We all know that in the lab there are often little tricks that are essential for experiments but that nobody talks about. After months of troubleshooting, those people who did not tell you that essential thing ask incredulously, “You seriously didn’t add 3 microliters of 5 mM star anise?” This is something I was expecting when I set out to make my first CRISPR/Cas9 gene edit. I wanted to inactivate the gene BRAF (a kinase implicated in several human cancers) in A549 cells (a human lung cancer cell line), armed only with viruses obtained through Addgene’s viral service and the methods sections of scientific articles (gasp). To my delight, not only was I able to make the edits without any reagent-grade endangered Martian chicory root, but considering this is a needle in a haystack type of objective, it was surprisingly easy. It’s true, I CRISPRed. In this post, I’ll summarize the basic steps and analyses, and give what I think are the main tips for each step of performing and analyzing a gene edit using Addgene’s lentiviral CRISPR tools.
Writing a review article is a wonderful way to develop and exercise your scientist skill set. If you dread the thought of writing a review, or if you’re currently stuck trying to write one, hopefully this post will help you get things moving - remember you're becoming an expert in your field and are the perfect person to be writing the review! Doing so is a great way to develop your ability to write, to read efficiently, to search the literature, and to synthesize a large volume of information: basically, a scientist’s tool kit.
High-throughput cloning, in a nutshell, is the systematic combination of different genetic sequences into plasmid DNA. In high throughput cloning techniques, although the specific sequences of the genetic elements may differ (e.g., a set of various mammalian promoters), the same cloning procedure can be used to incorporate each element into the final construct. This strategy can be used to build vectors with diverse functions, and thus, is used in many biological fields. In synthetic biology for example, high-throughput cloning can be used to combine the functions of different genetic elements to generate non-natural tools such as novel biological circuits or sensors. Given the expanding palette of fluorescent proteins and the availability of powerful imaging technologies, the combination of multiple fluorescent protein sequences to develop diverse fluorescent reporters is a useful application of high-throughput cloning. MXS Chaining is one such technique and has been used to produce complex fluorescent reporter constructs. These fluorescent reporters can be used to detect structure and protein localization, as well as cellular processes like gene expression and cell migration (Sladitschek and Neveu, 2015).
In July 2016, we launched our Viral Service and began delivering ready-to-use lentivirus and adeno-associated virus (AAV) to scientists around the world. We began with only a few inventory items offered domestically, but by the end of 2016, we expanded our viral inventory to 25 lentiviruses and 25 AAVs. These viruses have been distributed in over 200 packages to more than 20 countries. With this initial success, we will continue to provide and expand this diverse and useful collection of tools so that researchers around the world can accelerate their work. After all, as we like to sayat Addgene, productivity is infectious.
Curious which viruses researchers have found the most useful so far? We crunched the numbers on our Viral Service (and then we crunched them again) to find the most requested lentivirus and AAV of 2016.
The top viruses of 2016 were (drumroll please)...
An estimated 320,000 viruses can infect mammals. Even more abundant are the Earth’s estimated 1031 bacteriophages (viruses that infect bacteria), many of which are doing important work in our microbiomes. Given that viruses are everywhere and doing everything, it can be annoying when we try to use them in an experiment and they don't do anything.
Topics: Viral Vectors