal cell death was quantified by propidium iodide (PI) uptake, and visualised by Fluoro-Jade (FJ) staining. General cell death was monitored by lactate dehydrogenase (LDH) release into the culture medium. EC50 values for the different compounds, based on PI uptake after exposure for 48 hours in entire cultures, were: glutamate, 3.5 mM; DL-AAA, 2.3 mM; kainic acid, 13 microM; NMDA, 11 microM; and AMPA, 3.7 microM. In the slice cultures, the hippocampal subfields displayed the same differences in vulnerability as those observed in vivo. When subfield analysis was performed on the cultures, the CA1 subfield was most susceptible to glutamate, NMDA and AMPA, while CA3 was most susceptible to kainic acid. The amount of LDH release for DL-AAA was about four times that of L-glutamate, in accordance with the additional toxic effect on glial cells, which was also found by confocal microscopy to stain for FJ. In conclusion, it was found that organotypic brain slice culture, combined with standardised protocols and quantifiable markers, such as PI and FJ staining, is a relevant and feasible in vitro system for neurotoxicity testing. Considering the amount and quality of the available published data, it is recommended that the brain slice culture method could be subjected to pre-validation and formal validation for inclusion in a tiered in vitro neurotoxicity testing scheme to supplement and replace conventional animal tests.     

Oct4-induced reprogramming is required for adult brain neural stem cell differentiation into midbrain dopaminergic neurons

Neural stem cells (NSCs) lose their competency to generate region-specific neuronal populations at an early stage during embryonic brain development. Here we investigated whether epigenetic modifications can reverse the regional restriction of mouse adult brain subventricular zone (SVZ) NSCs. Using a variety of chemicals that interfere with DNA methylation and histone acetylation, we showed that such epigenetic modifications increased neuronal differentiation but did not enable specific regional patterning, such as midbrain dopaminergic (DA) neuron generation. Only after Oct-4 overexpression did adult NSCs acquire a pluripotent state that allowed differentiation into midbrain DA neurons. DA neurons derived from Oct4-reprogrammed NSCs improved behavioural motor deficits in a rat model of Parkinson's disease (PD) upon intrastriatal transplantation. Here we report for the first time the successful differentiation of SVZ adult NSCs into functional region-specific midbrain DA neurons, by means of Oct-4 induced pluripotency.     

A cell-survival factor (N-acetyl-L-cysteine) alters the in vivo fate of constitutively proliferating subependymal cells in the adult forebrain

The adult mouse brain contains a population of constitutively proliferating subependymal cells that surround the lateral ventricle and are the direct progeny of the neural stem cell. Constitutively proliferating cells divide rapidly; 6 days after labeling, 60% of their progeny undergo cell death, 25% migrate to the olfactory bulbs, and 15% continue to proliferate within the subependyma. We have intraventricularly infused a cell survival factor N-acetyl-L-cysteine (NAC), which is known to have survival effects without concomitant proliferative effects on cells in vitro, and examined the resulting fate of cells spared from the normally occurring cell death. NAC infusion for 5 days results in a five-fold increase in the number of retrovirally labeled subependymal cells compared to saline-infused controls. The increase in the number of subependymal cells is directly proportional to the amount of time during which NAC is present and is not due to increased proliferation. While NAC is able to keep all the normally dying progeny alive, the cells spared from death remain confined to the subependyma lining the lateral ventricles and do not migrate to the olfactory bulbs (one normal fate of constitutively proliferating progeny) or into the surrounding brain parenchyma. When animals survive for an additional 6 days following NAC infusion, the number of retrovirally labeled subependymal cells returns to control values, indicating that the continued presence of NAC is necessary for cell survival. These data suggest that preventing cell death is not sufficient to keep all of the progeny of these cells in a proliferative mode.     

Coagulation factor III (tissue factor) is required for vascularization in zebrafish embryos

Tissue factor (coagulation factor III) is a cell surface receptor for coagulation factor VII/VIIa; it was initially recognized as an initiator of the extrinsic coagulation pathway. Recently, the zebrafish tissue factor gene (TF) has been cloned. Paralogs encode coagulation factors IIIa and IIIb; both show remarkable sequence identity to the human and mouse coagulation factor III gene. It has been reported that TF could have additional properties that are essential for normal embryonic development, since knockout of the murine coagulation factor III gene resulted in 90% embryonic lethality. We examined the role of coagulation factor IIIb (f3b) during zebrafish embryonic development. Expression analysis revealed that endogenous f3b was chronologically expressed in the pectoral fins and in the vicinity of the pharynx. Knockout of f3b by injection of an f3b morpholino at the one-to-two cell stage caused distinctive morphological defects in embryos, including edema in the fourth brain ventricle at early embryonic stages and occasional bleeding at later stages. Furthermore, f3b morphants displayed abnormal vascular patterning. We conclude that f3b is required for brain vascular development and for development of part of the somatic vasculature during embryogenesis in the zebrafish.