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Past Issue in 2015
Volume: 5, Issue: 10
Cancer Biology
Proximity Ligation Assay (PLA) to Detect Protein-protein Interactions in Breast Cancer Cells
Protein-protein interaction networks provide a global picture of cellular function and biological processes, and the dysfunction of some interactions causes many diseases, including cancer. The in situ proximity ligation assay (PLA) is a powerful technology capable of detecting the interactions among proteins in fixed tissue and cell samples. The interaction between two proteins is detected using the corresponding two primary antibodies raised in different species. Species-specific secondary antibodies (PLA probes), each with a unique short DNA strand attached to it, bind to the primary antibodies. When the PLA probes are in close proximity (<40 nm), the DNA strands can interact through a subsequent addition of two other circle-forming DNA oligonucleotides. Several-hundredfold replication of the DNA circle can occur after the amplification reaction, and a fluorescent signal is generated by labelled complementary oligonucleotide probes. Therefore, each detected signal is visualized as an individual fluorescent dot, which can be quantified and assigned to a specific subcellular location based on microscopy images. This revolutionary technique enables us to study the protein complex formation with high specificity and sensitivity compared to the other traditional methods, such as co-immunoprecipitation (Co-IP).
Genome-Wide siRNA Screen for Anti-Cancer Drug Resistance in Adherent Cell Lines
The expression of genes is frequently manipulated in cell lines to study their cellular functions. The use of exogenous small Interfering RNAs (siRNAs) is a very efficient technique to temporarily downregulate the expression of genes of interest [reviewed by Hannon and Rossi (2004)]. A genome-wide siRNA library allows the user to study both the effect of each individual gene on a particular cell phenotype in a high throughput manner and also assess its phenotypic effect relative to all other genes targeted. Several factors that potentially influence the outcome of a screen need to be considered when performing a large siRNA screen (Jiang et al., 2011). Here we present a detailed protocol for a genome-wide screen to identify genes involved in anti-cancer drug resistance using the human siGENOME library from Dharmacon. In this protocol, we focus on resistance to treatment with the Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor (EGFR-TKI) erlotinib in the lung cancer cell line PC9, which is exquisitely sensitive to EGFR-TKIs (de Bruin et al., 2014). This protocol can be used for other cell lines and other drug treatments, as we expand in the Notes below.
Immunology
Murine Liver Myeloid Cell Isolation Protocol
In homeostasis, the liver is critical for the metabolism of nutrients including sugars, lipids, proteins and iron, for the clearance of toxins, and to induce immune tolerance to gut-derived antigens. These functions predispose the liver to infection by blood-borne pathogens, and to a variety of diseases ranging from toxin and medication-induced disorders (CCl4, acetaminophen) to metabolic disorders (steatohepatitis, alcoholic liver disease, biliary obstruction, cholestasis) or autoimmunity. Chronic liver injury often progresses to life threatening fibrosis and can end in liver cirrhosis and hepatocellular carcinoma (Pellicoro et al., 2014). The liver contains parenchymal cells or hepatocytes that make up the majority of hepatic cells. It also contains non-parenchymal structural cells such as sinusoidal endothelial cells and a large number of non-parenchymal innate immune cells, mainly monocytes, neutrophils, macrophages, DCs, NK and NKT cells that can trigger an adaptive immune response in the case of infections or other pathogenic insults (Jenne and Kubes, 2013). How this immune response is regulated determines the extent of acute and chronic liver injury (Stijlemans et al., 2014). In this context, liver macrophages have been demonstrated to play central but divergent (from initiating to resolving) functions in liver injury (Sica et al., 2014). It has become clear in the last years that hepatic macrophages consist of two classes, tissue-resident macrophages, the Kupffer cells (KCs) originating from yolk sac/fetal liver progenitors and tissue-infiltrating macrophages originating from bone marrow-derived Ly6CHi monocytes (Jinhoux and Jung, 2014; Tacke and Zimmerman, 2014). Distinguishing the activities of KCs from those of monocyte-derived macrophages during liver injury or repair is currently a frontline research topic in the macrophage field. Indeed, considering that clinical management of liver failure remains problematic, a better understanding of the immune mechanisms regulating liver injury is expected to allow the development of new therapeutic modalities. Here, we describe an isolation technique for liver non-parenchymal polymorphonuclear (PMN) and mononuclear myeloid cells permitting their molecular and functional characterization.
Isolation of Particles of Recombinant ASC and NLRP3
NLRP3 inflammasome is a multiprotein complex responsible for the activation of inflammatory caspase-1, resulting in processing and release of pro-inflammatory cytoquines IL-1β and IL-18 (Schroder and Tschopp, 2010). This inflammasome is composed of the sensor protein NLRP3 connected to caspase-1 through the adaptor protein ASC (apoptosis-associated speck-like protein with a caspase-recruitment domain) (Schroder and Tschopp, 2010). We and others have reported that upon inflammasome activation functional oligomeric inflammasome particles of NLRP3 and ASC were released from cells, acting as danger signals to amplify inflammation by promoting the activation of caspase-1 extracellularly (Baroja-Mazo et al., 2014; Franklin et al., 2014). Studying the extracellular function of oligomeric ASC and NLRP3 inflammasome particles was possible by purification of recombinant particles of ASC or the constitutively activated NLRP3 mutant associated with cryopyrin-associated periodic syndromes (CAPS, mutation p.D303N), both tagged with the yellow fluorescent protein (YFP) and expressed in HEK293 cells. The purification process was facilitated by the fact that expression of recombinant ASC or mutant NLRP3 in HEK293 cells resulted in their spontaneous aggregation into specks (Baroja-Mazo et al., 2014) and the protocol was originally adapted from Fernandes-Alnemri and Alnemri (2008).
Microbiology
Human, Bacterial and Fungal Amplicon Collection and Processing for Sequencing
Sequencing taxonomic marker genes is a powerful tool to interrogate the composition of microbial communities. For example, bacterial and fungal community composition can be evaluated in parallel using the 16S ribosomal RNA gene for bacteria or the internal transcribed spacer region in fungi. These are conserved regions that are universal to a taxonomic clade, yet have undergone some degree of evolution such that different lineages can be differentiated. Conserved regions are used for design of universal priming sites that allow amplification of the marker gene out of a mixed microbial community. Here, we describe our standard operating procedure to collect and sequence 16S rRNA and ITS1 amplicons from human skin. We use the 16S rRNA V1-V3 region for skin samples, as it has greater power for classifying common staphylococci in the skin. This protocol is adapted for 454 pyrosequencing of amplicons.
Determining the Relative Fitness Score of Mutant Viruses in a Population Using Illumina Paired-end Sequencing and Regression Analysis
Recent advances in DNA sequencing capacity to accurately quantify the copy number of individual variants in a large and diverse population allows in parallel determination of the phenotypic effects caused by each genetic modification. This systematic profiling approach is a combination of forward and reverse genetics, which we refer to as quantitative high-resolution genetics (qHRG). This protocol describes how to determine the relative fitness score of each variant compared to wild type (WT) virus based on its frequency determined by Illumina sequencing. Random mutagenesis techniques will be used to introduce randomization at each codon position of the targeted region, thereby generating a comprehensive input mutant library with substitutions at each position of interest (Qi et al., 2014; Wu et al., 2014a; Wu et al., 2014b). After selection, each selected library will be sequenced by Illumina paired-end sequencing and the frequency of each mutation will be determined. Based on the change in frequency, the relative fitness score of each mutant can be calculated with regression analysis.
Molecular Biology
RNA-binding Protein Immunoprecipitation (RIP) to Examine AUF1 Binding to Senescence-Associated Secretory Phenotype (SASP) Factor mRNA
Immunoprecipitation and subsequent isolation of nucleic acids allows for the investigation of protein:nucleic acid interactions. RNA-binding protein immunoprecipitation (RIP) is used for the analysis of protein interactions with mRNA. Combining RIP with quantitative real-time PCR (qRT-PCR) further enhances the RIP technique by allowing for the quantitative assessment of RNA-binding protein interactions with their target mRNAs, and how these interactions change in different cellular settings. Here, we describe the immunoprecipitation of the RNA-binding protein AUF1 with several different factors associated with the senescence-associated secretory phenotype (SASP) (Alspach and Stewart, 2013), specifically IL6 and IL8. This protocol was originally published in Alspach et al. (2014).
Plant Science
Establishment of a Symbiotic in vitro System between a Green Meadow Orchid and a Rhizoctonia-like Fungus
Symbiotic orchid seed germination in an in vitro system allows the growth of mycorrhizal protocorms and plantlets for scientific purposes. Orchids in nature need to establish a mycorrhizal symbiosis with fungal partners to germinate and develop into adult plants. Here we present a protocol for symbiotic germination of the terrestrial Mediterranean green meadow orchid Serapias vomeracea. The fungal symbiont Tulasnella calospora (T. calospora) (Basidiomycetes, Cantharellales) was chosen because of its common occurrence (Girlanda et al., 2011), its ability to grow in culture and compatibility in germination assays. T. calospora is one of the most common rhizoctonia-like fungi associated with terrestrial as well as epiphytic orchids.
Plant Materials and Growth Conditions of Japanese Morning Glory (Ipomoea nil cv. Violet)
Japanese morning glory (Ipomoea nil) is a summer annual vine that typically produces ephemeral flowers. This plant has been used extensively to investigate flowers, including studies on flowering, flower color, and petal senescence. Here we describe the materials and optimal growth conditions used to grow the Japanese morning glory cultivar, “Violet”, in a growth chamber. Violet plants are transformable with Agrobacterium (Rhizobium), and we have used this cultivar to examine petal senescence (Shibuya et al., 2009; Shibuya et al., 2014).
Histochemical Detection of Zn in Plant Tissues
Accumulation of metals in plant tissues, and occasionally, different cells of the same tissue, may be highly non-uniform (Seregin and Kozhevnikova, 2008). Easy-to-use histochemical methods may greatly help to investigate the distribution and accumulation of metals within and among plant tissues, and also provide information on their subcellular localization (Seregin and Kozhevnikova, 2011). The histochemical techniques of zinc (Zn) visualization are based on the formation of the blue-colored complex of Zn with the metallochrome indicator Zincon (C20H15N4NaO6S), or the green-fluorescent complex with Zinpyr-1 (C46H36Cl2N6O5) (Seregin et al., 2011; Seregin and Kozhevnikova, 2011). A method for histochemical Zn detection in plant tissues using Zinpyr-1 was first proposed by Sinclair et al. (2007), and later modified by Seregin et al. (2011), and Seregin and Kozhevnikova (2011). Histochemical data supplement the results of quantitative analysis, thus allowing a detailed study of the distribution, accumulation, and translocation pathways of Zn within the plant, which are important topics in modern plant physiology. These histochemical techniques have been successfully applied in different plant species, for example Zea mays (Seregin et al., 2011), Noccaea caerulescens and Thlaspi arvense (Kozhevnikova et al., 2014a), Capsella bursa-pastoris and Lepidium ruderale (Kozhevnikova et al., 2014b), in which Zn was detected in different root and shoot tissues. Here, we present the full staining protocols for these methods, developed or modified in our lab (Seregin and Kozhevnikova, 2011; Kozhevnikova et al., 2014a; Kozhevnikova et al., 2014b).
Pyrosequencing Approach for SNP Genotyping in Plants Using a M13 Biotinylated Primer
Single Nucleotide Polymorphisms (SNPs), which constitute single base-pair variations in the DNA sequence, are the most abundant molecular markers in plant and animal genomes. They are becoming the markers of choice for genotyping in all fields of molecular biology, as they are easily prone to automation and high throughput, for example through pyrosequencing. This technology is accurate, flexible and can be easily automated. However, the need for primers labelled with biotin, promptly rise the cost of any methodology employing a pyrosequencing approach. In this protocol we described an improved, efficient, reliable and cost-effective pyrosequencing protocol, based on a universal M13 biotinylated primer, for SNP genotyping in plants.
Fluorescence-based CAPS Multiplex Genotyping on Capillary Electrophoresis Systems
Recent advances in next-generation sequencing techniques allow the detection of a large number of SNPs and their use in a high throughput manner. However, Cleaved Amplified Polymorphic Sequences (CAPSs) still play a significant role as complement to other high throughput methods for SNP genotyping. Therefore, new methods focusing on the acceleration of this type of markers are highly desirable. The combination of the classical CAPS technique and a M13-tailed primer multiplexing assay was used to develop an agarose gel free protocol for the analysis of SNPs via restriction enzyme digestion. PCR products were fluorescence labeled with a universal M13 primer and subsequently digested with the appropriate restriction endonuclease. After mixing differently labeled products, they were detected on a capillary electrophoresis system. This method allows the cost-effective genotyping of several SNPs in a multiplexed manner at an overall low cost in a short period of time. Additionally, this method could be efficiently combined with the simultaneous detection of SSRs at the same electrophoresis run resulting in a procedure well suited for marker-based selection procedures, genotyping of mapping populations and the assay of genetic diversity.
Measurement of Mitochondrial Respiration Rate in Maize (Zea mays) Leaves
Mitochondria play essential roles in plant growth and development as they host the oxidative phosphorylation pathways, tricarboxylic acid cycle and other important metabolisms. Disruption of mitochondrial functions frequently leads to embryo lethality. Moreover, mitochondria play roles in programmed cell death, pathogen and stress responses in plants. In contrast to animal mitochondria, plant mitochondria possess an additional electron transport pathway, the cyanide-resistant alternative pathway catalyzed by a single alternative oxidase (AOX). Unlike cytochrome pathway that is coupled to oxidative phosphorylation via proton translocation, electron transport from ubiquinol to AOX is non-phosphorylating. It releases the energy as heat. Chlorolab II liquid-phase oxygen electrode (Hansatech) is a high-precise Clark type oxygen electrode, which is equipped with the powerful WINDOWS software and could record the oxygen changes in real time. Its electrode disc comprises a central platinum cathode and a concentric silver anode. The electrode disc is connected to an electrode control unit which applies a small polarising voltage between the platinum and silver electrodes. In the presence of oxygen, a small current is generated proportional to oxygen content in the sample. It could respond sensitively and rapidly to small changes of oxygen content in the sample. This protocol describes how to measure the mitochondrial total respiration rate, cytochrome pathway capacity as well as alternative pathway capacity in maize leaves with chlorolab II oxygen electrode.
Transient Transformation of Artemisia annua
Transient transformation of Artemisia annua does not depend on chromosomal integration of heterologous DNA, and recombinant DNA can be introduced into plant cells via Agrobacterium aided by vacuum. The leaves of 7th and 8th internode from 4-week-old seedlings were chosen as explants, a vacuum system was applied to facilitate agrobacteria into plant cells, the co-cultivation was in the dark at 25 °C for 36-72 h, then GUS or GFP maker genes were used for testing the efficiency of the transformation. The method is used for quick transferring of genes into Artemisia annua (A. annua) by transient transformation.