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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

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

Science Communication Snapshot: DayCon 2015

Posted by Mary Gearing on Jun 17, 2015 5:04:11 PM

SITN DayCon 2015 Team

Here at Addgene, we’re dedicated to advancing and sharing science! In association with the Harvard graduate student organization Science in the News (SITN), we recently sponsored a first-time event called DayCon. DayCon is a one-day conference aimed at the general public that provides an accessible introduction to various scientific topics. Over twenty graduate student volunteers worked hard to make this Saturday event a success, a true testament to the commitment of SITN members.

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Topics: Scientific Sharing, Science Communication

Back to Bacteria: CRISPR gRNA Multiplexing Using tRNAs

Posted by Mary Gearing on Jun 2, 2015 2:06:00 PM

In the short time since its development, CRISPR/Cas9 genome editing has been used to study the effect of gene knockout in vivo and in vitro, as well as to insert targeted mutations through homologous recombination. To further increase the utility of CRISPR/Cas9, it will be necessary to improve its multiplexing capacity. Multiplexing is key due to the natural redundancy of biological pathways;  to observe a phenotype, the modification of multiple genes is often necessary.

Guide RNAs (gRNAs) are commonly packaged in 400-500 bp cassettes containing the RNA pol III promoter, gRNA and pol III terminator. These relatively large cassettes (considering the gRNA itself is ~100 bases) limit the number of gRNAs that can be packaged together in a single vector. In addition, the pol III promoter is relatively weak, and low expression of gRNAs from these constructs could lower genome editing efficiency.

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Topics: Plasmid Technology, Genome Engineering, CRISPR

Cre-ating New Methods for Site-specific Recombination in Drosophila

Posted by Mary Gearing on May 12, 2015 9:32:10 AM

Cre-lox recombination is an incredibly useful molecular biology tool, but like any biological system, it has certain drawbacks. First, the efficiency of Cre recombination varies for different constructs and cell types. Second, Cre may induce recombination at pseudo- or cryptic loxP sites (estimated to occur at a frequency of 1.2 per megabase in mammals), leading to DNA damage and developmental aberrations. In multiple systems, Cre itself, without the presence of a floxed construct, may produce a phenotype. This problem is especially stark in Drosophila, where expression of Cre from the standard UAS/GAL4 system is toxic to proliferating cells. A Cre-estrogen receptor ligand binding domain-fusion can prevent this toxicity, but with the caveat of partial rather than complete recombination. If you’re looking to use site-specific recombination in Drosophila, read on to learn about new recombinases suitable for this system.

Gerald Rubin’s lab sought to make complex genome modifications in Drosophila using multiple recombinases. To make multiple, precise genome edits, the recombinases used must have high activity and specificity with low cross-reactivity, as well as low toxicity.

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Topics: Plasmid Technology, Cre-lox, Drosophila

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