Springer protocols feed by Pharmacology/Toxicology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Target Validation in Mice by Constitutive and Conditional RNAi
We describe three strategies to control gene silencing in mice that can be applied to any transcript of interest. This shRNA based approach enables either i) constitutive body-wide knockdown, ii) cell type-specific knockdown controlled by Cre recombinase, or iii) inducible body-wide knockdown controlled by doxycycline. For reliable expression the shRNA vector of interest is inserted into a Rosa26 docking site of ES cells by a site-specific recombinase. These ES cells can then be used to generate shRNA transgenic mice. This technology enables the production of adult knockdown mice within 11 months for an expedite in vivo va...
Source: Springer protocols feed by Pharmacology/Toxicology - February 26, 2013 Category: Drugs & Pharmacology Source Type: news
Genetically Engineered Animal Models for In Vivo Target Identification and Validation in Oncology
In vitro approaches using human cancer cell lines aimed to identify and validate oncology targets, have pinpointed a number of key targets and signalling pathways which control cell growth and cell death. However, tumors are more than insular masses of proliferating cancer cells. Instead they are complex tissues composed of multiple distinct cell types that participate in homotypic and heterotypic interactions and depend upon each other for their growth. Therefore, many targets in oncology need to be validated in the context of the whole animal. This review provides an overview on how animal models can be generated and use...
Source: Springer protocols feed by Pharmacology/Toxicology - February 26, 2013 Category: Drugs & Pharmacology Source Type: news
The Neurosphere Assay Applied to Neural Stem Cells and Cancer Stem Cells
The discovery of neural stem cells (NSCs) in the mammalian brain has raised many expectations as these unique cells might recapitulate different neurological diseases, including brain tumors, both from a functional and molecular perspective. Proper in vitro culturing of NSCs has emerged as a critical methodological issue, given that it should preserve the in vivo features of NSCs, with particular emphasis on cell heterogeneity. At the same time, the methodology for NSC culturing should allow the production of large amounts of cells to be exploited not only for prospective clinical applications, but also for drug screening....
Source: Springer protocols feed by Pharmacology/Toxicology - February 26, 2013 Category: Drugs & Pharmacology Source Type: news
Tumor Spheroid-Based Migration Assays for Evaluation of Therapeutic Agents
Cell migration is a key hallmark of malignant cells that contributes to the progression of cancers from a primary, localized mass to an invasive and/or metastatic phenotype. Traditional methods for the evaluation of tumor cell migration in vitro generally employ two-dimensional (2D), homogeneous cultures that do not take into account tumor heterogeneity, three-dimensional (3D) cell-cell contacts between tumor and/or host cells or interactions with extracellular matrix proteins. Here we describe a 3D tumor spheroid-based migration assay which more accurately reflects the solid tumor microenvironment and can accommodate both...
Source: Springer protocols feed by Pharmacology/Toxicology - February 26, 2013 Category: Drugs & Pharmacology Source Type: news
Two-Dimensional vs. Three-Dimensional In Vitro Tumor Migration and Invasion Assays
Motility and invasion are key hallmarks that distinguish benign from malignant tumors, enabling cells to cross tissue boundaries, disseminate in blood and lymph and establish metastases at distant sites. Similar properties are also utilized by activated endothelial cells during tumor-induced angiogenesis. It is now appreciated that these processes might provide a rich source of novel molecular targets with the potential for inhibitors to restrain both metastasis and neoangiogenesis. Such therapeutic strategies require assays that can rapidly and quantitatively measure cell movement and the ability to traverse physiological...
Source: Springer protocols feed by Pharmacology/Toxicology - February 26, 2013 Category: Drugs & Pharmacology Source Type: news
A Cell Culture System That Mimics Chronic Lymphocytic Leukemia Cells Microenvironment for Drug Screening and Characterization
Chronic Lymphocytic Leukaemia (CLL) is an incurable disease that warrants new therapeutic treatments. CLL cells accumulate in the peripheral blood, in the bone marrow and in secondary lymphoid organs. Unlike circulating CLL cells, CLL cells resident in these last two compartments display high chemoresistance and proliferative capacity. Given the importance of the microenvironment in this disease, strategies that aim to develop new therapeutic agents need to consider this critical factor. Various cell culture conditions have been described that attempt to emulate either the different types of microenvironments in which CLL ...
Source: Springer protocols feed by Pharmacology/Toxicology - February 26, 2013 Category: Drugs & Pharmacology Source Type: news
Reverse Phase Protein Microarrays and Their Utility in Drug Development
The majority of human diseases, including cancer, are characterized by abnormal protein function. Proteins regulate virtually every cellular process and exhibit multiple kinds of post-translational modification that modulate expression levels and activation states, such as phosphorylation by protein kinases. Additionally proteins interact with each other in complex regulatory networks and signal transduction pathways modulated by feedback mechanisms. These pathways are disrupted in disease and altered by therapeutic drugs. Reverse phase protein microarray (RPMA) technology allows simultaneous measurement of numerous phosph...
Source: Springer protocols feed by Pharmacology/Toxicology - February 26, 2013 Category: Drugs & Pharmacology Source Type: news
Systematic Analysis of Complex Signal Transduction Pathways Using Protein Fragment Complementation Assays
The state of a signal transduction pathway can be assessed by monitoring a given point, or a signaling node, of interest within that pathway by high content analysis. The activity at these nodes may be correlated with the general effect on cell number and morphology at the same time. Here we describe a method to analyze protein-protein interactions by protein fragment complementation assays. Complex signal transduction pathways become accessible by looking at them in their native cellular context, with all competitive and feedback mechanisms in place. Analyzing protein-protein interactions directly makes this method widely...
Source: Springer protocols feed by Pharmacology/Toxicology - February 26, 2013 Category: Drugs & Pharmacology Source Type: news
Validating Pharmacological Disruption of Protein–Protein Interactions by Acceptor Photobleaching FRET Imaging
Proteins are the major targets of drug discovery and many of the new drugs are designed to exert their effect by disrupting protein-protein interactions. Validation of the inhibition of molecular interactions is generally done by biochemical methods, however, these are often not feasible when the interaction is not stable enough. Fluorescence resonance energy transfer (FRET) is an excellent tool for determining direct molecular interactions between two molecules in the cell membrane or inside cells in their natural state. Although originally established as a flow cytometric approach, FRET has been adapted for microscopy, a...
Source: Springer protocols feed by Pharmacology/Toxicology - February 26, 2013 Category: Drugs & Pharmacology Source Type: news
Structure-Based Target Druggability Assessment
The focus of this chapter is on the important concepts behind the in silico techniques that are used today to assess target druggability. The first step of the assessment consists of finding cavity space in the protein using 2D and/or 3D topological concepts. These concepts underlie the geometry and energy-based pocketfinder algorithms. Analysis pursues on the physico-chemical complementarity between the binding site and the drug like molecule. Geometrical and molecular flexibility aspect are also included in this assessment. The presence of hot interaction spots are shown to be particularly important for targeting protein...
Source: Springer protocols feed by Pharmacology/Toxicology - February 26, 2013 Category: Drugs & Pharmacology Source Type: news
PARP Inhibition as a Prototype for Synthetic Lethal Screens
Although DNA damaging chemotherapy and radiation therapy remain the main stay of current treatments for cancer patient, these therapies usually have toxic side effect and narrow therapeutic window. One of the challenges in cancer drug discovery is how to identify drugs that selectively kill cancer cells while leaving the normal cell intact. Recently, synthetic lethality has been applied to cancer drug discovery in various settings, and has become a promising approach for identifying novel agents for the treatment of cancer. A prototypical example is the synthetic lethal interaction between PARP inhibition and BRCA deficien...
Source: Springer protocols feed by Pharmacology/Toxicology - February 26, 2013 Category: Drugs & Pharmacology Source Type: news
CellProfiler and KNIME: Open Source Tools for High Content Screening
We present and discuss the Open Source software CellProfiler for image analysis and KNIME for data analysis and data mining that provide software solutions which increase flexibility and keep costs low. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - February 26, 2013 Category: Drugs & Pharmacology Source Type: news
Integration of RNAi and Small Molecule Screens to Identify Targets for Drug Development
Cellular models for siRNA and small molecule high throughput screening have been widely used in the last decade to identify targets for drug discovery. As an example, we present a two-fold readout approach based on cell viability and multipolar phenotype. To maximize the discovery of potential targets and at the same time reduce the number of false positives in our dataset, we have combined focused and rationally designed custom siRNA libraries with small molecule inhibitor libraries. Here we describe a cellular model for centrosome amplification as an example of how to design and perform a multiple readout/multiple screen...
Source: Springer protocols feed by Pharmacology/Toxicology - February 26, 2013 Category: Drugs & Pharmacology Source Type: news
High-Throughput RNAi Screening for the Identification of Novel Targets
Gene silencing through RNA interference has provided researchers with an effective way to study gene function. High-throughput RNA interference (HT-RNAi) screening has further permitted researchers to identify functionally relevant mediators of cellular response on a large scale. These screens have greatly expedited the discovery of novel targets and pathway mediators. Here, we describe the methodology for performing HT-RNAi screening of HeLa cells transfected with short interfering RNA (siRNA) libraries in 384-well microplate format. Using this plate format, the HT-RNAi assay can be easily adapted to semi-automated or ful...
Source: Springer protocols feed by Pharmacology/Toxicology - February 26, 2013 Category: Drugs & Pharmacology Source Type: news
Western Blot Evaluation of siRNA Delivery by pH-Responsive Peptides
Gene silencing, via RNA interference (RNAi) technologies using small interfering RNA (siRNA), has been developed as an important tool for target identification and validation in drug discovery and has huge therapeutic potential. However, effective delivery into cells presents a major challenge to the use of siRNA. pH-responsive cell-penetrating peptides have attracted considerable attention in recent years as delivery vectors due to their ability to transport their cargos across the biological membrane and/or to promote endosomal escape and prevent lysosomal degradation. To evaluate the in vitro transfection efficiency of ...
Source: Springer protocols feed by Pharmacology/Toxicology - February 26, 2013 Category: Drugs & Pharmacology Source Type: news
