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Mary Gearing

Mary Gearing is a Scientist at Addgene. She got her start as a Science Communications Intern writing for the Addgene blog and website. As a full-time Addgenie, she still enjoys blogging about CRISPR and other cool plasmids!

Recent Posts

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

IDP and your PI: A Roadmap for Career Planning and Personal Development

Posted by Mary Gearing on Jul 21, 2015 10:30:00 AM

As we get closer to the start of another academic year, graduate students and post-docs alike are wondering where the time has gone. Are we any closer to graduating, publishing that key paper, or figuring out a career path? Many trainees are developing Individual Development Plans (IDP's) through Science Careers’ myIDP tool. Using myIDP, you can identify suitable careers based on your current interests and skillset. With this information in hand, you can then formulate a plan to further develop your transferable skills and reach your career goals.     

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Topics: Career, Lab Tips, Career Readiness

A Match Made in Heaven: CRISPR and AAV

Posted by Mary Gearing on Jul 14, 2015 10:30:00 AM

This post was updated on Dec 4, 2017.

CRISPR genome editing has quickly become the most popular system for in vitro and germline genome editing, but in vivo gene editing approaches have been limited by problems with Cas9 delivery. Adeno-associated viral vectors (AAV) are commonly used for in vivo gene delivery due to their low immunogenicity and range of serotypes allowing preferential infection of certain tissues. However, packaging Streptococcus pyogenes (SpCas9) and a chimeric sgRNA together (~4.2 kb) into an AAV vector is challenging due to the low packaging capacity of AAV (~4.5 kb.) While this approach has been proven feasible, it leaves little room for additional regulatory elements. Feng Zhang's group previously packaged Cas9 and multiple gRNAs into separate AAV vectors, increasing overall packaging capacity but necessitating purification and co-infection of two AAVs.

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Topics: CRISPR, Viral Vectors

Even more elegant: Single injection CRISPR/Cas9 in C. elegans

Posted by Mary Gearing on Jul 7, 2015 11:36:00 AM

In the summer of 2013, a remarkable nine papers describing CRISPR/Cas9 genome engineering methods for C. elegans were released, signaling a new era in C. elegans research. Homology directed repair (HDR), which enables insertion of custom genomic modifications, is very robust in C. elegans, and the methods for HDR-mediated modification continue to be improved. New work from Bob Goldstein’s lab at the University of North Carolina has made CRISPR in C. elegans even easier - now, one can generate a fluorescent protein fusion, transcriptional reporter, and loss-of-function allele in just one injection step! The entire protocol takes about 2-3 weeks but requires less than eight hours worth of hands-on time.

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Another Pathway into Cells: iTOP

Posted by Mary Gearing on Jun 23, 2015 4:37:00 PM

Primary cells recapitulate the natural biology of a cell type of interest better than immortalized lines derived from the same cell type; however, their usage has been limited by technical problems. For instance, it’s much more difficult to introduce a gene of interest into primary cells, so most primary cell lines require viral infection. A new paper from Niels Geijsen’s lab suggests that primary cells may be better transduced using only protein. Read on for a description of the lab’s iTOP protein-only transduction method and its potential applications to CRISPR/Cas9 genome editing.

 

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Topics: CRISPR, Techniques

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