Presented are the first genome wide, arrayed guide RNA screening libraries for CRISPR-Cas9. These libraries have been designed to cover 17166 human and 20430 mouse genes. To test the efficiency, we selected gRNAs targeting genes from the GPI anchor protein pathway. We observed that over 95% of these gRNAs successfully induced DNA cleavage and consequently gene loss of function. These libraries offer the prospect for performing high-throughput screens for genes of considerable scientific and therapeutic value in mouse and human cells
Methods for Validating a Gene Editing Approach to Treating Brain Disease with Zinc Fingers, TALEs, and CRISPR/Cas9
Gene editing – the ability to precisely alter the DNA information in living cells – is transforming the study and treatment of human disease. Advances in techniques have enabled modifications ranging from the “scarless” correction of a single base pair to the deletion of entire chromosomes. In this webinar, Dr. David J. Segal from UC-Davis will discuss targetable nucleases and transcription factors based on zinc fingers, TALEs and CRISPR/Cas9 that can precisely modify the sequence or expression of any gene with high efficiency. The description of an artificial transcription factor designed in the Segal lab to treat the rare neurodevelopmental disorder Angelman Syndrome will highlight the considerable delivery challenges – and present some solutions – for using gene editing tools to alter genetic and epigenetic information in the brain.
For years, molecular cell biologists have relied on Western blotting as an indispensable tool for validating the manipulation of target genes. Whether the focus is on engineering of new recombinant proteins or functional studies based on gene knock-out or knock-in, measuring the expression level of target proteins is an integral step in any molecular genetics project. In this webinar, Dr. Jun Park (MilliporeSigma) will describe methods for quantitative Westerns, and will share general guidelines for optimizing Western blot immunodetection.
Understanding the Key Epigenetic Processes in Cellular Reprogramming and Pluripotency-Building Future Therapeutics
The discovery of a defined set of transcription factors that can induce reprogramming of somatic cells to pluripotent stem cells (iPSCs) has had an unprecedented impact on our view on future cell transplantation-based tissue repair and restoration of faulty physiological functions. Somatic cell reprogramming is a several weeks long process through which cells reach pluripotency, the developmental state similar to embryonic stem cells. This cascade of events and the epigenetic driving forces behind the phenomenon are very poorly understood. It is, however, crucial to understand the fine details of this process in order to comprehend the true properties of iPSCs and better tailor their future use as viable therapeutic tools.
In this webinar, two leading experts in the field of cellular reprogramming and pluripotency of somatic cells will share exciting examples of their research and recent tools developed that examine the key factors that influence the reprogramming process at the epigenome, transcriptome and proteome levels.
Cancer stem cells represent an attractive model for in vitro tumor modeling, especially for closer, physiological screening of new anticancer compounds. Although much progress has been made in culturing cancer stem cells, technical challenges persist in the identification and characterization of cell types in a heterogeneous cellular population. In this webinar, our panelist discusses emerging tools and strategies for the characterization of stem cells derived from heterogeneous populations and applications of this model system for drug discovery.
Using Cellular Reprogramming and Live Cell Identification to Advance Cardiac Disease Modelling
Long QT Syndrome 2 is a genetic disorder that leads to an increased risk of developing fatal arrhythmias. While current heterologous disease models have provided some insights for this disease in humans, understanding its true mechanisms requires more accurate humanized models.
Human induced pluripotent stem cells derived from patient cells provide a source to generate various disease models that were previously unattainable. Furthermore, the ability of these pluripotent stem cells to differentiate into cardiac fate provides a powerful method for creating and understanding inherited cardiac diseases.
This webcast will discuss research on stem cell derived cardiomyocytes and the use of a novel probe-based method for identifying cardiac subtypes based on live cell RNA detection, confirming their disease states, and use in subsequent drug treatment studies.
Finding the miRNAs that Matter with Microcapillary Flow Cytometry
To elucidate the biological roles of microRNAs (miRNAs) and validate their utility as biomarkers, robust methods are needed to profile large sample cohorts. In this webinar, you will learn how guava easyCyte™ microcapillary flow cytometers enable miRNA profiling with exceptional throughput and high signal-to-noise ratios. Specifically, the guava easyCyte™ flow cytometers enable the new SmartRNAplex™ miRNA profiling assays, which can measure up to 68 targets simultaneously in a single well... More...
The Message in the Haystack: Screening Live Stem Cells Using RNA Detection Probes
This online seminar will provide an overview of new tools for screening stem cells — in particular, the use of RNA detection probes to detect pluripotency gene expression in live embryonic and induced pluripotent stem cells by fluorescence microscopy without the need for manipulation of the cells.
Since their introduction in 2012, MilliporeSigma's SmartFlare™ RNA detection probes have been cited in numerous publications, for studying gene expression in individual, live cells. This technique is widely applicable in dissecting heterogeneous cell populations across multiple cell types and research areas, including oncology, immunology, and cardiovascular research.
In this webinar, Don Weldon (Lead Scientist, MilliporeSigma) will first provide an overview of the SmartFlare™ technology and its ability to study the gene expression of population subsets. Next, Dr. Harald Lahm (Head of the Laboratory of Experimental Surgery, German Heart Center Munich) describes the detection of various pluripotency genes in live, murine embryonic stem cells using SmartFlare probes. The same probes were also successfully applied to iPS cells of murine, human and porcine origin.
You will also learn about Dr. Lahm’s use of SmartFlare™ probes as a live screening tool to identify truly reprogrammed murine iPS cells derived from murine tail-tip fibroblasts in situ, based on their fluorescence intensity.
As the messenger of genetic information, RNA plays an important regulatory role in cell and tissue development as well as during disease progression. Understanding the best ways to work with RNA and the various RNA detection methods can help scientists advance our understanding of gene expression patterns and elucidate the roles of different genomic elements in cellular function and dysfunction. A variety of RNA detection methods—including Northern blotting, in situ hybridization, and reverse transcription polymerase chain reaction—are already available; however, each comes with its own advantages and limitations in signal sensitivity, specificity, and stability. Additionally, most often detection is carried out on lysed cell samples, so a significant amount of information is lost. This webinar will bring together two experts who will share their knowledge and expertise in a variety of RNA detection methodologies, including one that utilizes live cells.
During the webinar the panelists will:
Discuss different RNA detection methods
Highlight the unique challenges and benefits of detecting RNA in live cells
Present some of their current research using RNA detection techniques
Answer audience questions during the live webinar!
Live-cell sorting by RNA markers: Unlocking the potential of native cells for use in downstream assays
Sorting live cells based on intracellular RNA markers, followed by functional assays using the sorted cells, provides a unique opportunity to connect gene expression with cell function in heterogeneous cell populations, such as in tumors and immune cells.
Traditional live cell sorting, using antibodies against cell surface proteins or transfected reporter constructs, has several disadvantages, including poor cell recovery, compromised cell integrity and perturbed cellular pathways. The ability to sort live cells based on RNA expression overcomes this limitation and unlocks the potential to sort populations based on RNA biomarkers.
In this webcast, we describe a recently published method for sorting cells based on expression of specific RNA markers, without any transfection reagents or intrusive sample preparation1. You’ll learn how to perform RNA-based, live sorting of multiple cell types, including epithelial cells, mesenchymal cells, monocytes and macrophages. We will then show that the sorted cells retain their native biological functions by demonstrating their use in post-sort migration and bacterial phagocytosis assays.
Advances in Single-Cell Genomics: Live Cell RNA and Circulating miRNA Detection
Data presented in this webinar illustrates the value of live cell analysis at the single-cell level to identify differences in expression levels across populations of cells. The cells remain intact for downstream analysis. Our experts also discuss the use of SmartFlare RNA detection probes for the direct quantification of circulating miRNAs with rapid processing of blood plasma/serum, which is done without the use of enzymes. Using circulating miRNAs with established roles in cancer and quality control, we can accurately detect these miRNAs in plasma using a microplate fluorometer within an hour after plasma preparation.