scholarly journals High-resolution in vivo identification of miRNA targets by Halo-Enhanced Ago2 Pulldown

2019 ◽  
Author(s):  
Xiaoyi Li ◽  
Yuri Pritykin ◽  
Carla P. Concepcion ◽  
Yuheng Lu ◽  
Gaspare La Rocca ◽  
...  
Keyword(s):  
Binding Sites ◽  
Embryonic Stem ◽  
Genetically Engineered ◽  
Lung Cancers ◽  
Mirna Targets ◽  
Pathological Conditions ◽  
Non Coding Rnas ◽  
Mrna Binding

SUMMARYThe identification of miRNA targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily translated to an in vivo setting. To overcome these limitations and to facilitate the investigation of miRNA functions in mice, we have developed a method (HEAP: for Halo-Enhanced Ago2 Pulldown) to map miRNA-mRNA binding sites. This method is based on a novel genetically engineered mouse harboring a conditional, Cre-regulated, Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be efficiently purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, in developing embryos, in adult tissues and in autochthonous mouse models of human brain and lung cancers.The tools and the datasets we have generated will serve as a valuable resource to the scientific community and will facilitate the characterization of miRNA functions under physiological and pathological conditions.

scholarly journals Generation and Characterization of Smac/DIABLO-Deficient Mice

2002 ◽  
Vol 22 (10) ◽  
pp. 3509-3517 ◽  
Author(s):  
Hitoshi Okada ◽  
Woong-Kyung Suh ◽  
Jianping Jin ◽  
Minna Woo ◽  
Chunying Du ◽  
...  
Keyword(s):  
Physiological Function ◽  
Es Cells ◽  
Embryonic Stem ◽  
Caspase Activation ◽  
Proapoptotic Protein ◽  
Apoptosis Proteins ◽  
Deficient Mice

ABSTRACT The mitochondrial proapoptotic protein Smac/DIABLO has recently been shown to potentiate apoptosis by counteracting the antiapoptotic function of the inhibitor of apoptosis proteins (IAPs). In response to apoptotic stimuli, Smac is released into the cytosol and promotes caspase activation by binding to IAPs, thereby blocking their function. These observations have suggested that Smac is a new regulator of apoptosis. To better understand the physiological function of Smac in normal cells, we generated Smac-deficient (Smac−/− ) mice by using homologous recombination in embryonic stem (ES) cells. Smac−/− mice were viable, grew, and matured normally and did not show any histological abnormalities. Although the cleavage in vitro of procaspase-3 was inhibited in lysates of Smac−/− cells, all types of cultured Smac−/− cells tested responded normally to all apoptotic stimuli applied. There were also no detectable differences in Fas-mediated apoptosis in the liver in vivo. Our data strongly suggest the existence of a redundant molecule or molecules capable of compensating for a loss of Smac function.

Analysis of in Vitro and in Vivo Characteristics of Human Embryonic Stem Cell-Derived Neural Precursors

2010 ◽  
Vol 19 (4) ◽  
pp. 471-486 ◽  
Author(s):  
Nataliya Kozubenko ◽  
Karolina Turnovcova ◽  
Miroslava Kapcalova ◽  
Olena Butenko ◽  
Miroslava Anderova ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Cellular Heterogeneity ◽  
Neural Cells ◽  
Lineage Specification ◽  
Neural Precursors ◽  
Selection Strategies

During the last decade, much progress has been made in developing protocols for the differentiation of human embryonic stem cells (hESCs) into a neural phenotype. The appropriate agent for cell therapy is neural precursors (NPs). Here, we demonstrate the derivation of highly enriched and expandable populations of proliferating NPs from the CCTL14 line of hESCs. These NPs could differentiate in vitro into functionally active neurons, as confirmed by immunohistochemical staining and electrophysiological analysis. Neural cells differentiated in vitro from hESCs exhibit broad cellular heterogeneity with respect to developmental stage and lineage specification. To analyze the population of the derived NPs, we used fluorescence-activated cell sorting (FACS) and characterized the expression of several pluripotent and neural markers, such as Nanog, SSEA-4, SSEA-1, TRA-1-60, CD24, CD133, CD56 (NCAM), β-III-tubulin, NF70, nestin, CD271 (NGFR), CD29, CD73, and CD105 during long-term propagation. The analyzed cells were used for transplantation into the injured rodent brain; the tumorigenicity of the transplanted cells was apparently eliminated following long-term culture. These results complete the characterization of the CCTL14 line of hESCs and provide a framework for developing cell selection strategies for neural cell-based therapies.

scholarly journals Generation and Characterization of Typhoid Toxin-Neutralizing Human Monoclonal Antibodies

2020 ◽  
Vol 88 (10) ◽  
Author(s):  
Xuyao Jiao ◽  
Sarah Smith ◽  
Gabrielle Stack ◽  
Qi Liang ◽  
Allan Bradley ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Typhoid Fever ◽  
Severe Disease ◽  
Genetically Engineered ◽  
Human Monoclonal Antibodies ◽  
Genetically Engineered Mice ◽  
Typhoid Toxin

ABSTRACT Typhoid toxin is a virulence factor of Salmonella enterica serovar Typhi, the causative agent of typhoid fever, and is thought to be responsible for the symptoms of severe disease. This toxin has a unique A2B5 architecture with two active subunits, the ADP ribosyl transferase PltA and the DNase CdtB, linked to a pentameric B subunit, which is alternatively made of PltB or PltC. Here, we describe the generation and characterization of typhoid toxin-neutralizing human monoclonal antibodies by immunizing genetically engineered mice that have a full set of human immunoglobulin variable region genes. We identified several monoclonal antibodies with strong in vitro and in vivo toxin-neutralizing activity and different mechanisms of toxin neutralization. These antibodies could serve as the basis for the development of novel therapeutic strategies against typhoid fever.

Myosin VIIA is specifically associated with calmodulin and microtubule-associated protein-2B (MAP-2B)

2001 ◽  
Vol 354 (2) ◽  
pp. 267-274 ◽  
Author(s):  
Penio T. TODOROV ◽  
Rachel E. HARDISTY ◽  
Steve D. M. BROWN
Keyword(s):  
Binding Sites ◽  
Strong Association ◽  
Mouse Kidney ◽  
Scatchard Analysis ◽  
Tail Region ◽  
Immunoblotting Analysis ◽  
Kd Values ◽  
Myosin Viia

Myosin VIIA is a motor molecule with a conserved head domain and tail region unique to myosin VIIA, which probably defines its unique function in vivo. In an attempt to further characterize myosin VIIA function we set out to identify molecule(s) that specifically associate with it. We demonstrate that 17 and 55kDa proteins from mouse kidney and cochlea co-purify with myosin VIIA on affinity columns carrying immobilized anti-myosin VIIA antibody. N-terminal sequencing and immunoblotting analysis identified the 17kDa protein as calmodulin, whereas MS and immunoblotting analysis identified the 55kDa protein as microtubule-associated protein-2B (MAP-2B). Myosin VIIA can also be co-immunoprecipitated from kidney homogenate using anti-calmodulin or anti-MAP2 (recognizing isoforms 2A and 2B) antibodies, confirming the strong association between calmodulin and myosin VIIA and between MAP-2B and myosin VIIA. Myosin VIIA binds to calmodulin with an apparent Kd of 10-9 M. Scatchard analysis of the binding of myosin VIIA to MAP-2B provided evidence for two binding sites, with Kd values of 10-10 and 10-9 M, which have been mapped to medial and C-terminal tail domains of myosin VIIA. The characterization of the interaction of calmodulin and MAP-2B with myosin VIIA provides new insights into the function of myosin VIIA.

Is the hypotensive effect of clonidine and related drugs due to imidazoline binding sites?

1997 ◽  
Vol 273 (5) ◽  
pp. R1580-R1584 ◽  
Author(s):  
Patrice G. Guyenet
Keyword(s):  
Binding Sites ◽  
Hypotensive Effect ◽  
Genetically Engineered ◽  
Therapeutic Effects ◽  
Genetically Engineered Mice ◽  
Wide Range ◽  
The Central Nervous System ◽  
Imidazoline Binding Sites ◽  
Cellular Components

Clonidine and related α2-adrenergic receptor (α2AR) agonists lower arterial pressure primarily by an action within the central nervous system. These drugs also have varying degrees of affinity for other cellular components called nonadrenergic imidazoline binding sites (NAIBS). For over 20 years, the α2AR agonist activity of clonidine-like drugs was thought to account for their therapeutic effects (α2 theory). However, several groups have recently proposed a competing “imidazoline theory” according to which the hypotensive effect of clonidine-like drugs would in fact owe more to their affinity for one type of NAIBS, called I1receptors. The α2-theory is strongly supported by four main types of congruent data. First, the hypotensive effect of systemically administered clonidine is blocked by α2AR antagonists that are without affinity for I1 NAIBs. Second, the hypotensive effect of intravenous clonidine is absent in genetically engineered mice in which a defective α2AAR has been substituted for the normal one. Third, the sympatholytic effect of clonidine is consistent with the presence of conventional inhibitory α2ARs on sympathetic preganglionic neurons and on their main excitatory inputs in the medulla oblongata. Fourth, the first I1 ligand without affinity for α2ARs was found to be biologically inactive. The imidazoline theory is supported by a limited repertoire of whole animal “in vivo” pharmacological experiments that remain open to a wide range of interpretations. In conclusion, the bulk of the evidence strongly supports a largely predominant role of α2AR mechanisms in the action of most clonidine-like agents at therapeutically relevant doses or concentrations. Even the small pharmacological differences between these agents cannot yet be linked with certainty to their relative affinity for I1 NAIBS.

Isolation and characterization of embryonic stem-like cells derived from in vivo-produced cat blastocysts

2008 ◽  
Vol 75 (9) ◽  
pp. 1426-1432 ◽  
Author(s):  
Xianfeng Yu ◽  
Guangzhen Jin ◽  
Xijun Yin ◽  
Sujin Cho ◽  
Jintae Jeon ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Isolation And Characterization

scholarly journals Characterization of Glial Cell Models andIn VitroManipulation of the Neuregulin1/ErbB System

2014 ◽  
Vol 2014 ◽  
pp. 1-15 ◽  
Author(s):  
Davide Pascal ◽  
Alessia Giovannelli ◽  
Sara Gnavi ◽  
Stefan Adriaan Hoyng ◽  
Fred de Winter ◽  
...  
Keyword(s):  
Cell Line ◽  
Schwann Cells ◽  
Peripheral Nerve Regeneration ◽  
Cell Behavior ◽  
Cell Models ◽  
Migratory Activity ◽  
Pathological Conditions

The neuregulin1/ErbB system plays an important role in Schwann cell behavior both in normal and pathological conditions. Upon investigation of the expression of the neuregulin1/ErbB systemin vitro, we explored the possibility to manipulate the system in order to increase the migration of Schwann cells, that play a fundamental role in the peripheral nerve regeneration. Comparison of primary cells and stable cell lines shows that both primary olfactory bulb ensheathing cells and a corresponding cell line express ErbB1-ErbB2 and neuregulin1, and that both primary Schwann cells and a corresponding cell line express ErbB2-ErbB3, while only primary Schwann cells express neuregulin1. To interfere with the neuregulin1/ErbB system, the soluble extracellular domain of the neuregulin1 receptor ErbB4 (ecto-ErbB4) was expressedin vitroin the neuregulin1 expressing cell line, and an unexpected increase in cell motility was observed.In vitroexperiments suggest that the back signaling mediated by the transmembrane neuregulin1 plays a role in the migratory activity induced by ecto-ErbB4. These results indicate that ecto-ErbB4 could be usedin vivoas a tool to manipulate the neuregulin1/ErbB system.

scholarly journals Elevated murine HB-EGF confers sensitivity to diphtheria toxin in EGFR-mutant lung adenocarcinoma

2021 ◽  
Author(s):  
Camila Robles-Oteiza ◽  
Deborah Ayeni ◽  
Stellar Levy ◽  
Robert J. Homer ◽  
Susan M. Kaech ◽  
...  
Keyword(s):  
Diphtheria Toxin ◽  
Tumor Regression ◽  
Genetically Engineered ◽  
Lung Cancers ◽  
Genetically Engineered Mice ◽  
Elevated Expression ◽  
Murine Tissue ◽  
Egfr Mutant ◽  
Lung Adenocarcinomas

Conditional ablation of defined cell populations in vivo can be achieved using genetically engineered mice in which the human diphtheria toxin (DT) receptor (DTR) is placed under control of a murine tissue-specific promotor, such that delivery of diphtheria toxin selectively ablates cells expressing the high-affinity human DTR. Cells expressing only the endogenous low-affinity mouse DTR are assumed to be unaffected. Surprisingly, we found that systemic DT administration induced rapid regression of murine EGFR-mutant lung adenocarcinomas in the absence of a transgenic allele containing human DTR. DT enzymatic activity was required for tumor regression, and EGFR-mutant tumor cells were the primary targets of DT toxicity. In FVB mice, EGFR-mutant tumors upregulated expression of HB-EGF, which is the DTR in mice and humans. HB-EGF blockade with CRM197, an enzymatically inactive DT mutant, partially abrogated DT-induced tumor regression. These results suggest that elevated expression of murine HB-EGF (low-affinity DTR) confers sensitivity to DT in EGFR-mutant tumors, demonstrating a biological effect of DT in mice lacking transgenic DTR alleles and highlighting a unique vulnerability of EGFR-mutant lung cancers.

scholarly journals lncRNA MALAT1 Mediates Osteogenic Differentiation and Apoptosis in Osteoporosis by Regulating the miR-485-5p/WNT7B Axis

2021 ◽  
Author(s):  
Yuan Zhou ◽  
Zhuo Xu ◽  
Yuanyi Wang ◽  
Qiang Song ◽  
Ruofeng Yin
Keyword(s):  
Osteogenic Differentiation ◽  
Cell Apoptosis ◽  
Binding Sites ◽  
Hindlimb Unloading ◽  
Cellular Functions ◽  
Alizarin Red ◽  
Lncrna Malat1 ◽  
Non Coding Rnas

Abstract Background: Accumulating evidence demonstrates that long non-coding RNAs (lncRNAs) are associated with the development of osteoporosis. This study aimed to investigate the effects of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) on osteogenic differentiation and cell apoptosis in osteoporosis. Methods: hindlimb unloading (HU) was performed to establish osteoporosis model in vivo. MicroCT was applied for pathological analysis. Microgravity (MG) was used to construct osteoporosis in vitro. The mRNA and miRNA expression was determined using RT-qPCR. Protein expression was determined using western blot. The binding sites between miR-485-5p and MALAT1/Wnt family member 7B (WNT7B) was predicted by bioinformatics analysis and verified by luciferase and RNA pull-down assays. Cellular functions were determined by ALP staining, Alizarin red staining, and flow cytometry assays. Results: MALAT1 expression was downregulated in HU mice and MG treated MC3T3-E1 cells. However, overexpression of MALAT1 upregulated the expression of Bmp4, Col1a1, Spp1, and enhanced ALP activity. Additionally, overexpression of MALAT1 inhibited apoptosis, decreased Bax and caspase-3 levels, and increased Bcl-2 level. Moreover, MALAT1 overexpression improved bone phenotype in vivo. MALAT1 functioned as a ceRNA to upregulate WNT7B. Overexpression of miR-485-5p rescued the promotion of osteogenic differentiation and the inhibition of apoptosis induced by MALAT1. Knockdown of WNT7B abolished the facilitation of osteogenic differentiation and the suppression of apoptosis induced by downregulation of miR-485-5p. Conclusion: In conclusion, MALAT1 promoted osteogenic differentiation and inhibited cell apoptosis through miR-485-5p/WNT7B axis, which suggested that MALAT1 is a potential target to alleviate osteoporosis.

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