... subtypes, thanks to viral serotype specificity during delivery (more on this later). Chemigenetic sensors... at the single neuron level in vitro. In vivo monitoring has also been achieved with these sensors, but...their fluorescence, which ultimately led to the engineering of the second type of these GEVIs: a FP-fused...component, the chemical factor, of a naturally occurring voltage indicator. To give an example, a genetically...

...not recognize the mouse or chicken homologs. By comparing the homologous sequences in these species, they...making the antibody more amenable to protein engineering. As a result, you can now find Myc1-9E10 in a...published a paper showing that the amino acids neighboring the Myc epitope influence 9E10’s affinity for...highly variable epitope recognition dependent on neighboring sequence context. Sci Signal 13. https://doi....

... Manuel Gonçalves; made with BioRender.   Delivering gRNA and Cas9 using adenoviral vectors As compared...published in Nature Methods the same year that delivering RNA-guided nucleases or TALENs together with ...the lab of Andrea Ventura at the Memorial Sloan Kettering Cancer Center did to create a model of Eml4–Alk-driven...Stanchina E, Lowe SW, Ventura A (2014) In vivo engineering of oncogenic chromosomal rearrangements with ...

...plasmids, or express a gene contained in a plasmid. During the propagation and/or transformation process, ...never receive a copy of the plasmid or will lose it during division. These non-plasmid-containing bacteria...while amp only affects the cell wall synthesis step during cell division. This is nifty because the recovery...it will induce mutations in the host/plasmid DNA during selection if it’s not completely bound up. While...

...find out! Homology-directed repair and genome engineering The easiest way to make a knock-in cell line ...built-in repair pathway cells already have for repairing DNA double strand breaks – (HR). HR is the dominant..., H., Ren, S., Yu, S., Pan, H., et al. Methods favoring homology-directed repair choice in response to.... A. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery....

...steps and outlined the considerations to be made during each step. Now that you know the basic principles...harsh - lipids and soluble proteins can be lost during fixation with these chemicals, which impacts sample...and attention.   * There are other methods for recovering epitope availability following cross-linking ...Immunofluorescence Staining. Methods in Molecular Biology. Springer New York, pp 299–311. https://doi.org/10.1007/...

...minigenome system Similar to other RNA viruses, EBOV brings its own RNA-dependent RNA polymerase into the infected...partially control expression levels of each gene by altering the amount transfected (see Technical Tips). Minigenome...changing the minigenome cis-acting sequences and monitoring effects on genome replication and transcription...strand RNA viruses: The power of reverse genetics. Springer Berlin Heidelberg, 2004. 1-41. PubMed PMID:15298166...

...eukaryotic transcriptional activation and cell biology. During this period, proteins were successfully expressed... own, but, if the two POI’s interact, this will bring the POI-TA fusion (commonly referred to as the “.... Lowered expression levels will increase the stringency of your binding. And most importantly: Independently...Ptashne, M. A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor....

...activity – it only requires the presence of oxygen during maturation. This meant that the protein could be...study of dynamic, physiological processes. Re-engineering GFP to increase its range of color and application...its sequence was elucidated, scientists began engineering new versions of GFP through mutagenesis in order...us how! And come back next week when we'll be featuring a guest blog post from Gal Haimovich of greenfluorescentblog...

...for the entire translation process. For example, during high growth rate conditions, E. coli preferentially...biosynthetic enzymes may be preferentially charged during amino acid starvation thus resulting in higher ...getting your genes of interest codon optimized by ordering plasmids containing them from Addgene. If a plasmid...that will generally segregate with the cell pellet during purification procedures. Even if you produce a ...

...but did not consider them for myself. I started picturing myself in a technical support role only when I...there is a second-line team that takes on cases requiring in-depth analysis, data review, in-house testing...are obtained. Or someone may heat an ELISA plate during incubation steps in order to enhance the OD signals...with other departments such as research and manufacturing, quality assurance, product management, marketing...

... not all, of these new experimental species. Discovering ways to culture, propagate, transform, store,...systems. How the EDGE Program promotes scientific sharing The EDGE Program is designed to expand the number..., in general, these labs all include materials sharing in their dissemination plans and in particular,...to mate with and if she decides to raise their offspring or leave them to their own devices. Chytrid fungi...

...phage-assisted continuous evolution and protein engineering to generate seven new prime editors, PE6a–g, ...(2023). Phage-assisted evolution and protein engineering yield compact, efficient prime editors. Cell,... increase the utility of this technique.  By engineering a light-sensitive LOV domain into the TurboID...mass spectrometry workflow identifies proteins bearing both labels (which had therefore been trafficked...

... Research Trust (RSRT) has an ambitious goal of curing Rett Syndrome and Addgene is thrilled to partner...methylated CpG dinucleotides and CAC trinucleotides during neuronal maturation (Tillotson & Bird, 2019) and...neurological deficits observed in Rett Syndrome. Restoring the MECP2 gene by excision of the Stop cassette...have shown exciting and promising results for repairing mutations in models of neurological disease. Klaus...

...neuronal loop which is activated and strengthened during memory formation and memory retrieval. In neuronal...recordings. The origins of these methods are naturally occurring viruses that infect, persist, and migrate within...modified and used as tools for tracing. With some engineering, RABV and HSV, but also others including pseudorabies...plasmids at Addgene). AAV vectors: A platform for delivering neuronal tracers AAV vectors have been widely...AAV1 – 9, with most of these serotypes capable of entering neurons (see table 1)(Choi et al., 2005; Taymans...

...biotin-streptavidin interaction (Figure 1a). This interaction brings the fluorophore in proximity to the target marker... used. These dyes are incorporated into the DNA during DNA synthesis and then diluted with subsequent ...negative live cells can still be easily gated on during the analysis. Conclusion Congratulations! You made...intracellular labeling protocol is necessary, requiring chemical treatment of the cells after surface ...

... allow cells to become persistently fluorescent during development. Stochastic multicolor labeling systems... tags instead of XFP to optimize it for tissue clearing, a method that makes tissues, such as the brain...visible. Chemical tags are more resistant to tissue clearing methods and can be labeled by synthetic fluorophores...Multicolor Brainbow imaging in zebrafish." Cold Spring Harbor Protocols 2011.1 (2011): pdb-prot5546. Richardson...

... the cut site were not efficiently incorporated during HDR. We decided to systematically characterize ...References 1. Cong, Le, et al. "Multiplex genome engineering using CRISPR/Cas systems." Science 339.6121 (...Mali, Prashant, et al. "RNA-guided human genome engineering via Cas9." Science 339.6121 (2013): 823-826. ...Fidelity Cas9 Variants How to Design Your gRNA Delivering CRISPR Ribonucleoproteins Resources on Addgene.org...

...), and in SNP analysis (to introduce naturally occuring SNPs in a plasmid context). The technique is also...sequence critical for Cas9 cleavage), thereby rendering the resulting construct resistant to Cas9 induced... successfully anneal to the plasmid of interest during the PCR reaction. Ideally, your primers should ...’ll want to steer clear of these strains when preparing the plasmid template for site directed mutagenesis...

...is a constantly evolving and demanding field, requiring a variety of biological and molecular tools. One... perfect for molecular cloning and metabolic engineering.  The E. coli genome has an extensive research...be analyzed to understand human disease alleles during and after embryogenesis. Addgene has over 600 plasmids...organism: quick development, a large number of offspring (produces many seeds), self fertilizing, a small...

...assembly work? The required homology between neighboring fragments can be created via PCR amplification...including regions of homology to the vector or neighboring fragment. Then you would amplify the fragments...required single-stranded annealing and priming of neighboring assembly fragments. Gibson assembly is commonly...modular overlap-directed assembly with linkers) which brings modularity to overlap-directed methods (Casini ...

...helpful from a biological standpoint, it makes delivering AAVs to the brain difficult. Traditionally, delivery...and/or its intermediate engineered precursor. Considering systemic capsids for your research? While systemic.../cJ and CBA/J mice. AAV1-X1 was developed by transferring the 7-mer peptide region from variable region...Fox AS. (2023). Advances in AAV technology for delivering genetically encoded cargo to the nonhuman primate...Bloom, J. M., Seed, C., & Deverman, B. E. (2019). Delivering genes across the blood-brain barrier: LY6A, a...AAV vectors to the central nervous system by engineering capsid–receptor interactions that enable crossing...

...An off-target rate of 5% is far too high when considering human patients and the diseases associated with...off-target frequency is (more on this below). Lowering off-target events There are several strategies...compatible with the bulkiness that accompanies mispairing of bases. To reduce off-targets, you’ll want ...which rely on insertion of DNA by the NHEJ pathway during DNA repair of the break to allow for site-specific...

...recorded plasmid multimerization in the naturally occurring E. coli plasmid ColE1 (Goebel & Helinski, 1968...whole plasmid sequencing of our plasmid samples during our quality control process. Since MiSeq involves...D, Berg DE. pBR322-derived multicopy plasmids harboring large inserts are often dimers in Escherichia ...Guttman A, Thompson L. Vaccine Plasmid Topology Monitoring by Capillary Gel Electrophoresis. Curr Mol Med...

...questions in your own lab. 2. Seek multiple mentors during your postdoc At the end of grad school, you searched...own experience, great mentors often can be found during conversations with senior investigators. Discussions...fundamental conflict. Science is about exploration, discovering new information. There are literally infinite...well practiced or too skilled at presenting and answering questions. Organizing a thoughtful engaging talk...

...the five is different. To identify significant pairings, additional tests, referred to as post-hoc analysis...temperature, humidity, sunlight exposure time, and watering volume on total crop production (though you would... error is the probability of a false positive occurring when many comparisons are performed. For example...drawbacks to these tests as they can be quite stringent if many samples are assessed. However, if many...

...minimize recombination that are typically employed during viral plasmid amplification (Fischer, KB. 2019)... showing the mechanism of spontaneous reversion during AAV plasmid amplification. On the left homologous...  Some classes of neurons transiently express TH during development or have low levels of TH mRNA without...time, frustration, and funds in the long-run by ensuring you are minimizing any off-target expression that...

...insertions were positive or negative selected for during the experiment (Sandoval-Villegas et al., 2021)...Targeting) system. INTEGRATE is based on a naturally occurring Tn7 transposon found in Vibrio cholerae that encodes...Sleeping Beauty, piggyBac and Tol2 for Genome Engineering. IJMS 22:5084. https://doi.org/10.3390/ijms22105084...about the INTEGRATE system for bacterial genome engineering  Read about the Sleeping Beauty DNA transposon...

...evidence,” and to speak out in unprecedented force during the 2015 federal election.   Even as we marched... of the few members of Congress who holds an engineering degree. It was a great, positive event that I...a PhD Candidate studying Nuclear Science and Engineering at Rensselaer Polytechnic Institute. He also ...face of evidence. Stephanie Hays is moving from engineering synthetic microbial communities at Harvard University...

... best known reversible PA-FP is the naturally occurring Dronpa protein (named after dron, a ninja term...dynamics (fission, fusion, etc). For example, by engineering PA-GFP tagged to a mitochondrial matrix protein...PMCID:PMC528746. 5.Gurskaya, Nadya G., et al. "Engineering of a monomeric green-to-red photoactivatable ...organelles shows that mitochondrial fusion is blocked during the Bax activation phase of apoptosis." J Cell ...

...letters and be able to guess whether or not that string of letters is a part of the word you’re searching...monoclonal antibodies, which come from naturally-occurring immunoglobulin genes, recombinant antibodies are...Y, Srinivas S, Zhang Y, Lin M, Wang S (2013) Engineering production of functional scFv antibody in E. ... the Addgene blog: Read about another reagent sharing platform for antibodies, the Developmental Studies...

...Getting a Faculty Position Author: Erik Lee SnappSharing is Caring, But How to Distribute Open Hardware? Author... holiday celebrations! We're kicking it off by sharing all the incredible guest posts and stories featured...

...breakdown Intro Chapter 1 - Introduction to Mark Springel, Olivia Ho-Shing, and the Journal of Emerging ...Many thanks to our guests,Olivia Ho-Shing, Mark Springel, Arooba Ahmed, Jiachen Lee, and Jillian Parker...to introduce students to this amazing genome enegineering technology Resources at Addgene.org Find basic...

...University of Amsterdam. In addition to creating and sharing many great fluorescent protein tools, Professor...and pay attention to the details. Chapter 3 - Answering interesting biological questions using microscopy... genome engineering...

...a GIPHY page! The best part is, we’re not only sharing them with you, but you can share them with your...labmates, and friends as well!   Addgene is sharing so much more than plasmids these days. via GIPHY...out everything we’ve uploaded so far, and start sharing the science love today!        ...

...completing her PhD in Chemistry and Chemical Engineering with Harry Gray at Caltech, Crystal moved on ...arise. Tips on applying for industry jobs and deciphering job listings. Mobility between different positions...Addgene Find more management resources in our Mentoring for Scientists Series Learn about Turning Your...

...prizes!), polls, and shout outs, and we’ll be featuring the work of the Addgene community. Perhaps you...Friday: Wrap up! To show the magnitude of plasmid sharing over the years, we crunched the numbers (shout ...news of deposits coming from two new countries bringing the total to 51 countries with plasmid deposits...

... What if after ordering a plasmid you didn't have to grow the bacteria and prep the plasmid before you...preparative experiments.  We’re excited to begin offering this service to further contribute to our mission... community and possibly expand the collection.  During this period, we’d love to hear from you if you’...

...and a key step for that is sharing the data when an AAVs doesn't work. Sharing your negative data helps ...others and, when published, will meet FAIR data-sharing guidelines. But what if my AAVs didn't work?  ...

... the neuroscience community pulls off such an inspiring, thought-provoking, collaborative event – all ... to help their labs save time and resources by storing and distributing their plasmids with the scientific...of this speaks to the importance of scientific sharing to the neuroscience community. The scientists we've...

...control mice into depression. In case you were wondering, depression shows up in mice as poorer performance...kynurenic acid can’t cross the blood-brain barrier. By lowering circulating levels of kynurenine in the body, ...metabolites that are dynamically secreted by muscle during exercise to meet the brain’s physiological demands...

...has opened up a world of possibilities in the engineering of complex gene networks. A comparable system...assemble designer PUF domains for RNA specificity engineering. “The RNA binding domain is interesting because...report published in The Journal of Biological Engineering in March is fused to a translational repressor...

...legal complexities can arise when scientists are sharing their materials. Questions about intellectual property...material transfers. When Addgene entered the plasmid sharing space, we heard over and over again that the legal...were one of the biggest hurdles for scientific sharing. So we set out to streamline this process. We take...

...figured this internship would be a perfect way of exploring my different interests but also a way of challenging...sciences, I knew I would run into some challenges during my internship, but nothing could have prepared ...challenges would be from doing a remote internship during a pandemic. Before I knew how bad COVID was getting...

...no secret that one of the best things you can do during your training is meet interesting people doing ...that position such as a direct connection to the hiring manager. They might say yes and they might say ...connections is your university’s alumni database. Sharing a school is a little connection that many people...

...earlier, but the researchers in the directory are volunteering their time and resources and there is a chance...regulations from country to country when it comes to administering phage therapy. The laws behind phage therapy... open science Find blog posts about scientific sharing Find blog posts about networking Additional resources...

...ago, Melina Fan had an idea and her family helped bring it to fruition. The concept, though relatively simple...was once a complicated process of requesting and sharing plasmids has become simplified through Addgene.... our impressive stats from 10 Years of Plasmid Sharing Or read more about what happens Inside Addgene ...

... how he would go home in the afternoon during the child-rearing years, learning “lots of interesting things...enjoys each other’s company outside of the lab by sharing meals, going on trips, and going to New Hampshire...

...your experiments. Get organized now! Start by gathering that pile of lab notebooks and binder on your ...frequently. Copy these into an electronic file sharing tool, such as Google Drive or Dropbox, and file...to organize your cryo freezer. No more issues deciphering handwriting or trying to find to where the binder...

...much easier to get around our site.  Community, sharing, and fun Along with improving search and navigation...the values that make Addgene unique: community, sharing, and fun. The Addgene repository is built on the..., fun, and promote our values of community and sharing.  One big feature upgrade? Blugene now has a full...