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Past Issue in 2014
Volume: 4, Issue: 6
Biochemistry
Cellular Translational Reporter Assay
The method described here allows measuring the effect of exogenously introduced modifications to in vitro-transcribed mRNA on the translation in cells. Using cells derived from knockout mice and control littermates, this method enables to compare the results in the presence or absence of specific gene products. In our lab, we used this protocol to check whether the exogenous addition of 5’ capping and 2’-O methylation to in vitro-mRNA affects the translational efficiency. Here we describe the details of our experiments.
Synthesis of the Adenosine A2A Receptor Fluorescent Agonist MRS5424
MRS5424 is a functional fluorescent agonist for the adenosine A2A receptor (A2AR) in which the fluorescent dye Alexa Fluor 532 is covalently attached to the A2AR agonist 2-[[2-[4-[2-(2-aminoethyl)-aminocarbonyl]ethyl]phenyl]ethylamino]-5'-N-ethyl-carboxamidoadenosine (APEC). This easy-to-synthesize new A2AR fluorescent ligand was shown to be extremely useful for determining the binding kinetic constants of A2AR in a real-time mode (Fernandez-Duenas et al., 2012). In addition, this fluorescent A2AR ligand is compatible with ligand-receptor interaction studies using fluorescent plate readers. Finally, it is important to mention that even though the sensitivity of this A2AR fluorescent ligand may not be as high as that observed for the marketed A2AR radioactive compounds, the use of such fluorescent derivative may have some advantages over radioactive probes, for example its safe delivery, manipulation and disposal, the short signal acquisition times, the feasibility to automate and to miniaturize, and finally its cost.
Cancer Biology
In vitro Regulatory T cells Differentiation From Naïve T Cells
In the past years, a subset of regulatory T cells (Tregs) expressing CD4, CD25 and the transcription factor FoxP3 has gained considerable attention as key regulators of T-cell tolerance and homeostasis (Sakaguchi, 2004). This population of T cells is specifically engaged in the maintenance of immune self-tolerance and the control of aberrant immune responses to foreign antigens. Remarkably, regulatory T cells have been implicated in tumor cell evasion of immune responses (Curiel et al., 2004; Zou, 2006) by suppressing T cell mediated antitumor immunity. The study of the signals that promote the differentiation of this suppressive population in the tumor microenvironment has become a central issue. Here we described a detailed method to in vitro differentiate Tregs using tumor cells conditioned media from mouse naïve T cells and to identify them based on their specifics markers (Dalotto-Moreno et al., 2013).
Immunology
Measurement of CD8 and CD4 T Cell Responses in Mouse Lungs
Study of the adaptive immune response to a viral challenge in an animal model often includes analysis of the T cell response. Here we discuss in detail the methods that are used to characterize the CD8 and CD4 T cell response following viral challenge in the lung.
Microbiology
RNA Isolation and Northern Blot Analysis
The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA in a sample. With northern blotting it is possible to observe particular gene expression levels during differentiation, morphogenesis, as well as abnormal or diseased conditions. Here, we examine ATF3, ATF4, and GADD153 gene expression profiles by northern blot in Vero cells and H1299 cells after IBV infection. RNA was extracted in IBV (infectious bronchitis virus) infected cells and electrophoresis was used to separate the RNA sample. RNA was transferred from the electrophoresis gel to the blotting membrane by capillary transfer. Specific mRNA was detected with hybridization probes complementary to part of target sequence. The probes were prepared by RT-PCR and labeled by digoxigenin (DIG) using DIG labeling kit.
Catalase Activity Assay in Candida glabrata
Commensal and pathogenic fungi are exposed to hydrogen peroxide (H2O2) produced by macrophages of the host. Pathogenic fungi counteract the harmful effects of H2O2 with the enzyme catalase (EC 1.11.1.6), which decomposes two molecules of H2O2 to two molecules of H2O and O2. Contribution of antioxidant systems on fungal virulence is actively studied. Measurement of catalase activity can contribute to the elucidation of the factors that influence the regulation of this pivotal enzyme. Here we describe a simple spectrophotometric method in which the activity of catalase is measured in total yeast extracts. Decomposition of H2O2 by the yeast extract is followed by the decrease in absorbance at 240 nm. The difference in absorbance through time (ΔA240) is inferred as the measure of catalase activity.
Helicase Assays
Helicases are a class of enzymes which are motor proteins using energy derived from ATP hydrolysis to move directionally along a nucliec acid phosphodiester backbone (such as DNA, RNA and DNA-RNA hybrids) and separate two annealed nucleic acid strands. Many cellular processes, such as transcription, DNA replication, recombination and DNA repair involve helicase activity. Here, we provide a protocol to analyze helicase activities in vitro. In this protocol, the DNA helicase protein Merkel cell polyomavirus large T-antigen was expressed in the mammalian cell line HEK293 and immoblized on an IgG resin. The helicase assay is performing while the protein is immoblized on IgG resin.
Virus Infection and Titration of SARS-CoV in Mouse Lung
Two critical steps when investigating an animal model of a virus infection are consistently successfully infecting animals and accurately determining viral titers in tissue throughout the course of infection. Here we discuss in detail how to infect mice with SARS-CoV and then quantify the titer of virus in the lung.
Western Blotting for Staphylococcus aureus AgrA
Staphylococcus aureus has a quorum sensing system to regulate the expression of various virulence factors, which is exerted by the agr locus that encodes agrBDCA and a regulatory RNA called RNAIII. AgrB, AgrD, and AgrC proteins are involved in producing and recognizing extracellular quorum sensing molecules and transduce the signal by altering the phosphorylation status of AgrA, which is a positive transcription factor, to regulate cytolysin genes as well as the RNAIII gene. RNAIII regulates the expression of various virulence genes. Expression of the agr locus has been examined in depth at the transcriptional level, but investigations of translational expression are limited, because immunoglobulin G used to detect a specific protein highly reacts to S. aureus protein A. Here, we report a method to detect AgrA that is the transcription factor encoded by the agr regulatory system. Although this is a specific protocol for western blotting of S. aureus AgrA protein, it can also be used for other S. aureus proteins by using the appropriate antibody.
Human Astrovirus Propagation, Purification and Quantification
Astrovirus are small, nonenveloped, single-stranded RNA viruses that cause diarrhea in a wide variety of mammals and birds. Here, we describe astrovirus propagation, purification and titration. The Caco-2 human intestinal adenocarcinoma cell line is most widely used for studying astrovirus, although other cell lines, such as 293, T84 and LLC-MK2 can be used for propagation. However, Caco2 cells are desirable for their ability to form a differentiated intestinal epithelium, mimicking the human intestine and providing a realistic model for astrovirus growth and propagation.
Determination of Mutation Frequency During Viral DNA Replication
This protocol is a simple method for evaluating mutation frequency during African swine fever virus (ASFV) replication, although it could be used also for other DNA viruses (poxvirus, herpesvirus, mimivirus, etc) with minor modifications. In the original Carrascosa et al. (1982), the protocol was carried out with two cloned viruses, BA71Vc (a purified clone from BA71V wild type strain) and vΔpolX (lacking the reparative polymerase, pol X, gene), and two different cell types that can be infected by ASFV, Vero cells and swine macrophages. To facilitate the sequence comparison, a genome fragment containing the B646L gene was amplified by PCR and blunt-end cloned. This gene codes for the major capsid protein (p72) and multiple sequences can be found in the database, so the mutations found could be compared with natural gene variations. The cloned fragment can be either sequenced directly from bacteria colonies or from miniprep purified DNA.
Measuring Genetic Robustness in Vesicular Stomatitis Virus
Genetic robustness is the ability of a genome to incorporate mutations with the result of no fitness changes. Thus, more robust viruses have an increased neutral mutation rate. This property is particularly important in RNA viruses due to their high mutation rates. The most direct way of measuring robustness in vesicular stomatitis virus (VSV) is to carry out clonal analysis of populations: randomly isolating individual VSV strains (plaques), measuring the fitness of each one and generating fitness distributions (Novella et al., 2010). A second possibility is to carry out multiple replicates of repeated plaque-to-plaque passages, determining fitness in progeny populations and generating fitness distributions (Novella et al., 2010). Depending on the expected differences, the former may require hundreds of determinations, while the latter may require tens of determinations. A third approach consists of increasing the mutation rate of populations under analysis to magnify any differences that may exist and, instead of measuring fitness, measuring survival (Novella et al., 2013). One caveat of this method is that changes in survival can also be explained by changes in polymerase fidelity. For that reason, it is important to perform complementary experiments, in this case quantifying mutant frequency.
Fitness Determinations in Vesicular Stomatitis Virus
Fitness is defined as the overall replicative ability. Testing whether a mutation (or combination of mutations) has an effect on fitness often relays on determining virus production as a surrogate measurement. However, viruses do not usually replicate in a void, and evolutionary speaking, it is key to determine replicative ability compared to other viruses, e.g. the relative fitness. John Holland developed a method for vesicular stomatitis virus based on the use of a neutral genetic marker that allows to distinguish two competitors and to measure accurately the relative ratio between the two during competition (Holland et al., 1991). The marker is a mutation in the external G glycoprotein that has no effect on the virus other than conferring resistance to a monoclonal antibody, I1. To measure fitness a marked test strain is mixed with a reference unmarked strain and the mixture is allowed to infect a cell monolayer. Ratios before and after competition are measured by plaque assay in the presence and absence of I1 antibody, and changes in ratio give the fitness value.
Neuroscience
Isolation and Culture of Neurospheres for the Study of Pathogenesis of Prion Disease
Neurosphere contains neural stem cells that are capable of self-renewal and multilineage differentiation including neurons, astrocytes, and oligodendrocytes (Gage, 2000). Cell culture model using differentiated neurosphere cultures are suggested to be a valuable tool for studying the pathogenesis of prion disease at the cellular level (Iwamaru et al., 2013). This protocol describes the procedure for a culture of whole brain-derived neurospheres from newborn mouse brains. Neurosphere formation steadily occurs within a week from the cultures of neonatal whole brains and these cells have stem cell properties.
Neural Stem Cell Differentiation and Prion Infection
Prion diseases are transmissible, fatal, neurodegenerative diseases in human and animals. The molecular basis of neurodegeneration in prion diseases is largely unclear. Developing a cellular model capable of monitoring prion-induced cytotoxicity would be a promising approach for better understanding the prion pathogenesis. One candidate cellular assay is a model based on neurospheres, which contains neural stem cells (NSCs). Both undifferentiated and differentiated NSCs have been demonstrated to be permissive to prion infection, and prion-induced cytopathic changes in differentiated neruosphere cultures were reported (Iwamaru et al., 2013). This protocol describes the procedure to induce differentiation of NSCs from transgenic mice overexpressing prion protein (tga20 mice) into cultures susceptible for prion infection.
Adenosine A2A Receptor Ligand Binding Experiments by Using Real-time Single-cell FRET
We designed a fluorescence resonance energy transfer (FRET)-based approach to study the ligand binding constants of the adenosine A2A receptor (A2AR). Our assay is based in the interaction of a fluorescent A2AR agonist ligand (MRS5424) with an A2AR tagged with the cyan fluorescent protein (CFP) at the N-terminus (i.e. A2ARCFP) and expressed in living cells. Thus, upon fast superfusion of the A2ARCFP expressing cells with MRS5424, the ligand-receptor interaction is determined by single-cell FRET in a real-time mode. Accordingly, our approach allowed immediate ‘real-time’ readout of the ligand-receptor interaction, thus allowing kinetic binding experiments, a feature impossible to achieve using conventional radioisotope-labelled ligands. In addition, since our assay permitted the visual confirmation of receptor localization it also allowed localized saturation binding experiments.
Transpharyngeal Exposure of GnRH Neurons
The neurons secreting gonadotropin-releasing hormone (GnRH) control fertility in all mammalian species. However, investigations of this neuronal population are difficult as the cell bodies of the approximately 800 GnRH neurons are scattered through the basal forebrain ranging from the olfactory bulbs through to the base of the hypothalamus. While acute brain slice preparations enable the electrophysiological characteristics of these cells to be determined in vitro, their topography has made investigations in vivo extremely difficult. We detail here a surgical approach that allows GnRH neurons at the base of the hypothalamus to be assessed in vivo in the anesthetized mouse (Figure 1). This procedure enables electrical recordings to be made from neurons located on the ventral surface of the mouse brain (Constantin et al., 2013).