... are two types of Class 1 TEs: those with long terminal repeats (LTRs) and those without (non-LTR TEs)...contain two open reading frames (ORFs) which often terminate with a poly(A). ORF2 encodes endonuclease and ...transposase enzyme binds the transposon’s long terminal repeats (LTRs), induces a double-stranded break...synthetic transposable element developed from inactivated Tc1/mariner transposons found in fish (Sandoval-Villegas...it leaves behind the CAG DNA footprint from its terminal sequences at the excision site after cleavage ...medaka fish because its insertion into the fish’s tyrosinase gene caused albinism (Koga et al., 1996). Unlike...Tol2 or miniTol2 is a truncated version of the original Tol2 and has ~3-fold increase in transposition...

...protocols, including vector-insert joining for recombinant plasmid construction (restriction cloning), adaptor...can reliably detect single-base differences. The original creators of this technique visualized the ligation... have been generally found to have a higher discrimination at the upstream side of the ligation junction...including Taq DNA ligase, are naturally able to discriminate against ligating substrates containing base ... at elevated temperatures, allowing further discrimination by incubating the ligation at a temperature... fidelity ligation can be achieved through a combination of the intrinsic selectivity of the ligase active...method allowed screening of all possible base combinations (Watson-Crick and mismatched) around the ligation...

...CRISPR/Cas9 gene edit. I wanted to inactivate the gene BRAF (a kinase implicated in several human cancers... really need to use FACS (like they did in the original publication). I thought I could see GFP expression...against GFP expression, but I didn’t. 2. In the original publication they see GFP expression in about 20%... cells at an MOI of 0.3, as recommended by the original publication. Pro-Tip: 1. Use multiple gRNAs to...human line, using methods based on homologous recombination (Rago et al., 2007). Using CRISPR, I was able... active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Doench JG, Hartenian E, Graham DB, Tothova ...

...inventory of recombinases and recombinase-dependent vectors. We recently released several recombinases in various...Calcium sensors and GABA sensors Optogenetics Recombinases And more! Control AAV For many AAV experiments...densely-labeled neurons because background fluorescence from inactive neurons is reduced. This variant has an approximately...pAAV-CamKIIa-ChrimsonR-mScarlet-KV2.1 (124651-AAV9) Recombinase AAV Targeting and manipulating specific, genetically-defined...

...scientific support staff receives – in fact, the blog originally started as a way to easily store and share in-depth... everything goes through an editing process and final check before publication.   Do research! Nearly...that’s not enough, we consult with experts, watch webinars, or use other research methods. In the case of...to make sure absolutely everything was correct. Final touches We try to use figures and images to help...communicator "just sketch a western blot out quickly." Final design (right). Immediate and immense regret of ...image sharing repositories. Then we format, do a final review to catch those pesky typos, and hit publish...

... Bryan Roth’s lab from the University of North Carolina, Connie Cepko’s lab from Harvard Medical School...provided valuable insight into AAV production. Finally, we are blessed to have an incredibly supportive...” Under the leadership of Addgene’s co-founder, Melina Fan, we created a new team to push this project...project involved every Addgenie in the company. Our Finance, Business and Legal teams had to pave the way for...export and shipping on dry ice. The Business and Finance Teams continue to facilitate the formation of partnerships...dedication to do this well ... because it did. So finally, a big thank you to the Addgenies who went the ...

...generated with 4 different, binary 2in1 vectors with all possible combinations of tag orientation (NN, NC...activity.   With the addition of blue light, the terminal helices of the LOV2 domain unfold, disrupting ...responds to blue light quickly. Within minutes of inactivating the CASANOVA complex, fluorescent CRISPR complexes...the brain compute using electrical impulses to coordinate emotions, thoughts, and behavior. Historically..., where insertion of the GOI occurs through recombination. The 2in1 cloning system is well suited for...

...others you’ll encounter include monoclonal and recombinant antibodies). By the end, you’ll have a better...column and from the target protein. Pre-adsorption Finally, polyclonal antibodies may be pre-adsorbed to remove...batch variability. Once an animal has undergone a terminal bleed to acquire all of its serum, there is no...polyclonal antibody, or select a monoclonal or recombinant antibody instead. Otherwise, you might experience...of background noise compared to monoclonal or recombinant antibodies. This is because the polyclonal antibody...antibodies Learn about the advantages of using recombinant plasmid-based antibodies Resources on Addgene.org...

...websites, Facebook and Gmail, were launched. And Melina Fan, a cell biology graduate student, was frustrated... make sharing reagents and protocols so easy!” — Nina Jain, Harvard Medical School. We want to give a ... really like this award because you don’t get nominated for it, it just reflects how useful the reagents... we could use the same principles to provide recombinant antibodies, another plasmid-based product that... In March 2022, we launched our ready-to-use recombinant antibodies service, part of the Neuroscience ... A blast from the past: Cofounder and CSO Melina Fan setting up Addgene's very first in-house NGS...

...depending on the combination of fluorescent proteins expressed. CreLite-mediated recombination was found in...editing efficiencies in the mouse brain, liver, retina, heart, and skeletal muscle. The dosing required...cyanobacteria. This kit contains 96 parts including terminators and promoters, CRISPRi parts, selectable markers...longer present, the fusion proteins separate, inactivating Cre. This is critical for reducing Cre toxicity...various fluorescent proteins controlled by Cre recombination. Each cell can have multiple copies of the transgene...

...general availability of their recombinant antibodies. Since recombinant antibodies are a newer technology...chose monoclonal antibodies (mAbs) and their recombinant forms (rAbs). mAbs and rAbs have higher homogeneity...produce the same results even to a different sample. Finally, as a lab we discuss the results together, explaining...Zhuhao Wu at the Icahn School of Medicine at Mount Sinai.  References and resources References Baker M. ...ValidationSelecting the Right AntibodyPlasmid-based Recombinant Antibodies    ...

...two non-functional fragments: N-terminal BirA* (N-BirA*) and C-terminal BiraA* (C-BirA*). These two fragments...(PPIs). These interactions are often modeled as binary relationships, i.e. protein A interacts with protein...Split-BioID is that it allows for the validation of a binary PPI as well as the identification of additional...complementation assay that can be used to study binary protein interactions. However, FRET generally only...proteins were tagged with. It’s best to test all combinations and pick the conditions that give the strongest...

...-specific diagnostics for the Ebola virus. Our original proof-of-concept work was exciting because it ...the spread of the virus by the Aedes mosquito (Messina et al, 2016). We quickly assembled a team of students...Asian-, African- and American-lineages). Strain discrimination is especially important in the current outbreak...setting. Molecular diagnostics for the Zika virus predominantly look at viral load in patient blood and urine...self-assembly for applications in health, energy, and sustainability.   References 1. Pardee, Keith, et al. "Rapid...25417167. PubMed Central PMCID: PMC4243060. 4. Messina, Jane P., et al. "Mapping global environmental ...

...Validated recombinant antibody toolbox for aminoacyl tRNA synthetases By Kate Harten Recombinant antibodies...library to study antibody escape in SARS-CoV-2 Recombinant antibodies for aminoacyl tRNA synthetases Nanocompartments... axonal projections and putative pre-synaptic terminals with one vector. AAV hSyn FLEx mGFP-2A-Synaptophysin-mRuby...Synaptophysin-tagged GCaMP6s is enriched in the pre-synaptic terminal providing better signal to noise for imaging calcium...dynamics in fine structures. We have expanded our recombinase-dependent virus selection to now include Flp-...

...training, going from intermediate to advanced and finally, to expert.   Figure 1. Visual representation...subcutaneous injections take some practice to get the coordination down. Subcutaneous injections are most often...delivered to the central nervous system via cerebral spinal fluid. The injection site is the space in and around...minimally invasive. Restraint: anesthesia Location: spinal cord Gauge: 30 Volume: up to 0.01 ml (10 µl) Skill...what method you’d like to use, remember that the final decision will likely come down to experimental necessity... 26 < 0.05 mL Advanced Intrathecal Anesthesia Spinal cord 30 up to 0.01 mL Expert Intratracheal Anesthesia...

...°C instead of 37 °C and then screen for ITR recombination with a SmaI or XmaI restriction enzyme digest...RecA, a protein that aids in the homologous recombination of duplicated regions like the ITRs. Restriction... concentrations of iodixanol to separate out contaminants from an impure AAV preparation. 15%, 25%, 40%...standard and AAV transfer plasmids. It’s easy to contaminate a workspace with plasmid, not that I would know...adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Human gene therapy methods, ...R.A., & Samulski, R.J. (2007). Production of recombinant adeno-associated viral vectors for in vitro and...

...cells and eliminate them from the body. While effective, the immune system does not always eliminate every...with the goal of being able to discriminately and effectively eliminate cancerous cells from the body..... What is cancer, really? While cancer is extraordinarily complex at the molecular level, at the cellular...commit itself to the apoptotic pathway and be eliminated from the body. In the case of cancer, however...

...stability and yield. If a plasmid is prone to recombination Use a strain optimized for plasmids with repeat...elements, such as viral plasmids with inverted terminal repeats. We use NEB Stable to transform and prep...Stbl3 have been engineered to reduce plasmid recombination, but are also endA+. endA encodes a thermostable...volume of culture used for inoculation if we get inadequate plasmid DNA yields. Why would volume make a difference...recommended range of the filter or you might inadvertently lower the amount of DNA you purify! Additionally...should be determined for each plasmid/strain combination individually, based on determining the cell density...

...developments.  Further, this post may contain technical inaccuracies or typographical errors.  No action should be...patents and letters of recommendation. EB-1A extraordinary ability category The EB-1A - is similar to national...journal editor) of the work of those in your field. Original contributions to the field that have had a major...include the fact that you are a classically trained ballerina who has performed at Lincoln Center. Your case...of permanent, tenured or indefinite (no fixed termination date) employment at a university or institution... the same or allied academic field Evidence of original scientific or scholarly research contributions...management, alliance management, business development, finance, and operations. To learn more, visit www.propelcareers.com...

...alterations. These studies utilize a catalytically inactive form of Cas9 known as dCas9 (Jinek et al., 2012... mammalian cells by expressing dCas9 with an N-terminal 3xFLAG tag (3xFLAG-dCas9) and a gRNA targeting...this form, the CRISPR complex, consisting of a recombinant dCas9 protein and synthetic gRNA, is incubated...DUX4, a transcriptional regulator that when inappropriately expressed can cause facioscapulohumeral muscular...non-coding RNAs associated with telomeres using a combination of enChIP and RNA sequencing." PLoS One 10.4 ...chromatin by in vitro enCh IP technology using recombinant CRISPR ribonucleoproteins." Genes to Cells 21.4...

...sure that the sample is pure and free of any contaminants that could affect the measurements. You'll need... if not, since buffer components can affect the final readout. When preparing a standard curve, change...even when the same protocol is being followed.  Finally, standard curves must be validated for each run...software to calculate the data’s coefficient of determination, R2. R2 is the square of the correlation between... to increase the accuracy of your experiment.  Finally, carefully analyze the data and use the trendline...Addgene's Ready-to-Use Antibody collection Addgene's Recombinant Antibody Plasmids collection  ...

...was over 300 years ago. Teaching by example No examination of the state of science would be complete without...story has its own Franklin in Doug Prasher, who originally isolated the GFP gene but was not included amongst...spends some time on Addgene’s role in the rapid dissemination of these technologies, proposing that “Addgene...undermining of the scientific process and the marginalization of science, particularly public health and ... observations and suggestions are not so much inaccurate as already somewhat dated. Zimmer could not have...continue to be a “critical consequence of ...the marginalization of science,” we know now in the summer of 2020...

...a wash solution removes contaminants while nucleic acids stick around. Finally, purified total nucleic...sample can be treated with RNase to remove RNA contamination and you can use a phenol chloroform extraction...extraction to remove remaining proteins and RNase. Finally, DNA is precipitated with alcohol and resuspended...into this mixture to absorb the nucleic acids. Containinants bind cellulose less tightly than nucleic acids...with 1M phosphoric acid and re-used, but trace contamination could interfere with interpreting the results...

... digital PCR is a sensitive assay, so cross contamination into a no template control (NTC) well is common...reduce aerosols that could potentially lead to contamination. Optimize your PCR For a good starting place...place on what PCR parameters to use, see Lock’s seminal paper on AAV titration (Lock et al., 2014). If you...tool for titration and integrity evaluation of recombinant adeno-associated viral vectors." Human Gene Therapy...PMID: 29548643. Lock, Martin, et al. "Absolute determination of single-stranded and self-complementary adeno-associated... PubMed Central PMCID: PMC5915492. Witte, Anna Kristina, et al. "A systematic investigation of parameters...

...gene-transfer tools in the study of the brain and spinal cord, which together are known as the central nervous...transduction of both the CNS and PNS.That is until the Gradinaru Lab at Caltech stepped up to the challenge. Find...is the CREATE method?  CREATE stands for Cre Recombinase-based AAV Targeted Evolution. In this technique...short DNA sequences that contain all possible combinations into a region of the cap gene. In this special... one RGB AAV and red, green, or blue are the predominate colors expressed (figure 4c).   AAV Vector...therapy treatments for neurological diseases). The Gradinaru Lab has a lot going on. Here are the key takeaways...

... input and polling, Blugene's new virus friend finally has a name! We thank you for your participation...* Aavery! Welcome to Addgene, Aavery!       Original post: In December, we introduced you to Blugene...names and we’ve recently chosen four names as finalists. Now, we’re asking you to help us make the decision...meaningful names…. Watch the video to find out the final four! (Note: For cases where the same name was submitted...submitted by more than one person, we selected the finalist based on the first time of submission.)     ...

...cytosine or adenosine deaminases to facilitate a single base pair edit. (Cytosine deaminases edit C-to-T and...and adenosine deaminases edit A-to-G.) In short, the impaired Cas9 localizes the deaminase to where the...This post was originally written by Joel McDade and significantly updated in 2022 by Susanna Stroik.  ...choose this system.  Fig. 1: Cytidine deamination takes place on the free strand of DNA and converts...

... of these CRISPR systems. The original pCas/pTargetF system Our original pCas/pTargetF system consists...Originally published Apr 13, 2021 and last updated Aug 12, 2021. This post was contributed by guest bloggers... to yield pEcCas. In pTargetF, we replaced the original 20 bp specific sequences on pTargetF with the ...of the pEcCas/pEcgRNA system Compared with the original system, the pEcCas/pEcgRNA is superior in several.... The pDonor plasmid pRE57-Ter with multiple terminators can serve to interrupt genes (or gene clusters...

...packages navigated customs and arrived at their destination in a timely manner and at the correct temperature...have partnered with six distributors (Table 1), in China, Korea, Japan, Brazil, India, and Mexico. Table ... Country Served Beijing Zhongyuan 2012 China Summit Pharmaceuticals International 2013 ... e-nnovation Life Sciences 2020 India Sinapse Biotecnologia 2022 Brazil   In some cases...agreement with a distributor for that country. In China, direct shipments weren’t able to clear import control...materials and accept deposits from scientists in China. They were also able to help with sharing information...

...growing amount of interdisciplinary research while at the same time increasing interdisciplinary citations in...continue to rely on printed journal articles as the dominant tool for sharing ideas. This leads to everyday... solutions to complex challenges. As a cross-disciplinary researcher, the challenge of keeping up on studies...interactive chart where you can see the yearly interdisciplinarity index of research for single or multiple ...2009): 858-862. 2. Van Noorden, Richard. "Interdisciplinary research by the numbers." Nature 525.7569 ...