...zebrafish, and mice. Second Generation iGluSnFR: SF-iGluSnFR In the second generation of iGluSnFR, the Looger... for Synaptic Imaging However, the first two generations of iGluSnFR had limitations: they were not optimal...extrasynaptic glutamate release. Building on the previous generation of the SF-Venus-iGluSnFR (itself an improvement...not neurons). iGluSnFR3 is also available in several ready-to-use viral vectors.    References and ...

...encoded fluorescent executioner caspase reporters. Several apoptosis reporters have been developed, such as... and fluorescence based reporters however have several limitations. First, FRET signals are often difficult...They then optimized the length of both linkers to generate a reporter that would fluoresce: FlipCherry. FlipCherry...array of diseases and pathologies such as neurodegenerative diseases, autoimmune disorders, and cancer...Cabantous, Stéphanie, et al. "A new protein-protein interaction sensor based on tripartite split-GFP association...

...SCF-TIR1 ubiquitin ligase complex, promoting its interaction with the degron sequence and ultimately its degradation... plant protein TIR1 in non-plant cells, which interacts with the cells’ endogenous ubiquitin ligase, the...with mAID. Using this method, the lab was able to generate conditional alleles of a few essential proteins...attempted to engineer loops to create genomic interactions that wouldn’t normally occur. The synthetic ...enhancer. What makes LADL unique from previous iterations is the addition of CRY2, another Arabidoposis...

...to edit. These cells are then transfected with several different constructs for the reporter to work and...your CRISPR experiments. But how? There have been several systems to do this but most are labor-intensive... the native methionine start site. This first iteration was named GEmCherry1, due to the “genomic fragment...and 21 to create GEmCherry2. The Denham lab made several other small changes creating 2 other versions but...  Find GEmCherry2 reporter plasmids at Addgene Overall SRIRACHA and GEmCherry2 reporter systems provide...

...the target molecule. Although it’s possible to generate antibodies against many types of molecules including...recognize proteins. Figure 1: Steps in the generation of hybridomas for monoclonal antibody production...19420862.2018.1445456  Holzlöhner P, Hanack K (2017) Generation of Murine Monoclonal Antibodies by Hybridoma ...(DC): National Academies Press (US); 1999. 1, Generation of Hybridomas: Permanent Cell Lines Secreting...hybridomas  Learn about monoclonal antibodies for immunotherapy Resource on Addgene.org Check out Addgene’...

...and grades that serve as automated filters of consideration. If you are worried about any eligibility provision... under two pages unless you are applying for a Federal Government opportunity, which tends to be more ...on grammatical errors, sentence structures, and overall format (fonts, font size, legibility, and more)...set of common questions with a peer or mentor. Several days prior to the interview, inspect your technology...come along for the ride, and consider applying to several opportunities. In an age of scarcity, the rational...

... software development team was working on lab operations software for next-gen sequencing (NGS), and high-throughput...did not. It was my job to translate complex lab operations workflows into a format the developers could ...could understand, so they could code the best operations software possible. Similarly, when the developers...a good fit for you if you are energized from interacting with others, and have strong communication skills... get software product management internships, preferably on teams that run within the agile framework ...

...cleavage promotes collateral cleavage of other RNAs. As with other Cas13s, this collateral cleavage does ...and Yan et al. describes new Cas13d enzymes that average only 2.8 kb in size and are easy to package in ...possibilities. To identify Cas13d, both groups used very general bioinformatic screens that looked for a CRISPR ...accessory protein RspWYL1 increases both targeted and collateral RNA degradation by RspCas13d. RspWYL1 also increased...

...successful mutation of your favourite gene, there are several different methods available. Thankfully, there ...validation, such as this "CRISPR 101" post. Next-generation sequencing technologies are the gold standard...emerging validation technology. Digital PCR has several advantages over mismatch nuclease assays that will...desired genome edit. Since exploitation of NHEJ to generate translational reading frame-altering indels for...validation methods As mentioned, these assays have several major advantages over the commonly employed mismatch...

...components of these vectors are also split across several plasmids (often three), for safety purposes. The...The 5’ LTR and 3’ LTR, respectively, act as RNA polymerase II promoter and terminator regions and are slightly...factors displayed on the outside of the particle to interact with the host. This is the role of Gag proteins...of lab evolution. There are 1st, 2nd, and 3rd generation plasmids for their production. As such, there...replication, with E2B being the primary viral polymerase. E4/E1A activate transcription of other relevant...

...previously a large barrier for gene therapy. In fact, AAV gene therapies have already been approved in the...non-integrating) as lenti and retro viruses do. They are generally used to achieve transient, high expression of ... Broad Tropism Only Infects Dividing Cells Moderate Immunogenicity Insertional Mutagenesis Possible...Lentivirus Listen to Connie Cepko Discuss Gene Therapy with AAV Figure out How to Get the Most from Your...

...and fostering team success. This shift requires deliberate preparation and a willingness to embrace new ...the room," understanding team motivations, and leveraging tools like the Myers-Briggs Type Indicator or...technical and managerial roles, and I've witnessed several colleagues do this while changing industries and...Courses: Leadership programs on platforms like Coursera, LinkedIn Learning, or edX. Podcasts: HBR IdeaCast...Scientists: What Makes a Good Leader Anyways?  Transferable Skills Guide: Managing a Team  ...

... a structural precursor protein, and Pol is a polymerase. Envelope gene (may be pseudotyped to alter infectivity...γ-retroviral packaging plasmids are not interchangeable. General envelope plasmids, such as VSV-G (Vesicular Stomatitis...packaging cell line. For example, Phoenix, a second generation γ-retrovirus packaging cell line developed by...region of the 3’ LTR and are primarily used in gene therapy research to reduce promoter interference and/or...More resources on addgene.org Addgene Protocols: General Transfection The Addgene Retrovirus Guide  ...

...single color controls On a flow cytometer, you generally want your positive cells to read between 104 and...provided by the antibody companies or check the literature. Pro tip! Early gates, like your live/dead markers...population (b). There is some debate in the literature as to when you need isotype controls. Our lab...isotype controls, while others don’t. However, the general consensus seems to be that if you are looking for...research background in CD8+ T cells and cancer immunotherapies. She is currently a research technician in ...

...can I use it? CasPEDIA operates like many search engines, featuring both a general search bar tool and an... trans activity. AsCas12a, on the other hand, generates staggered DNA nicks at its target in cis and cleaves...resources throughout the site. In the Experimental Considerations section on each enzyme page, you'll find a ...packaging limitations. Maybe you want to read literature on the less common applications of an enzyme ...

...publication in a scientific journal requires several considerations. First, it is important to establish the...whether your paper is in scope for the journal’s coverage. These guidelines also provide information on ...delay publication and manuscripts may go through several rounds of revision before acceptance. F1000Research...prerequisites of the journal. Ask colleagues (preferably native English speakers) to proofread your manuscript...

...on DNA purification without a kit that outlined several reasons why doing something without a kit has advantages...everything, including your skin. Here are some general tips for working with RNA, even if you are using...method, so biotechnology companies have marketed several products that help to further streamline this process...quantitative PCR to assess gene expression. There are several things that researchers can do to combat this issue...and proteins. Lastly, using a DNase (there are several DNase enzyme products on the market to choose from...

...secondary considerations to validation, application specificity, and experimental considerations - no other...rarely work for every assay. Antibodies that are generated against a linear antigen tend to work very well...permeabilization will influence how the antibody interacts with its target and consequently the quality of...isolate an antibody’s immune complexes and confirm interacting partners by mass spectrometry (Uhlen, 2016). ...

...studied a synaptic protein that is linked to neurodegeneration. The level of this protein is decreased in...method for the visualization of protein-protein interactions in situ. In the standard PLA assay, primary ...endogenous proteins of interests, suspected to interact, are added to a cellular sample. Both antibodies...visualization of the location of the protein-protein interaction. To study protein levels in situ, the Schumann...protein plasmids Use FRET to study protein-protein interactions Deposit plasmids with Addgene! ...

... cloning, you’re probably aware that there are several methodologies currently available for approaching... and Gateway® cloning technologies, as well as several others. Each method is unique and relies on specific...to propagate because CcdA wasn’t available to counteract the toxin. If, however, an investigator were ...answer lies in special strains of E. coli that tolerate the expression of the toxin gene. One such strain...plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes.” J Mol Biol. 1992 Aug 5;226(3)...