Methods in molecular biology (Clifton, N.J.) (Meth Mol Biol )

Publisher: Humana Press

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Current impact factor: 1.29

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Other titles Methods in molecular biology (Clifton, N.J.), Methods in molecular biology
ISSN 1940-6029
OCLC 24839341
Material type Series
Document type Journal / Magazine / Newspaper

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Humana Press

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    • 'Humana Press' is an imprint of 'Springer Verlag (Germany)'
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Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: As obligate intracellular parasites, viruses need to cross the plasma membrane and deliver their genome inside the cell. This step is initiated by the recognition of receptors present on the host cell surface. Receptors can be major determinants of tropism, host range, and pathogenesis. Identifying virus receptors can give clues to these aspects and can lead to the design of intervention strategies. Interfering with receptor recognition is an attractive antiviral therapy, since it occurs before the viral genome has reached the relative safe haven within the cell. This chapter describes the use of an immunoprecipitation approach with Fc-tagged viral spike proteins followed by mass spectrometry to identify and characterize the receptor for the Middle East respiratory syndrome coronavirus. This technique can be adapted to identify other viral receptors.
    Methods in molecular biology (Clifton, N.J.) 03/2015; 1282(165):82.
  • [Show abstract] [Hide abstract]
    ABSTRACT: As obligate intracellular parasites, viruses need to cross the plasma membrane and deliver their genome inside the cell. This step is initiated by the recognition of receptors present on the host cell surface. Receptors can be major determinants of tropism, host range, and pathogenesis. Identifying virus receptors can give clues to these aspects and can lead to the design of intervention strategies. Interfering with receptor recognition is an attractive antiviral therapy, since it occurs before the viral genome has reached the relative safe haven within the cell. This chapter describes the use of an immunoprecipitation approach with Fc-tagged viral spike proteins followed by mass spectrometry to identify and characterize the receptor for the Middle East respiratory syndrome coronavirus. This technique can be adapted to identify other viral receptors.
    Methods in molecular biology (Clifton, N.J.) 03/2015; 1282:165-82.
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    ABSTRACT: Human embryonic stem cells hold great promise for future biomedical applications such as disease modeling and regenerative medicine. However, these cells are notoriously difficult to culture and are refractory to common means of genetic manipulation, thereby limiting their range of applications. In this protocol, we present an easy and robust method of gene repression in human embryonic stem cells using lipofection of small interfering RNA (siRNA).
    Methods in molecular biology (Clifton, N.J.) 02/2015;
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    ABSTRACT: Mouse embryonic stem cells (ESCs) derive from the inner cell mass (ICM) of a blastocyst at E3.5 while mouse epiblast stem cells (EpiSCs) derive from the late epiblast of a post-implantation embryo at E5.5-E7.5. Both cells are able to differentiate into derivatives of the three germs layers but only ESCs are able to produce chimeras when they are introduced into a blastocyst. To support the naive state of pluripotency, ESC culture requires Leukemia inhibitory factor (Lif) and either serum or inhibitors of Erk and Gsk3 pathways (2i) while the primed pluripotency of EpiSCs is maintained using Activin A and Fibroblast Growth Factor 2 (FGF2). It is possible to obtain EpiSCs in vitro starting from ESCs but also to induce ESCs starting from EpiSCs even if this second process is very difficult and inefficient. In this protocol we describe how we perform the process of conversion from ESCs to EpiSCs.
    Methods in molecular biology (Clifton, N.J.) 02/2015;
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    ABSTRACT: Mouse embryonic stem cells (ESCs) derive from the inner cell mass (ICM) of a blastocyst. These cells are pluripotent and thus able to generate both somatic and germinal lineages. It is possible to maintain ESCs in different pluripotent states depending on the in vitro culture conditions. Classically, ESCs are cultured in the presence of serum and LIF, which sustain the naive state of pluripotency but in this metastable state cells exhibit a large degree of heterogeneity. In the last few years, it has been discovered that when ESCs are cultured in a chemically defined medium (without serum), in the presence of LIF and with the addition of two small molecules (in particular the inhibitors of MAPK and Gsk-3 pathways), they reach a ground state of pluripotency where cells are more homogeneous and more "ICM-like." In this protocol, we describe how we culture mouse ESCs and the way we switch them from naive to ground state.
    Methods in molecular biology (Clifton, N.J.) 02/2015;
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    ABSTRACT: Reprogramming somatic cells into a pluripotent state involves the overexpression of transcription factors leading to a series of changes that end in the formation of induced pluripotent stem cells (iPSCs). These iPSCs have a wide range of potential uses from drug testing and in vitro disease modelling to personalized cell therapies for patients. While viral methods for reprogramming factor delivery have been traditionally preferred due to their high efficiency, it is now possible to generate iPSCs using nonviral methods at similar efficiencies. We developed a robust reprogramming strategy that combines episomal plasmids and the use of commercially available animal free reagents that can be easily adapted for the GMP manufacture of clinical grade cells.
    Methods in molecular biology (Clifton, N.J.) 02/2015;
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    ABSTRACT: Human induced pluripotent stem cells (hiPSCs) provide a platform for studying human disease in vitro, increase our understanding of human embryonic development, and provide clinically relevant cell types for transplantation, drug testing, and toxicology studies. Since their discovery, numerous advances have been made in order to eliminate issues such as vector integration into the host genome, low reprogramming efficiency, incomplete reprogramming and acquisition of genomic instabilities. One of the ways to achieve integration-free reprogramming is by using RNA-based Sendai virus. Here we describe a method to generate hiPSCs with Sendai virus in both feeder-free and feeder-dependent culture systems. Additionally, we illustrate methods by which to validate pluripotency of the resulting stem cell population.
    Methods in molecular biology (Clifton, N.J.) 02/2015;
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    ABSTRACT: Stem cells have the unique properties of self-renewal and multipotency (producing progeny belonging to two or more lineages). Induced pluripotent stem (iPS) cells can be generated from somatic cells by simultaneous expression of pluripotent factors (Oct3/4, Klf4, Sox2, and c-Myc). They share the same properties as embryonic stem (ES) cells and can differentiate into several tissue cells, i.e., neurons, hematopoietic cells, and liver cells. Therefore, iPS cells are suitable candidate cells for regenerative medicine and analyses of disease mechanisms.The liver is the major organ that regulates a multitude of metabolic functions. Hepatocytes are the major cell type populating the liver parenchyma and express several metabolic enzymes that are necessary for liver functions. Although hepatocytes are essential for maintaining homeostasis, it is difficult to alter artificial and transplanted cells because of their multifunctionality, donor shortage, and immunorejection risk. During liver development, hepatic progenitor cells in the fetal liver differentiate into both mature hepatocytes and cholangiocytes. As hepatic progenitor cells have bipotency and high proliferation ability, they could present a potential source for generating transplantable cells or as a liver study model. Here we describe the induction and purification of hepatic progenitor cells derived from human iPS cells. These cells can proliferate for a long term under suitable culture conditions.
    Methods in molecular biology (Clifton, N.J.) 02/2015;
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    ABSTRACT: Somatic reprogramming to generate induced pluripotent stem cells, or iPSC, is a powerful tool in developmental biology, disease modeling, and regenerative medicine. microRNAs have been shown to regulate many key pathways in iPSC induction. Here we describe a microRNA mimic enhanced somatic reprogramming process starting from mouse embryonic fibroblast isolation to iPSC induction to colony derivation and characterization.
    Methods in molecular biology (Clifton, N.J.) 02/2015;
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    ABSTRACT: The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) using a combination of defined transcription factors has become one of the most widely used techniques in stem cell biology. A critical, early event in iPSC reprogramming is the induction of the endogenous transcription factor network that maintains pluripotency in iPSCs. Here we describe using a transgenic, conditional Oct4-Cre construct to investigate the spatial and temporal induction of endogenous Oct4 expression during the reprogramming of mouse fibroblasts into iPS cells.
    Methods in molecular biology (Clifton, N.J.) 02/2015;
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    ABSTRACT: The loss of neurological function is closely related to axonal damage. Neurofilament subunits are concentrated in neurons and axons and have emerged as promising biomarkers for neurodegeneration. Electrochemiluminescence (ECL) based assays are known to be of superior sensitivity and require less sample volume than conventional ELISAs. Here, we describe a highly sensitive ECL based immunoassay for quantification of neurofilament light chain (NfL) in blood and CSF.
    Methods in molecular biology (Clifton, N.J.) 02/2015;
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    ABSTRACT: The laboratory rat (Rattus norvegicus) is now on the leading edge used as a laboratory model system to study pharmacology, toxicology, immunology, nutrition, behavior, and numerous other topics. Therefore, generation of rat induced pluripotent stem cells (iPSCs) through somatic cells reprogramming is a powerful tool for establishing in vitro disease model, development of new protocols for treatment of different diseases, and creating transgenic rat models. Here, we describe a simple adopted protocol for establishing rat iPSCs from different types of somatic cells including rat primary ear fibroblast (PEF) and primary bone marrow cells (BMC).
    Methods in molecular biology (Clifton, N.J.) 02/2015;
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    ABSTRACT: This protocol describes the efficient isolation of peripheral blood mononuclear cells from circulating blood via density gradient centrifugation and subsequent generation of integration-free human induced pluripotent stem cells. Peripheral blood mononuclear cells are cultured for 9 days to allow expansion of the erythroblast population. The erythroblasts are then used to derive human induced pluripotent stem cells using Sendai viral vectors, each expressing one of the four reprogramming factors Oct4, Sox2, Klf4, and c-Myc.
    Methods in molecular biology (Clifton, N.J.) 02/2015;
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    ABSTRACT: In this report, we introduce a standard protocol for stem cell self-renewal in vitro. Both fundamental and major procedures of stem cell manipulation, which are required for somatic cell coculture and self-renewal, are briefly described since they are important for stabilization and data normalization. In this chapter, information on the basic preparation of stem cell culture such as labware washing, equipment sanitization, microbe control, and mycoplasmosis prevention is provided. In addition, protocols for cell retrieval and preservation, proliferation assays, and basic manipulation techniques for the coculture of stem cells with somatic cells are described.
    Methods in molecular biology (Clifton, N.J.) 02/2015;
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    ABSTRACT: Transposon mutagenesis is a genetic process that involves the random insertion of transposons into a genome resulting in the disruption of function of the genes in which they insert. Identification of the insertion sites through DNA sequencing allows for the identification of the genes disrupted and the creation of "libraries" containing a collection of mutants in which a large number of the nonessential genes have been disrupted. These mutant libraries have been a great resource for investigators to understand the various biological functions of individual genes, including those involved in metabolism, antibiotic susceptibility, and pathogenesis. Here, we describe the detailed methodologies for constructing a sequence defined transposon mutant library in both Staphylococcus aureus and S. epidermidis using the mariner-based transposon, bursa aurealis.
    Methods in molecular biology (Clifton, N.J.) 02/2015;
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    ABSTRACT: Many methods exist to extract DNA from bacteria. Indeed, there is no shortage of kits available from manufacturers that allow for isolation of highly purified DNA. However, for many applications samples do not need to be extremely pure (i.e., free of contaminating proteins or RNA). Furthermore, for quick genetic screening, it is often useful to have a rapid and inexpensive option for DNA isolation from small samples. For these occasions, the method found in this chapter provides a cost-efficient, yet rapid, isolation of DNA.
    Methods in molecular biology (Clifton, N.J.) 02/2015;
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    ABSTRACT: Pulse Field Gel Electrophoresis (PFGE) is a powerful genotyping technique used for the separation of large DNA molecules (entire genomic DNA) after digesting it with unique restriction enzymes and applying to a gel matrix under the electric field that periodically changes direction. PFGE is a variation of agarose gel electrophoresis that permits analysis of bacterial DNA fragments over an order of magnitude larger than that with conventional restriction enzyme analysis. It provides a good representation of the entire bacterial chromosome in a single gel with a highly reproducible restriction profile, providing clearly distinct and well-resolved DNA fragments.
    Methods in molecular biology (Clifton, N.J.) 02/2015;
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    ABSTRACT: There is increasing evidence that exposure to air pollutants is associated with human disease and may act through epigenetic modification of the nuclear genome, but there have been few publications describing their impact upon the mitochondrial genome. Mitochondrial DNA may be more susceptible to pollutant-induced changes via increased oxidative stress in the cell, and therefore this field of research is of growing interest. Many techniques employed to study DNA methylation of the nuclear genome are also applicable to mitochondrial epigenetic studies. In this chapter, we describe a protocol for the isolation of mitochondrial DNA from peripheral blood samples and the analysis of 5-methylcytosine content by bisulfite pyrosequencing.
    Methods in molecular biology (Clifton, N.J.) 02/2015; 1265.
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    ABSTRACT: Familial transthyretin amyloidosis (ATTR) is an autosomal dominant protein-folding disorder caused by over 100 distinct mutations in the transthyretin (TTR) gene. In ATTR, protein secreted from the liver aggregates and forms fibrils in target organs, chiefly the heart and peripheral nervous system, highlighting the need for a model capable of recapitulating the multisystem complexity of this clinically variable disease. Here, we describe detailed methodologies for the directed differentiation of protein folding disease-specific iPSCs into hepatocytes that produce mutant protein, and neural-lineage cells often targeted in disease. Methodologies are also described for the construction of multisystem models and drug screening using iPSCs.
    Methods in molecular biology (Clifton, N.J.) 02/2015;