Plasmids for Endogenous Gene Tagging in Human Cells

Posted by Guest Blogger on Apr 6, 2017 9:02:59 AM

This post was contributed by the gene editing team at the Allen Institute for Cell Science. Learn more by visiting the Allen Cell Explorer at allencell.org and the Allen Institute website at alleninstitute.org.

A classic challenge in cell biology is making sure that what we observe through the microscope represents reality as accurately as possible. This is especially true in the case of protein tagging to elucidate cellular structures. Overexpression methods flood the cell with protein, which can both interfere with a cell’s normal function and result in a ubiquitous background signal that makes it hard to visualize the precise location of the protein or structure of interest.

Endogenous gene tagging is an ideal solution because it allows for tagging and visualization of specific, individual proteins under endogenous regulatory control. But even with the advent of CRISPR/Cas9 technology, inserting large tags into a precise location in the genome is still inefficient, particularly in human cell lines. Furthermore, the quality control necessary to ensure the edited cells are behaving normally can be prohibitively expensive for many labs.

Read More >

Topics: Plasmid How To, CRISPR, Techniques

Screening for Successful Genome Editing with Digital PCR

Posted by Guest Blogger on Mar 30, 2017 10:30:00 AM

This post was contributed by Scott Findlay, a Postdoctoral Fellow at the University of Alberta.

If you’re like many researchers these days, you are ready to take (if you haven’t already) the plunge into the world of precision genome editing. When it comes time to (hopefully) validate successful mutation of your favourite gene, there are several different methods available. Thankfully, there are many great resources available to help guide you through the rough waters of mutation validation, such as this "CRISPR 101" post. Next-generation sequencing technologies are the gold standard but they remain cost-prohibitive for many labs, and are often impractical for small projects. Most researchers instead turn to so-called “mismatch nuclease” assays (e.g. Surveyor® or T7E1) for mutation detection. While these methods paved the way for mutation validation, we found these assays frustrating to work with, time consuming, and minimally informative. In this blog post, we’ll introduce digital PCR as an emerging validation technology. Digital PCR has several advantages over mismatch nuclease assays that will be elaborated below.

Read More >

Plasmids 101: SunTag and Fluorescent Imaging

Posted by Mary Gearing on Mar 28, 2017 10:30:00 AM

Quick Announcement from the Plasmids 101 Team: In preparation for the release of Addgene's Fluorescent Protein eBook - our next couple of plasmids 101 posts will gain a healthy, fluorescent glow. Stay tuned for more fluorescence-based Plasmid 101 posts in the coming weeks!

In biology as in life, more is often better. More transcription factor binding sites in a promoter lead to higher transcriptional activation. Multiple nuclear localization signals (NLS) increase protein import into the nucleus. In developing their SunTag technology, the Vale and Weissman labs took this biological lesson and created a system to amplify fluorescent signals. Named for the "stellar explosion SUperNova," SunTag can help you turn up the brightness in your fluorescent imaging experiments.

Read More >

Topics: Hot Plasmids, Plasmids 101, Fluorescent Proteins

Addgene’s Viral Service - Why Virus? Why Now?

Posted by Joanne Kamens on Mar 23, 2017 10:30:00 AM

In the middle of 2016 Addgene started distributing a small but growing catalog of ready-made AAV and Lentiviral preps. This new Viral Service represents Addgene’s largest new initiative since we started distributing plasmids in 2004. We’ve already distributed over 500 viral samples to scientists all over the world. Now that the service is successfully launched, I would like to thank some of the people and organizations who helped us reach this milestone.

Read More >

Topics: Viral Vectors

Quick Guide to All Things Lentivirus

Posted by Benoit Giquel on Mar 21, 2017 10:30:00 AM

If you are interested in using lentiviral vectors to introduce your favourite gene into your favourite cell line or into primary cells, this blog article will give you some tips to plan your experiment using the lentiviral vector system.

Viral vectors have been increasingly popular in fundamental and applied research since their first use in the early 90’s to genetically modify primary cells. Amongst the different vectors used, lentiviral vector constructs have proven very useful due to their ability to infect both dividing and non-dividing cells, including stem cells. These properties make lentiviral vectors fantastic options for delivering shRNA, CRISPR/Cas9 components, and fluorescent sensors.

Read More >

Topics: Viral Vectors

Addgene blog logo

Subscribe to Our Blog