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  • Establishment and applications of epstein-barr virus-based episomal vectors in human embryonic stem cells. 16410388

    Human embryonic stem (hES) cells are capable of unlimited cell proliferation yet maintain the potential to differentiate into many cell types. Here we reported an Epstein-Barr virus (EBV)-based vector system used to improve transfection efficiency in hES cells. Plasmids containing oriP, the latent replication origin of EBV, can be propagated stably as episomal DNA in human cells that express the EBV nuclear antigen 1 (EBNA1), which binds to oriP and functions as the trans-acting replication initiator. It was reported that the EBV replicon could harbor a DNA fragment of up to 330 kilobase pairs. Plasmids containing an enhanced green fluorescent protein (EGFP)/puromycin resistance gene cassette along with or without oriP were used to transfect hES cells that stably express EBNA1. The presence of oriP moderately increased the transient transfection efficiency and more importantly it elevated the stable transfection efficiency by approximately 1,000-fold as compared with oriP-minus plasmids. The oriP plasmid as episomal DNA and green fluorescent protein expression in hES cells was maintained for months in the presence of drug selection and gradually lost (2%-4% per cell doubling) in the absence of selection. The presence of EBNA1 did not interfere with the hES cell properties or differentiation we tested and could maintain stable EGFP expression during differentiation. In addition to transgene expression, the EBV vector system could effectively enhance the RNA interference efficiency in hES cells. Thus, the EBV vector system that allows a large DNA insert and sustained expression of transgene or small hairpin RNA will enhance basic and translational research using hES cells.
    Document Type:
    Reference
    Product Catalog Number:
    S7700
    Product Catalog Name:
    TRAPeze® Telomerase Detection Kit

  • Establishment and characterization of a human retinal pericyte line: a novel tool for the study of diabetic retinopathy. 19212656

    Loss of pericytes from retinal microvessels is one of the key events in the natural history of diabetic retinopathy. Cultured human retinal pericytes would constitute an extremely useful tool for the study of the early events in the pathogenesis of this complication, but, due to legal and ethical issues, pericytes of animal origin have been mostly used so far for in vitro assays. We aimed at establishing an immortalized human retinal pericyte (HRP) line, as a species-specific model to investigate the pericyte-related aspects of diabetic retinopathy. Primary human retinal pericytes (WT-HRP) were immortalized through electroporation with a plasmid vector containing the Bmi-1 oncogene that induces telomerase activity, resulting in the establishment of a permanent pericyte line (Bmi-HRP), which showed telomerase activity and facilitated propagation. The immortalized cells were characterized for typical pericyte morphology and marker expression. Immunofluorescence studies demonstrated that Bmi-HRP maintain the same morphology and express the typical markers of wild-type pericytes. The response of the cell line to high glucose damaging stimulus was also evaluated, as senescence-associated beta-galactosidase activity and cell proliferation and a clear negative effect of high glucose on Bmi-HRP proliferation and senescence, in line with the characteristic response of wild-type cells, was observed. The combination of infinite proliferation capability and stable differentiation potential makes our Bmi-HRP line a promising candidate model to study pathogenic mechanisms and therapeutic applications in diabetic retinopathy.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Establishment and characterization of novel porcine embryonic stem cell lines expressing hrGFP. 19508116

    The purpose of this study was to establish transgenic porcine embryonic stem (pES) cell lines that can stably express report gene. Established pES cell line at passage 44 was transfected with pAAV-hrGFP Control Plasmid by electroporation-mediated, viral vector-mediated, and liposome-mediated strategies. Although there were several pES colonies expressing green fluorescent protein (GFP) obtained from the retrovirus-mediated and liposome-mediated transfection methods, no stable GFP-expressing pES cell line was then derived. A total of 28 GFP-expressing pES cell colonies were obtained following electroporation with two DC pulses of 150 V/cm for 10 msec and three GFP-expressing pES (pES/GFP(+)) cell lines were established. These pES/GFP(+) cell lines stably expressed exogenous GFP and continuously proliferated in vitro for more than 90 passages in 20 months. They maintained normal karyotype of 36 + XX and typical characteristics of pluripotent stem cells, including expression of pluripotent markers Oct-4, AP, SSEA-4, TRA-1-60, and TRA-1-81, formation of embryoid bodies under suspension culture. They were able to differentiate in vitro into neural and cardiomyocytic lineage, respectively, under suitable induction. To our knowledge, there has been no report of establishing GFP-expressing pES cell lines. These novel pES/GFP(+) cell lines established in this study might serve as a nonrodent model and would benefit to the studies involving ES cell transplantation, cell replacement therapy, and tissue regeneration due to their traceable capacity.
    Document Type:
    Reference
    Product Catalog Number:
    MAB4360
    Product Catalog Name:
    Anti-TRA-1-60 Antibody, clone TRA-1-60

  • Design of a single AAV vector for coexpression of TH and GCH1 to establish continuous DOPA synthesis in a rat model of Parkinson's disease. 22294150

    Preclinical efficacy of continuous delivery of 3,4-dihydroxyphenylalanine (DOPA) with adeno-associated viral (AAV) vectors has recently been documented in animal models of Parkinson's disease (PD). So far, all studies have utilized a mix of two monocistronic vectors expressing either of the two genes, tyrosine hydroxylase (TH) and GTP cyclohydrolase-1 (GCH1), needed for DOPA production. Here, we present a novel vector design that enables efficient DOPA production from a single AAV vector in rats with complete unilateral dopamine (DA) lesions. Functional efficacy was assessed with drug-induced and spontaneous motor behavioral tests where vector-treated animals showed near complete and stable recovery within 1 month. Recovery of motor function was associated with restoration of extracellular DA levels as assessed by online microdialysis. Histological analysis showed robust transgene expression not only in the striatum but also in overlying cortical areas. In globus pallidus, we noted loss of NeuN staining, which might be due to different sensitivity in neuronal populations to transgene expression. Taken together, we present a single AAV vector design that result in efficient DOPA production and wide-spread transduction. This is a favorable starting point for continued translation toward a therapeutic application, although future studies need to carefully review target region, vector spread and dilution with this approach.
    Document Type:
    Reference
    Product Catalog Number:
    MAB377
    Product Catalog Name:
    Anti-NeuN Antibody, clone A60

  • The assessment of CD146-based cell sorting and telomere length analysis for establishing the identity of mesenchymal stem cells in human umbilical cord. 25232467

    Adult stem cells are characterised by longer telomeres compared to mature cells from the same tissue. In this study, candidate CD146 (+) umbilical cord (UC) mesenchymal stem cells (MSCs) were purified by cell sorting from UC tissue digests and their telomere lengths were measured in comparison to donor-matched CD146-negative fraction. UC tissue fragments were enzymatically treated with collagenase and the cells were used for cell sorting, colony-forming fibroblast (CFU-F) assay or for long-term MSC cultivation. Telomere lengths were measured by qPCR in both culture-expanded MSCs and candidate native UC MSCs. Immunohistochemistry was undertaken to study the topography of CD146 (+) cells. Culture-expanded UC MSCs had a stable expression of CD73, CD90 and CD105, whereas CD146 declined in later passages which correlated with the shortening of telomeres in the same cultures. In five out of seven donors, telomeres in candidate native UC MSCs (CD45 (-)CD235α (-)CD31 (-)CD146 (+)) were longer compared to donor-matched CD146 (-) population (CD45 (-)CD235α (-)CD31 (-)CD146 (-)). The frequency of CD45 (-)CD235α (-)CD31 (-)CD146 (+) cells measured by flow cytometry was ~1000-fold above that of CFU-Fs (means 10.4% and 0.01%, respectively). CD146 (+) cells were also abundant in situ having a broad topography including high levels of positivity in muscle areas in addition to vessels. Although qPCR-based telomere length analysis in sorted populations could be limited in its sensitivity, very high frequency of CD146 (+) cells in UC tissue suggests that CD146 expression alone is unlikely to be sufficient to identify and purify native MSCs from the UC tissue.
    Document Type:
    Reference
    Product Catalog Number:
    MAB16985
    Product Catalog Name:
    Anti-MCAM Antibody, clone P1H12

  • Transcriptional integration of Wnt and Nodal pathways in establishment of the Spemann organizer. 22627292

    Signaling inputs from multiple pathways are essential for the establishment of distinct cell and tissue types in the embryo. Therefore, multiple signals must be integrated to activate gene expression and confer cell fate, but little is known about how this occurs at the level of target gene promoters. During early embryogenesis, Wnt and Nodal signals are required for formation of the Spemann organizer, which is essential for germ layer patterning and axis formation. Signaling by both Wnt and Nodal pathways is required for the expression of multiple organizer genes, suggesting that integration of these signals is required for organizer formation. Here, we demonstrate transcriptional cooperation between the Wnt and Nodal pathways in the activation of the organizer genes Goosecoid (Gsc), Cerberus (Cer), and Chordin (Chd). Combined Wnt and Nodal signaling synergistically activates transcription of these organizer genes. Effectors of both pathways occupy the Gsc, Cer and Chd promoters and effector occupancy is enhanced with active Wnt and Nodal signaling. This suggests that, at organizer gene promoters, a stable transcriptional complex containing effectors of both pathways forms in response to combined Wnt and Nodal signaling. Consistent with this idea, the histone acetyltransferase p300 is recruited to organizer promoters in a Wnt and Nodal effector-dependent manner. Taken together, these results offer a mechanism for spatial and temporal restriction of organizer gene transcription by the integration of two major signaling pathways, thus establishing the Spemann organizer domain.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Chromatin signature of embryonic pluripotency is established during genome activation. 20336069

    After fertilization the embryonic genome is inactive until transcription is initiated during the maternal-zygotic transition. This transition coincides with the formation of pluripotent cells, which in mammals can be used to generate embryonic stem cells. To study the changes in chromatin structure that accompany pluripotency and genome activation, we mapped the genomic locations of histone H3 molecules bearing lysine trimethylation modifications before and after the maternal-zygotic transition in zebrafish. Histone H3 lysine 27 trimethylation (H3K27me3), which is repressive, and H3K4me3, which is activating, were not detected before the transition. After genome activation, more than 80% of genes were marked by H3K4me3, including many inactive developmental regulatory genes that were also marked by H3K27me3. Sequential chromatin immunoprecipitation demonstrated that the same promoter regions had both trimethylation marks. Such bivalent chromatin domains also exist in embryonic stem cells and are thought to poise genes for activation while keeping them repressed. Furthermore, we found many inactive genes that were uniquely marked by H3K4me3. Despite this activating modification, these monovalent genes were neither expressed nor stably bound by RNA polymerase II. Inspection of published data sets revealed similar monovalent domains in embryonic stem cells. Moreover, H3K4me3 marks could form in the absence of both sequence-specific transcriptional activators and stable association of RNA polymerase II, as indicated by the analysis of an inducible transgene. These results indicate that bivalent and monovalent domains might poise embryonic genes for activation and that the chromatin profile associated with pluripotency is established during the maternal-zygotic transition.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • A comprehensive characterization of pancreatic ductal carcinoma cell lines: towards the establishment of an in vitro research platform. 12692724

    There are a large number of stable pancreatic ductal carcinoma cell lines that are used by researchers worldwide. Detailed data about their differentiation status and growth features are, however, often lacking. We therefore attempted to classify commonly used pancreatic carcinoma cell lines according to defined cell biological criteria. Twelve pancreatic ductal adenocarcinoma cell lines were cultured as monolayers and spheroids and graded according to their ultrastructural features. The grading system was based on the integrity of membrane structures and on the presence of mucin granules, cell organelles, nuclear and cellular polymorphism, cell polarity, and lumen formation. On the basis of the resulting scores the cell lines were classified as well, moderately, or poorly differentiated. In addition, immunocytochemistry was performed for the markers cytokeratin 7, 8, 18, 19, carcinoembryonic antigen, MUC1 MUC2, MUC5, and MUC6. The population doubling time of monolayer cultures, determined by a tetrazolium salt based proliferation assay was correlated with the ultrastructural grade. The grading of the ultrastructural features of the monolayers, and particularly of the spheroids, revealed that Capan-1 and Capan-2 cells were well differentiated; Colo357, HPAF-2, Aspc-1, A818-4, BxPc3, and Panc89 cells were moderately differentiated and PancTu-I, Panc1, Pt45P1, and MiaPaCa-2 cells poorly differentiated. Membrane-bound MUC1 staining was a characteristic of well differentiated cell lines. The population doubling time of the monolayer cultures was related to the differentiation grade. No relationship was found between the p53, K-ras, DPC4/Smad4, or p16(INK4a) mutation status and the grade of differentiation. We conclude that the proposed ultrastructural grading system combined with the proliferative activity provides a basis for further comparative studies of pancreatic ductal adenocarcinoma cell lines.
    Document Type:
    Reference
    Product Catalog Number:
    MAB2011
    Product Catalog Name:
    Anti-Mucin MUC5AC Antibody, clone CLH2

  • β3 integrin is dispensable for conditioned fear and Hebbian forms of plasticity in the hippocampus. 22748100

    Integrins play key roles in the developing and mature nervous system, from promoting neuronal process outgrowth to facilitating synaptic plasticity. Recently, in hippocampal pyramidal neurons, β3 integrin (ITGβ3) was shown to stabilise synaptic AMPA receptors (AMPARs) and to be required for homeostatic scaling of AMPARs elicited by chronic activity suppression. To probe the physiological function for ITGβ3-dependent processes in the brain, we examined whether the loss of ITGβ3 affected fear-related behaviours in mice. ITGβ3-knockout (KO) mice showed normal conditioned fear responses that were similar to those of control wild-type mice. However, anxiety-like behaviour appeared substantially compromised and could be reversed to control levels by lentivirus-mediated re-expression of ITGβ3 bilaterally in the ventral hippocampus. In hippocampal slices, the loss of ITGβ3 activity did not compromise Hebbian forms of plasticity - neither acute pharmacological disruption of ITGβ3 ligand interactions nor genetic deletion of ITGβ3 altered long-term potentiation (LTP) or long-term depression (LTD). Moreover, we did not detect any changes in short-term synaptic plasticity upon loss of ITGβ3 activity. In contrast, acutely disrupting ITGβ1-ligand interactions or genetic deletion of ITGβ1 selectively interfered with LTP stabilisation whereas LTD remained unaltered. These findings indicate a lack of requirement for ITGβ3 in the two robust forms of hippocampal long-term synaptic plasticity, LTP and LTD, and suggest differential roles for ITGβ1 and ITGβ3 in supporting hippocampal circuit functions.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1501
    Product Catalog Name:
    Anti-Actin Antibody, clone C4

  • Pathogenetic mechanisms of parkin in Parkinson's disease. 15325839

    CONTEXT: The cause and pathogenesis of Parkinson's disease remain unknown; mitochondrial dysfunction, oxidative damage, environmental factors, and genetic predisposition might all be involved. Identification of the causative genes for familial Parkinson's diseases allow study of the pathogenesis of the disease at the molecular level. STARTING POINT: Katja Hedrich and colleagues studied 75 Serbian patients with early-onset Parkinson's disease for DJ-1 mutations (Neurology 2004; 62: 389-94). One patient was a compound heterozygote and another had a heterozygous exon deletion. DJ-1 mutations seem to be rare in this European population. By contrast, parkin mutations have been found in about 50% of familial cases and in 10-20% of cases without a positive family history. WHERE NEXT: The fact that parkin is a ubiquitin ligase gives special meaning to the molecular mechanism of neurodegeneration in general. In Parkinson's disease, Lewy bodies are immunoreactive for ubiquitin. Accumulation of abnormal proteins has also been seen in other neurodegenerative disorders. Disturbance of protein degradation by the ubiquitin-proteasome system might have a critical role in neurodegeneration. Although alpha-synuclein mutations are infrequent, alpha-synuclein accumulates in Lewy bodies, and alpha-synuclein fibrils impair the 26S proteasome function. UCH-L1 is also an abundant deubiquitylating enzyme, and its mutation is linked to PARK5. Furthermore, DJ-1 might interact with SUMO-1 (small ubiquitin-like modifier), which can counteract ubiquitin and stabilise proteins against degradation by the 26S proteasome. Uncovering the mechanisms of protein degradation should add importantly to understanding the neurodegenerative process in these neurodegenerative diseases.
    Document Type:
    Reference
    Product Catalog Number:
    05-882
    Product Catalog Name:
    Anti-Parkin Antibody, clone PRK8