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  • Robust activation of the human but not mouse telomerase gene during the induction of pluripotency. 20354136

    Pluripotent stem cells (PSCs) express telomerase and have unlimited proliferative potential. To study telomerase activation during reprogramming, 3 classes of embryonic stem cell (ESC)-like clones were isolated from mouse fibroblasts containing a transgenic hTERT reporter. Class I expressed few pluripotency markers, whereas class II contained many, but not Oct4, Nanog, and Sox2. Neither class of cells differentiated efficiently. Class III cells, the fully reprogrammed induced PSCs (iPSCs), expressed all pluripotency markers, formed teratomas indistinguishable from those of mESCs, and underwent efficient osteogenic differentiation in vitro. Interestingly, whereas the endogenous mTERT gene expression was only moderately increased during reprogramming, the hTERT promoter was strongly activated in class II cells and was further elevated in class III cells. Treatment of class II cells with chemical inhibitors of MEKs and glycogen synthase kinase 3 resulted in their further reprogramming into class III cells, accompanied by a strong activation of hTERT promoter. In reprogrammed human cells, the endogenous telomerase level, although variable among different clones, was dramatically elevated. Only in cells with the highest telomerase were telomeres restored to the lengths in hESCs. Our data, for the first time, demonstrated that the hTERT promoter was strongly activated in discrete steps, revealing a critical difference in human and mouse cell reprogramming. Because telomere elongation is crucial for self-renewal of hPSCs and replicative aging of their differentiated progeny, these findings have important implications in the generation and applications of iPSCs.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Early-onset and robust amyloid pathology in a new homozygous mouse model of Alzheimer's disease. 19936202

    Transgenic mice expressing mutated amyloid precursor protein (APP) and presenilin (PS)-1 or -2 have been successfully used to model cerebral beta-amyloidosis, one of the characteristic hallmarks of Alzheimer's disease (AD) pathology. However, the use of many transgenic lines is limited by premature death, low breeding efficiencies and late onset and high inter-animal variability of the pathology, creating a need for improved animal models. Here we describe the detailed characterization of a new homozygous double-transgenic mouse line that addresses most of these issues.The transgenic mouse line (ARTE10) was generated by co-integration of two transgenes carrying the K670N/M671L mutated amyloid precursor protein (APP(swe)) and the M146V mutated presenilin 1 (PS1) both under control of a neuron-specific promoter. Mice, hemi- as well as homozygous for both transgenes, are viable and fertile with good breeding capabilities and a low rate of premature death. They develop robust AD-like cerebral beta-amyloid plaque pathology with glial inflammation, signs of neuritic dystrophy and cerebral amyloid angiopathy. Using our novel image analysis algorithm for semi-automatic quantification of plaque burden, we demonstrate an early onset and progressive plaque deposition starting at 3 months of age in homozygous mice with low inter-animal variability and 100%-penetrance of the phenotype. The plaques are readily detected in vivo by PiB, the standard human PET tracer for AD. In addition, ARTE10 mice display early loss of synaptic markers and age-related cognitive deficits. By applying a gamma-secretase inhibitor we show a dose dependent reduction of soluble amyloid beta levels in the brain.ARTE10 mice develop a cerebral beta-amyloidosis closely resembling the beta-amyloid-related aspects of human AD neuropathology. Unifying several advantages of previous transgenic models, this line particularly qualifies for the use in target validation and for evaluating potential diagnostic or therapeutic agents targeting the amyloid pathology of AD.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • NLRC5 interacts with RIG-I to induce a robust antiviral response against influenza virus infection. 25404059

    The NLR protein, NLRC5, is an important regulator of MHC I gene expression, however, the role of NLRC5 in other innate immune responses is less well defined. In the present study, we report that NLRC5 binds RIG-I and that this interaction is critical for robust antiviral responses against influenza virus. Overexpression of NLRC5 in the human lung epithelial cell line, A549, and normal human bronchial epithelial (NHBE) cells resulted in impaired replication of influenza virus A/Puerto Rico/8/34 virus (PR8) and enhanced IFN-β expression. Influenza virus leads to induction of IFN-β that drives RIG-I and NLRC5 expression in host cells. Our results suggest that NLRC5 extends and stabilizes influenza virus-induced RIG-I expression and delays expression of the viral inhibitor protein NS1. We show that NS1 binds to NLRC5 to suppress its function. Interaction domain mapping revealed that NLRC5 interacts with RIG-I via its N-terminal death domain and that NLRC5 enhanced antiviral activity in a LRR-domain-independent manner. Taken together, our findings identify a novel role for NLRC5 in RIG-I-mediated antiviral host responses against influenza virus infection, distinguished from the role of NLRC5 in MHC class I gene regulation. This article is protected by copyright. All rights reserved.
    Document Type:
    Reference
    Product Catalog Number:
    MABF260
    Product Catalog Name:
    Anti-NLRC5 Antibody, clone 3H8

  • Excision of viral reprogramming cassettes by Cre protein transduction enables rapid, robust and efficient derivation of transgene-free human induced pluripotent stem cell ... 24713299

    Integrating viruses represent robust tools for cellular reprogramming, however, the presence of viral transgenes in induced pluripotent stem cells (iPSCs) is deleterious as it holds the risk of insertional mutagenesis leading to malignant transformation. Here, we combine the robustness of lentiviral reprogramming with the efficacy of Cre recombinase protein transduction to derive iPSCs devoid of transgenes. By genome-wide analysis and targeted differentiation towards the cardiomyocyte lineage, we show that transgene-free iPSCs are superior to iPSCs before Cre transduction. Our study provides a simple, rapid and robust protocol for the generation of clinical-grade iPSCs suitable for disease modeling, tissue engineering and cell replacement therapies.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Robust pluripotent stem cell expansion and cardiomyocyte differentiation via geometric patterning. 24141327

    Geometric factors including the size, shape, density, and spacing of pluripotent stem cell colonies play a significant role in the maintenance of pluripotency and in cell fate determination. These factors are impossible to control using standard tissue culture methods. As such, there can be substantial batch-to-batch variability in cell line maintenance and differentiation yield. Here, we demonstrate a simple, robust technique for pluripotent stem cell expansion and cardiomyocyte differentiation by patterning cell colonies with a silicone stencil. We have observed that patterning human induced pluripotent stem cell (hiPSC) colonies improves the uniformity and repeatability of their size, density, and shape. Uniformity of colony geometry leads to improved homogeneity in the expression of pluripotency markers SSEA4 and Nanog as compared with conventional clump passaging. Patterned cell colonies are capable of undergoing directed differentiation into spontaneously beating cardiomyocyte clusters with improved yield and repeatability over unpatterned cultures seeded either as cell clumps or uniform single cell suspensions. Circular patterns result in a highly repeatable 3D ring-shaped band of cardiomyocytes which electrically couple and lead to propagating contraction waves around the ring. Because of these advantages, geometrically patterning stem cells using stencils may offer greater repeatability from batch-to-batch and person-to-person, an increase in differentiation yield, a faster experimental workflow, and a simpler protocol to communicate and follow. Furthermore, the ability to control where cardiomyocytes arise across a culture well during differentiation could greatly aid the design of electrophysiological assays for drug-screening.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1691
    Product Catalog Name:
    Anti-Troponin I Antibody, a.a. 186-192, clone C5

  • A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples. 21192942

    Phagocytosis can be induced via the engagement of Fcγ receptors by antibody-opsonized material. Furthermore, the efficiency of antibody-induced effector functions has been shown to be dramatically modulated by changes in antibody glycosylation. Because infection can modulate antibody glycans, which in turn modulate antibody functions, assays capable of determining the induction of effector functions rather than neutralization or titer provide a valuable opportunity to more fully characterize the quality of the adaptive immune response. Here we describe a robust and high-throughput flow cytometric assay to define the phagocytic activity of antigen-specific antibodies from clinical samples. This assay employs a monocytic cell line that expresses numerous Fc receptors: including inhibitory and activating, and high and low affinity receptors--allowing complex phenotypes to be studied. We demonstrate the adaptability of this high-throughput, flow-based assay to measure antigen-specific antibody-mediated phagocytosis against an array of viruses, including influenza, HIV, and dengue. The phagocytosis assay format further allows for simultaneous analysis of cytokine release, as well as determination of the role of specific Fcγ-receptor subtypes, making it a highly useful system for parsing differences in the ability of clinical and vaccine induced antibody samples to recruit this critical effector function.
    Document Type:
    Reference
    Product Catalog Number:
    MAB10226
    Product Catalog Name:
    Anti-Dengue Virus-2 Antibody, clone 3H5-1 | MAB10226

  • Robust, high-throughput solution for blood group genotyping. 20560530

    With the concomitant increase of blood transfusions and safety rules, there is a growing need to integrate high-throughput and multiparametric assays within blood qualification centers. Using a robust and automated solution, we describe a new method for extended blood group genotyping (HiFi-Blood 96) bringing together the throughput possibilities of complete automation and the microarray multiplexed analysis potential. Our approach provides a useful resource for upgrading blood qualification center facilities. A set of six single-nucleotide polymorphisms (SNPs) associated with clinically important blood group antigens (Kell, Kidd, Duffy, and MNS systems) were selected and the corresponding genotyping assays developed. A panel of 293 blood samples was used to validate the approach. The resulting genotypes were compared to phenotypes previously determined by standard serologic techniques, and excellent correlations were found for five SNPs out of six. For the Kell, Kidd, Duffy, and MNS3/MNS4 systems, high matching percentages of 100%, 98.9%, 97.7%, and 97.4% were obtained, respectively, whereas a concordance percentage of 83.3% only was attained for the MNS1/MNS2 polymorphism.
    Document Type:
    Reference
    Product Catalog Number:
    MSPLRS096

  • Robust tonic GABA currents can inhibit cell firing in mouse newborn neocortical pyramidal cells. 20846324

    Within the hippocampus and neocortex, GABA is considered to be excitatory in early development due to a relatively depolarized Cl(-) reversal potential (E(Cl)). Although the depolarizing nature of synaptic GABAergic events has been well established, it is unknown whether cortical tonic currents mediated by extrasynaptically located GABA(A) receptors (GABA(A) Rs) are also excitatory. Here we examined the development of tonic currents in the neocortex and their effect on neuronal excitability. Mean tonic current, recorded from layer 5 (L5) pyramidal cells of the mouse somatosensory cortex, is robust in newborns [postnatal day (P)2-4] then decreases dramatically by the second postnatal week (P7-10 and P30-40). Pharmacological studies, in combination with Western blot analysis, show that neonatal tonic currents are partially mediated by the GABA(A) R α5 subunit, and probably the δ subunit. In newborns, the charge due to tonic current accounts for nearly 100% of the total GABA charge, a contribution that decreases to < 50% in mature tissue. Current clamp recordings show that tonic current contributes to large fluctuations in the membrane potential that may disrupt its stability. Bath application of 5 μM GABA, to induce tonic currents, markedly decreased cell firing frequency in most recorded cells while increasing it in others. Gramicidin perforated patch recordings show heterogeneity in E(Cl) recorded from P2-5 L5 pyramidal cells. Together, these findings demonstrate that tonic currents activated by low GABA concentrations can dominate GABAergic transmission in newborn neocortical pyramidal cells and that tonic currents can exert heterogeneous effects on neuronal excitability.
    Document Type:
    Reference
    Product Catalog Number:
    AB9752

  • Robust internal elastic lamina fenestration in skeletal muscle arteries. 23359815

    Holes within the internal elastic lamina (IEL) of blood vessels are sites of fenestration allowing for passage of diffusible vasoactive substances and interface of endothelial cell membrane projections with underlying vascular smooth muscle. Endothelial projections are sites of dynamic Ca(2+) events leading to endothelium dependent hyperpolarization (EDH)-mediated relaxations and the activity of these events increase as vessel diameter decreases. We tested the hypothesis that IEL fenestration is greater in distal vs. proximal arteries in skeletal muscle, and is unlike other vascular beds (mesentery). We also determined ion channel protein composition within the endothelium of intramuscular and non-intramuscular skeletal muscle arteries. Popliteal arteries, subsequent gastrocnemius feed arteries, and first and second order intramuscular arterioles from rat hindlimb were isolated, cut longitudinally, fixed, and imaged using confocal microscopy. Quantitative analysis revealed a significantly larger total fenestration area in second and first order arterioles vs. feed and popliteal arteries (58% and 16% vs. 5% and 3%; N = 10 images/artery), due to a noticeably greater average size of holes (9.5 and 3.9 µm(2) vs 1.5 and 1.9 µm(2)). Next, we investigated via immunolabeling procedures whether proteins involved in EDH often embedded in endothelial cell projections were disparate between arterial segments. Specific proteins involved in EDH, such as inositol trisphosphate receptors, small and intermediate conductance Ca(2+)-activated K(+) channels, and the canonical (C) transient receptor potential (TRP) channel TRPC3 were present in both popliteal and first order intramuscular arterioles. However due to larger IEL fenestration in first order arterioles, a larger spanning area of EDH proteins is observed proximal to the smooth muscle cell plasma membrane. These observations highlight the robust area of fenestration within intramuscular arterioles and indicate that the anatomical architecture and endothelial cell hyperpolarizing apparatus for distinct vasodilatory signaling is potentially present.
    Document Type:
    Reference
    Product Catalog Number:
    07-1210
    Product Catalog Name:
    Anti-IP3 Receptor Antibody