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AAV: A Versatile Viral Tool for Gene Expression in Mammals

Posted by Guest Blogger on Jul 26, 2016 10:30:00 AM

This post was contributed by guest blogger Didem Goz Ayturk, a Postdoctoral Fellow in Connie Cepko’s Lab at Harvard Medical School with edits from Addgenie Karen Guerin.

Adeno-associated virus (AAV) has emerged as a favorite viral tool for both research and clinical applications. AAV can be used to transiently express a gene of interest in a variety of cell types. It was first described about 50 years ago as a contaminant of adenoviral preparations, hence the name (Atchison et al., 1965) AAV is a single stranded, DNA virus belonging to the family Parvoviridae. It has a "simple" genome packaged in an icosahedral capsid. It does not have a lipid coat, also called an envelope, and thus cannot support the addition of a glycoprotein, such as VSV-G, to its surface. In research applications, the genome is typically gutted so that precious cargo space is opened for gene delivery, and for safety. You can easily complement the virus in a tissue culture setting, in other words “in trans”, by supplying the genes that encode the replicase functions and capsid proteins. This gives researchers the ability to produce more virus in a controlled setting. Even though AAV is isolated from a wide range of organisms, it has not been associated with disease, and it is considered a biosafety level 1 (BSL1) viral agent.

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Topics: Viral Vectors

Gendered Innovations: Why Does Sex of the Cell Matter?

Posted by Guest Blogger on Jul 21, 2016 10:30:00 AM

The post was contributed by guest blogger Londa Schiebinger, PhD, Hinds Professor of History of Science, Stanford University.

Sex and gender are critical components of biological research that are often forgotten or ignored. If we wish to conduct research that fails less and helps more people, we need to take sex into account. Gendered Innovations is an international, collaborative project—funded by the European Commission, the US National Science Foundation, and Stanford University—that harnesses the creative power of sex and gender analysis for innovation and discovery.

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Topics: Lab Tips

Special Delivery: Fluorophore Targeting for FRET Studies

Posted by Guest Blogger on Jul 19, 2016 10:30:00 AM

This post was contributed by guest blogger James D. Fessenden, an Assistant Professor at Brigham and Women’s Hospital.

Biochemists often struggle to understand how a protein of interest actually behaves. How large is it? What parts of it move when you feed it substrate or add an essential cofactor? How many binding partners does it have and how do they come off and on in a cellular environment? If these are pressing issues in your laboratory, then FRET experiments are a viable biophysical path to answers.

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Topics: Lab Tips, Fluorescent Proteins

Generating Mouse Models Using CRISPR/Cas9

Posted by Guest Blogger on Jul 12, 2016 10:30:00 AM

This post was contributed by guest bloggers, Wenning Qin and Haoyi Wang.

CRISPR/Cas9 is revolutionizing the mouse gene-targeting field. Mice have long been extremely useful in the lab – they are relatively small and easy to work with, making them the go-to choice for studying mammalian biology. Similar to any model, mice are not without their problems, but much genetic and physiological data have been accumulated over the years using them. Indeed, the future of mouse work is bright as it is now easier than ever to manipulate the mouse genome using CRISPR/Cas9.

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Topics: Genome Engineering, CRISPR

Tips for CRISPR Gene Editing in Mice

Posted by Guest Blogger on Jun 28, 2016 6:59:27 AM

This post was contributed by guest blogger Samantha Young.

The use of CRISPR/Cas9 for gene editing has expanded since its adaptation for use in mammalian cells in 2012-2013. Researchers are now using this system in ever more creative ways, (Wang et al., 2013, Cho et al., 2014). There are several variants of the CRISPR/Cas9 system floating around, and many pre-designed plasmids containing these variants ready for purchase. But what is the easiest and fastest way to use the system in mice? We'll have a post that goes into the mouse genome editing process in a bit more detail in the coming weeks, but, in this post, we will outline a simple method for selecting the guide RNA, validating its efficacy in vitro, and using it in mouse embryos to generate gene modified mouse lines. Hopefully this post will help get your in vivo research up and running as soon as possible!

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Topics: Plasmid Technology, Genome Engineering, Lab Tips, CRISPR

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