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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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Walking beside Academia and giving it a high five: My transition from graduate work to a position at Addgene

Posted by Tyler Ford on Jul 2, 2015 4:37:00 PM

Just over a month ago I finished up my PhD in Biological and Biomedical Sciences at Harvard University and entered a new role here at Addgene as an Outreach Scientist. I used to spend my days (and often my nights :D) engineering E. coli to produce biofuels in Pamela Silver’s Lab (plasmid page here). Now I spend much less time wrangling bacteria and instead help Addgene fulfill its mission of helping scientists share information and, of course, plasmids. My primary duties are to manage this very blog (you’ll have to let me know how I’m doing a few months down the line!) and to visit scientists in person to figure out ways we can make Addgene better and make scientists’ lives easier.

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Topics: Career

Sleeping Beauty Awakens for Genome Engineering

Posted by Emma Markham on Jun 30, 2015 10:00:00 AM

Transposons are sequences of DNA that can move around in a genome. In a laboratory setting, transposons can be used to both introduce genes into an organism’s genome (see figure) and to disrupt endogenous genes at the site of insertion. In both of these cases, transposons combine the advantages of viruses and naked DNA while eliminating some of the drawbacks. Specifically, viruses are able to infect and replicate in host cells, but they are susceptible to cells’ defense mechanisms. The use of non-viral vectors, like transposons, avoids many, though not all, of these defenses. For some applications of genome engineering - such as certain forms of gene therapy - avoiding the use of viruses is also important for social and regulatory reasons.

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Topics: Plasmid Technology, Genome Engineering

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

Easing the Protein Purification Process with pCri

Posted by Mary Gearing on Jun 19, 2015 11:08:00 AM

Protein purification can be one of the most stressful lab activities. Working with proteins requires a substantial amount of properly folded, relatively pure protein, but getting to this stage is often much easier said than done. As reviewed in our Plasmids 101 series, proteins are overexpressed from a plasmid construct, most often in special E. coli strains designed for protein expression. Cultures are then lysed and the protein of interest is purified using an affinity tag. Additional tags may be used to improve protein stability and solubility.

Determining the best way to express one’s protein of interest can save a lot of time later.

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Topics: Hot Plasmids, Plasmid Kits

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