...infectious disease once limited to small pockets in Africa has spread to urban areas and across the ocean....percent of the most recent cases, primarily in West African countries. Even that grim estimate might prove ...protein present on the virion surface – making it critical for viral attachment and fusion. Once inside a...glycoprotein’s role in infection also makes it a critical component of vaccines and the target of neutralizing...

...the user. This approach is useful for several applications as will be discussed below. The software could...has a maximum of one gene knocked out. In this genetically heterogeneous population, cells behaving differently... you could majorly accelerate progression into clinical trials. Secondary screens  If you have generated...genes you’d like to test for phenotypic effects. Typically the phenotype would be studied in vitro, but there...contain multiple gRNAs targeting all genes in the indicated species and were suggested to be the best gRNAs...

...analyzing your cells using flow cytometry, you are typically measuring the presence or absence of certain markers...reporter, such as GFP or mCherry, through genetic modification. As your protein of interest will only occur...free amines on and inside the cells and are thus typically used for distinguishing live from dead cells. ...describe a basic panel (Table 2, Figure 5) for a hypothetical experiment in the lab. Let's say I am culturing...Associate in the lab of Arlene Sharpe at Harvard Medical School. His projects include investigating negative...

...Antibody characterization is critical to enhance reproducibility in biomedical research. eLife, 13, e100211...tell only part of the story. This year, we made significant strides in making science more accessible and...launched AAV Packaged on Request, a service that dramatically expands and simplifies your viral vector options...

...promoter. It can be used to drive Cre expression specifically in neurons. AAV Delivered Cre - A useful tool...Indeed, this AAV provides you with a means to specifically activate expression of any Cre-dependent gene...pmSyn1-EBFP-Cre Of course there are also more complicated ways to use this particular AAV. For instance...Additionally, you could use the retrograde form to specifically express Cre in neurons with axons that extend...

...service. A small subset of AAV plasmids will have technical or legal restrictions that make them ineligible...be reviewed by Addgene for legal approvals and technical compatibility with our production process. This...request is approved, you will receive an email notification that your request is ready to order. An “Add...found in our in-stock preps. If a request has technical considerations that may affect expected titers...

...incredibly useful molecular biology tool, but like any biological system, it has certain drawbacks. First, the ...Gerald Rubin’s lab sought to make complex genome modifications in Drosophila using multiple recombinases. To...offspring carried both the recombinase and GAL4, indicating that the recombinases were inherited normally...Chinese hamster ovary (CHO) cells, as well as in cortical neurons in layer 2/3 of the mouse brain. Rubin...

...  “This enables you to do customized genetic modification on a scale that was really not possible before...genome engineering with CRISPRs was limited to modification of a few loci at a time,” Wang said. “It was... lentiviral libraries and their demonstrated application offer proof-of-principle that CRISPR-Cas9 can...aimed for 300 cells per construct, but in some biological systems one “might need larger numbers.” Further...

...: Find sequences for plasmids assembled from publications and commercially available sources, as well ... as expression patterns, post-translational modifications and polymorphisms Codon optimization tool: To...making it ready to copy and paste into other applications Primer design tools Many of the above-listed... well as primer design for different cloning applications. Primer3 and Primer3Plus: For general primer...

...time for me to complete an online social media certification on social media data, and I was given a fantastic... occasional TikTok. I was also responsible for typical entry level tasks such as stuffing new depositor...connections with coworkers seem, in retrospect, nonsensical. I have found the people at Addgene have become... at Northeastern and Addgene co-op studying communications, marketing, and global fashion studies. She...

...symposia on Sunday afternoon were dedicated to science communication through art. Addgene Advisory Board...page and, just a few weeks ago, we attended the American Society for Microbiology's annual meeting (ASM... Jessica Welch...

...service organization like the Red Cross or the American Cancer Society, or maybe you think about a local...services we provide like quality control, curation, technical service and open-access educational resources....decisions at Addgene If you’re ever curious what specifically drives the decision making process at Addgene...Gizela, Henry, Ina, Inna, Ishrat, Jane, Jason, Jessica, Joel, Jonathan, Justine, Karen, Kate, Kayla, Kelly...

... perspective – a plasmid-containing cell must replicate the plasmid in addition to its own chromosomal...How else can antibiotics be used in the lab? Historically, antibiotics have also been used to disrupt ...cloning. In these instances, the cloning vector typically has two separate resistance cassettes and your...and can be used in place of Ampicillin in most applications. Antibiotics vary in their sensitivitly to heat...

...preparations have worked well in my lab, including in applications where we have delivered them in vivo to the ...Roth’s lab are already available allowing you to chemically control neuronal activity while studying your...distributing new types of materials. At the time of publication we’ve already fulfilled more than 140 orders ... service as we continue to develop it. More communication and sharing will only help research advance....

... lab at ETH Zürich. “Instead of a graphical abstract, it’s a musical abstract,” says Di Ventura, a professor...in @nchembio Title: Bye bye L-arabinose drive. Dedicated to Edo, Armin and @KhammashLab. I hope you all...reached the level of expression needed for most applications.   Figure 1: With blue light, the VVD...

...physiology. Ribosome profiling Ribosome profiling, a biochemical method developed in 2009, takes a “snapshot” ...translation While ribosome profiling co-opts the physical location of the ribosome, TRICK (Translating RNA...development, the osk-TRICK reporter was double labeled, indicating translational repression. During later stages...studying translation in various cell types and physiological states. References 1. Ingolia, Nicholas T.,...

...represent the fraction of that total with the indicated function. One huge reason CRISPR has become such...Chavez, researcher at the Wyss Institute for Biologically Inspired Engineering and member of the Addgene...achieving the gRNA representation recommended in the publications associated with these libraries. Please note...particular, the Church lab at the Wyss Institute for Biologically Inspired Engineering and the Zhang and Doench...

...microorganisms based on metagenomic data. MerMAIDs (Metagenomically discovered Marine, Anion-conducting and Intensely...publicly available metagenomic datasets with biophysical studies and the potential of mining datasets ...plasmids at Addgene Opperman J et al., Nature Communications, 2019. PubMed PMID: 31346176. Using LADL to...CIBN), which they call the LADL Anchor. Using specifically designed gRNAs, they can then recruit these ...

...ll need a few controls specific to flow as an application. These controls will allow you to distinguish...sample. The isotype antibody will be virtually identical to the antibody for your marker of interest. It...commercial antibody pages. Flow cytometry can be a complicated and daunting process. Controls like single color...research technician in Arlene Sharpe's lab at Harvard Medical School.   More resources on the Addgene blog Introduction...

...cell types so it is useful in a wide range of applications. This plasmid was the top requested plasmid ...notice the “V2.0” at the end of this plasmid which indicates that there was a prior version of this plasmid...plasmid was called pLKO-Tet-On in the original publication but its name was subsequently changed to clarify...generation lentiviral backbone that must be used with lentiCas9-Blast or with cell lines already expressing Cas9...

...study it. The process is fairly straightforward. Basically, you place your gene of interest in a plasmid ...genetic crosses with balancer chromosomes to make genetically stable fly lines. After a few weeks of waiting...throughout the lifespan of the fly to determine the implications of your genetic manipulations. You can produce... aware of at least some of the possibilities. Basically, if you can devise an experimental assay that ...

...Immunosurveillance and Defense With increasingly sophisticated visualization techniques it is possible to follow... and correlating them with tumor mutations and clinical responses. This platform has the potential to ...the use of optogenetic tools is expanding to immunological research. Here the Zhou group presented Opto-CRAC... et al. "Caloric restriction mimetics enhance anticancer immunosurveillance." Cancer cell 30.1 (2016):...

...of interest and, for in vivo work, generate a dedicated transgenic line. Using zebrafish as a model organism... for example).  Given that the liver is an immunologically active organ, this advance is likely to afford...off-target mutations. These plasmids contain catalytically inactive dCas9 and microbial SSAPs (dCas9-SSAP...clonal multiplexing of cells in live imaging application. Each visual barcode consists of a fluorescent...

...neurons and specific behaviors. Chemogenetics uses genetically engineered receptors to interact with small molecules... to optogenetics but instead of light, these genetically modified receptors are activated by small molecules...some GPCRs further to respond to specific, pharmacologically-inert, small molecules. These new receptors...Kadowaki S, Haga T, Takaesu H, Mitaku S (2002) Identification of G protein-coupled receptor genes from the...

... light-mediated? The researchers split the proteolytically active light chain of BoNT/B into two fragments...that undergo light-induced dimerization. Upon application of blue light, iLID undergoes a conformational...rely heavily on fluorescent protein genes. These optical reporters have limited deep-tissue performance,...the field. Arlene Sharpe’s group at the Harvard Medical School developed a solution: CHIME, a CRISPR-Cas9...

...α-complementation. The system described above was put to practical use in the following way. Scientists engineered...insert. All colonies on this plate should be blue, indicating that your IPTG and x-gal are working as they ...of false positives: Blue-white screening only indicates the presence of AN insert, not necessarily YOUR... Jessica Welch...

...missing or damaged nucleotides that are common to biological sources of DSBs, and which need to be processed...generated in the course of repair by NHEJ are typically small (1-10 bp) but extremely heterogeneous. There.... NHEJ can also be engaged by variants of the canonical Cas9 approach. A pair of CRISPR guides that flank...Translocations in Human Cells Are Generated by Canonical Nonhomologous End-Joining. Molecular Cell 55:829...

.... Listen to our podcast interview with Harvard Medical School researcher Connie Cepko to learn about her...mentoring programs. The groups in these programs typically consist of 1-2 mentors and 3-5 mentees who meet...This is a chance to practice leadership and communication skills. The leader also provides materials such...present formally often enough) and give honest, critical feedback for improvement. Go to a science or career...

...produce genetically modified cells in less than a month's time. Addgene: What are the implications of this...Musunuru: The kit includes 832 plasmids containing prefabricated pieces that allow you to build a DNA-binding...diseases. We are knocking out genes that have been implicated in disease or, in cases where particular variants...

...time it takes to make a plasmid could increase dramatically if you are piecing together several PCR products...to make plasmids that already exist, and we significantly reduce costs of producing plasmids with high-throughput...The faster methods, like Gibson assembly, are typically more expensive, so you’ll have to find a balance...calculations for any of the numbers in this post is a mathematical story well beyond my ability to tell. But even...