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Light Sheet Fluorescence Microscopy

Posted by Guest Blogger on Jul 16, 2015 10:30:00 AM

This post was contributed by Jae Lee and Pantelis Tsoulfas of the Department of Neurological Surgery at the University of Miami.

The beginning of this century has seen some major advances in light microscopy, particularly related to the neurosciences.  These developments in microscopy coupled with techniques that make tissues transparent are enabling microscopes to visualize the cellular architecture of whole tissues in 3D with unprecedented detail.  One of these advances in microscopy has been light sheet fluorescence microscopy (LSFM). The underlying method was developed in 1902 by Richard Zsigmondy and Henry Siedentopf to enhance the microscopic resolution for studying colloidal gold (1).  The method was based on using a thin plane (sheet) of light generated by sunlight to observe single gold particles with diameters less than 4nm.

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Topics: Imaging, Fluorescent Proteins

Transgenic Organisms, Cas9 Gene Drives, and Appropriate Safeguards

Posted by Guest Blogger on May 22, 2015 12:58:12 PM

This post was contributed by Kevin Esvelt, a Wyss Technology Development Fellow at the Wyss Institute and Harvard Medical School.

Scientists making transgenic organisms with Cas9 should be aware of the potential hazards of creating “gene drives” capable of spreading through wild populations. Whereas most genomic changes impose a fitness cost and are eliminated by natural selection, gene drives distort inheritance in their favor and consequently can spread even when costly.

If even a single organism carrying a synthetic gene drive were to escape the laboratory, the drive could eventually spread through the entire wild population with unpredictable ecological effects. Because the consequences of such a mistake would necessarily extend far beyond the laboratory and seriously damage public trust in scientists, experiments involving potential gene drives should be conducted with extreme caution.

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Topics: Plasmid Technology, Lab Tips, CRISPR, CRISPR 101

Writing Scientific Manuscripts: Literature Searching, Reading, & Organizing

Posted by Guest Blogger on May 5, 2015 11:54:00 AM

This post was contributed by Johnna Roose. This post was originally published on Johnna's New Under The Sun Blog and is part of her larger tutorial series, A Beginner’s Guide to Writing Scientific Manuscripts.

Any scientific manuscript will require numerous other references to scientific literature to substantiate the facts upon which it builds. This means you have to become familiar with a body of literature related to the topic. Finding reliable references and sorting out what they mean is no small task. As a scientist, it is useful to make literature searching and reading a regular part of your routine. Set a goal to read a certain number of papers each week to keep up with the research in your area. When you are in ‘writing-mode’ for a grant or a scientific manuscript, the reading will likely be more intense, but it is a general good practice to keep up with the scientific literature a little bit at a time.

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Topics: Career, Science Communication

CRISPR 101: Non-Homologous End Joining

Posted by Guest Blogger on Apr 16, 2015 11:45:08 AM

This post was contributed by David Wyatt and Dale Ramsden, UNC at Chapel Hill.

One advantage to using the CRISPR/Cas system for genome engineering is the fact that Cas9 can be easily programmed to make a DNA double strand break (DSB) in the genome wherever the user chooses. After the initial cut, the next steps in the process involve repairing chromosomal DSBs. It is important to know that cells possess two major repair pathways  Non-Homologous End Joining (NHEJ) and Homology Directed Repair (HDR) – and how these pathways work, as this could be relevant when planning your experiment. This blog has previously considered the HDR pathway; below we’ll discuss NHEJ, and how it impacts what happens to Cas9-mediated DSBs in the genome.

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Topics: Genome Engineering, CRISPR, CRISPR 101

Rewiring Metabolic Circuitry with CRISPR RNA Scaffolds [Video]

Posted by Guest Blogger on Apr 7, 2015 12:21:00 PM

This post was contributed by Adam Chin-Fatt, a Ph.D. student at the University of Western Ontario. Adam summarizes Zalatan JG, et al.'s recent paper, "Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds." Adam has also created a video to help scientists visualize the concepts discussed in the paper.

The transcriptional control of multiple loci is deftly coordinated by the eukaryotic cell for the execution of many complex cellular behaviors, such as differentiation or metabolism. Our attempts to manipulate these cellular behaviors often fall short with the generation of various flux imbalances. The conventional approach has typically been to either systematically delete/overexpress endogenous genes or to introduce heterologous genes, but the trend of research has shifted in recent years toward tinkering with regulatory networks and multiplex gene control. However, these approaches are often met with the challenges of regulatory bottlenecks and their scope is limited by the lack of well characterized inducible promoters. Far removed from the bio-industry’s vision of ‘biofactories’, most successes in metabolic engineering have been limited to the overexpression of various metabolites in Escherichia coli or Saccharomyces cerevisiae with few techniques that are easily transferrable across host species or metabolic pathways. A new study takes us one step closer to the vision of metabolic biofactories by demonstrating the use of CRISPR-based RNA scaffolds to mimic natural transcriptional programs on multiple genes.

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Topics: Plasmid Technology, Genome Engineering, Synthetic Biology, CRISPR

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