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Mapping the 4D nucleome with CRISPR/Cas9

Posted by Mary Gearing on Aug 11, 2015 10:30:00 AM

It seems that there’s a new CRISPR advance or technique published every week! One of the newest applications is a colorful system that uses fluorescently labeled Cas9 to label multiple genomic loci in live cells. While other systems can be used to label loci, such as fluorescence in situ hybridization (FISH) or fluorescently labeled TALEs, CRISPR/Cas9’s ease of use and ability to label live cells make this system truly advantageous. This new technique, developed in Thoru Pederson’s lab, brings us one step closer to mapping the 4D nucleome, the organization of the nucleus in space and time, and to understanding how nuclear organization varies across the life of a cell, or how organization may be altered in disease states.

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Topics: CRISPR, Imaging, Fluorescent Proteins

Seeing Red: Simple GFP Photoconversion

Posted by Mary Gearing on Aug 4, 2015 10:30:00 AM

Since the first research applications of GFP were published in the 1990s, biologists have spent a lot of time making things glow. Chances are you’ve used a GFP derivative to conduct subcellular localization studies or make a reporter construct. Fluorescent proteins (FPs) are also the foundation of multiple important technologies, including FRET and optogenetics. Even though GFP has been so thoroughly characterized, it turns out this protein has a few more secrets - during a collaboration, members of Maureen Hanson’s and Rima Menassa's labs made the accidental discovery that laser treatment can photoconvert GFP from green to RED! This simple technique has been shown to work in plant, Drosophila and mammalian cells, and it may find wide use in biological research.

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Topics: Fluorescent Proteins

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

Evolution of Brainbow: Using Cre-lox for Multicolor Labeling of Neurons

Posted by Mary Gearing on Apr 24, 2015 10:39:00 AM

CRISPR-Cas9 genome editing may be the hot new way to manipulate gene expression, but other gene manipulation systems remain valuable to biology. Cre-lox recombination, discovered in the 1980s, is one of the most important ways to spatially and temporally control gene expression, especially in in vivo models, and new Cre-lox based technologies are still being developed today. In this post, I will highlight the evolution of the  Brainbow multicolor labeling system - a perfect example of the continued utility of Cre-lox. Check out our previous blog post, Plasmids 101: Cre-lox, if you need a quick primer on how Cre-lox recombination works.

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Topics: Plasmid Technology, Genome Engineering, Fluorescent Proteins

Tips for Using FRET in Your Experiments

Posted by Benoit Giquel on Nov 5, 2014 10:41:00 AM

The first time I heard about FRET during a journal club, my guitarist brain automatically thought about the raised element found on the neck of my guitar...not really useful for a biologist you would say. The student was of course talking about the now well-known FRET, aka Fluorescence (Förster) Resonance Energy Transfer, technique which allows the detection of molecules' interactions, modifications or dissociations in situ. Used since the mid-90s, this technique has revolutionised the way we apprehend molecular complexes and is still a very useful tool.   

Like a guitar hero (that I’m not), FRET loves playing “live”. Indeed, FRET was one of the first techniques which enabled the measurement of single molecule interactions in living cells using a microscope. Historically, molecular interactions were detected by indirect means often using probes with the potential to target several molecules. By analogy, it was like pointing out a group of students in a university hall but not knowing if these students know or interact with each other. FRET reduced the scale of our perception about molecular interactions.

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Topics: Imaging, Fluorescent Proteins

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