...the sample for entry into Addgene’s repository. For long-term storage in our facilities, every sample is...used for long term storage (one is kept at Addgene and another is sent to a storage facility). The third...is the Laboratory Operations Manager at Addgene.  In addition to helping with daily laboratory operations...courier and then the journey of transformation and storage begins. Some samples are submitted as bacterial...through barcoded tubes that are scanned into our Laboratory Information Management System (LIMS), a database...sample can be tracked easily at each step in the storage process. After the original sample is barcoded,...different types of samples at Addgene - DNA sent from depositors, bacterial glycerol stocks, prepped DNA ready...

...relies on the restoration of a frame-shift in mCherry that restores fluorescence to monitor APOBEC-Cas-9...with the BFP variant, a BE4am-Cas9 base editor, and sgRNA vectors for the BFP-to-GFP conversion and to target...Base editors create specific point mutations in the genome, but they’re inefficient compared to CRISPR...editing reporters that can help you do just that. Monitoring APOBEC and Cas9-mediated editing in real time...time The Harris lab created ACE, a reporter that monitors APOBEC (a family of cytidine deaminase proteins...that disrupts fluorescence due to a frameshift. Restoration of fluorescence can only occur through editing...results in two DSBs that can be repaired by NHEJ restoring mCherry expression. Cells can then be sorted using...

...including terminators and promoters, CRISPRi parts, selectable markers, transformation acceptor vectors, and...therapeutic potential for the split base editors. Find the split base editors at Addgene. Levy et al., Nature ...voltage indicator Article contributed by Shreya Vedantam Genetically encoded voltage indicators (GEVIs)...broad use. This kit is designed for use with acceptor vectors from the MoClo Toolkit from Sylvestre Marillonnet...of Phytochrome B interacting factor 6 (PIF6APB), respectively. The cofactor analog, phycocyanobilin (PCB...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles...sign up here.        AAV delivery of split base editors Article contributed by Jennifer Tsang AAVs are ...

...you out. What is the Open Repository of CRISPR Screens? The Open Repository of CRISPR Screens (ORCS) database...This post was contributed by the Open Repository of CRISPR Screens. Imagine you’ve just discovered that...research. Fortunately, there’s a growing open repository of CRISPR screen data that might be able to help... resource developed by the Biological General Repository for Interaction Datasets (BioGRID) in order to...biologists. Figure 1: The BioGRID Open Repository of CRISPR Screens (ORCS) homepage. Screenshot...Screenshot updated 2024.   Using the Open Repository of CRISPR Screens BioGRID ORCS currently houses over ...metadata for each screen is captured by BioGRID curators, including the cell line/tissue type, enzyme system...

...some good news with you: with contributions from depositors around the globe, we’re now sharing over 100,...100,000 different plasmids available from the repository (currently at 98,276 plasmids as of April 8, ...

...pH. We compared the dry ice-stored samples to controls that were only stored in a -80°C freezer and not...of tubes: the ones we use to store lentivirus and the ones we use to store AAV. Our results showed that...mitigating the pH effects of dry ice storage. To do this, we stored samples on dry ice for 72 hours and...reading a publication describing how short term storage on dry ice can shift sample pH, we wondered whether...medium, which is buffered and contains the pH indicator phenol red. We froze samples in a -80°C freezer...freezer and then moved them to a closed dry ice storage container for 48 hours (to replicate how our viruses...from the dry ice). After 48 hours of CO2/dry ice storage, we then thawed the samples at room temperature...

.... We’re here to help with that! Four important factors to consider for any experiment are: Speed Cost...and colleagues. Here you simply PCR-amplify your vector and your insert, DpnI digest away the templates... time is of the essence. This method requires a vector backbone containing a topoisomerase I cut site,...the type IIS enzymes in the reaction to redigest vector reseals and other incorrect products. This feature...use of toxic ccdB to prevent any non-recombinant vectors from propagating. The ligation-independent methods...Addgene.org0 Check out protocols for plasmid cloning Find vector backbones Read our molecular biology reference ...

...or lentiviral vectors in our previous blog posts.  Viral vector injection Viral vectors allow a tight ...neuronal function directly such as receptor subunits or neurotrophic factors. Another option uses optogentics...is cloned into a viral shRNA expression vector and the vector particles are delivered into the brain region.... On the other hand, lentiviral vectors are superior packaging vectors for larger genes - they can hold...around 1012-1013 vg/ml (vector genomes per milliliter) and lentiviral vectors around 108 IU/ml (infectious...using viral vectors By modulating neuronal gene expression and activity, you can use viral vectors to study...viral vectors blog posts Find tips for preparing AAVs View our Beginner's Guide to Viral Vectors Resources...

...and local movements predominate (e.g. a basal progenitor cell migrating to the epithelium). At this stage...fluorescent proteins can and have been used to monitor cellular movements in the immune system. The techniques...cell imaging, giving researchers the ability to monitor cellular movements in real time. This technique...host-pathogen interactions leading to the understanding of effector function in infected tissues. Before the use of...pathogens and fluorescent cells enables scientists to monitor cell interactions in diverse tissues, including...reveals limited antigen presentation and T cell effector function in mycobacterial granulomas." Immunity...PMCID: PMC4401519. 13. Auffray, Cedric, et al. "Monitoring of blood vessels and tissues by a population ...

... taking up a position on the Addgene Board of Directors. We're excited to watch both Addgene and Sample...How to Be A Good Manager from Addgene Executive Director Joanne Kamens Resources on the Addgene Website... Check Out the Other Members of our Board of Directors Become a Part of the Addgene Community ...

... a Good Mentor? Choosing a Good Mentor for Scientists Will You Be My Mentor?   I Have a Mentor: What now...your own peer mentoring circle. In a Peer Mentoring Group all participants act as both mentors and mentees...Addgene Blog Mentoring for Scientists Series. I have been thinking a lot about Mentoring for over 10 years...mentees. So I experimented with a group mentoring format where 1 mentor met with a group of mentees to get ...her mentoring style. While working on this project, I read the book “Every Other Thursday: Stories and...success. Their stories made it clear to me that, executed correctly, the group mentoring format could work...Science (AWIS) mentoring programs. The groups in these programs typically consist of 1-2 mentors and 3-5 mentees...

...adenine base editors with improved editing in cell lines Nicole Gaudelli, the Associate Director and Head ...Adenine base editors (ABE) mediate A•T-to-G•C base changes (Figure 1), but it can be challenging to make...to create the eighth generation of adenine base editors, called ABE8 (Gaudelli et al., 2020). The generated...to increase the targeting scope of adenine base editors The most commonly used S. pyogenes Cas9 requires...targeting scope for base editing.  Try ABE8 base editors for your research! Base editing in primary human...human cells  Cell therapy approaches can use base editors to correct mutations in stem cells and return the...Reducing off-target editing in ABE8s While base editors are precise molecular tools, they do have undesired...

...transcription factors to increase gene expression. CRISPRa methods vary in their transcriptional activators: some...potent transcriptional activators (p65 and Rta). These transcriptional activators work in tandem to recruit...generally thought of as the “first generation” CRISPR activator. While it requires a relatively simple construct...modest gene activation. Synergistic Activation Mediator (SAM) Description SAM uses specially engineered...levels of gene activation compared to other CRISPR activators, making it a popular method for gene activation... SunTag performs better than first generation activators while showing lower activation levels than SAM...transcription. This complex consists of the VP64 activator used in other CRISPR activation methods, as well...

...A newly established all-in-one vector construction system for CRISPR/Cas9-mediated multiplex genome engineering...systematically established way of making an all-in-one vector for multiplex genome engineering.” Now, they’...ease and efficiency for all-in-one CRISPR/Cas9 vectors expressing up to seven guide RNAs (gRNAs). Sakuma...Schematic overview of the all-in-one CRISPR/Cas9 vector construction system for multiplex genome engineering...expression cassettes of the gRNAs into a single vector in tandem. They went on to show that their CRISPR...CRISPR/Cas9-nuclease and CRISPR/Cas9-nickase vectors work successfully to target multiple sites in cultured...it has not yet been demonstrated, they say the vectors are also likely to work in animal embryos based...

...increasing CRISPR editing efficiency, new CRISPR base editors, DIY DNA ladders. You can find additional hot plasmids... Ouabain Selection  Hot Plasmid 3: CRISPR base editors with enhanced range and specificity. Listen to ...to to the Segmennt of Improved CRISPR Base Editors Hot Plasmid 4: pPSU Plasmids for generating DNA molecular...Out All of Our Hot Plasmids Articles Browse the Repository  ...

...unlikely receptor for PHP.B, and how the Deverman and Wilson labs determined LY6A was a receptor used by....B uses the LY6A receptor to gain access to the CNS. The bottom line of viral vector permissiveness While...pathogens to the CNS, but this also stops viral vectors, such as adeno-associated virus (AAV), from reaching...injection into the bloodstream, but requires a high vector dose and is unable to reach all regions of the ...identified lymphocyte antigen 6 complex (LY6A) as a receptor for PHP.B. Read on to learn how the design of ...also known as stem cell antigen-1 (SCA-1), is the receptor used by PHP.B to reach the CNS; yet LY6A is an...Wilson Labs independently identified LY6A as a receptor for PHP.B by looking for a unique sequence of ...

...voltage indicator Archon Article contributed by Shreya Vedantam  Listen to the voltage indicator podcast... genetically encoded voltage indicators. To expand the voltage indicator toolbox, Ed Boyden’s lab developed... cloned into an entry vector. The 2in1 cloning system contains two entry vectors (pUC57-L1L4 and pUC57...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles...name their most potent Acr-LOV fusion protein inhibitor CASANOVA for “CRISPR-Cas9 activity switching via...CASANOVA? It works with the S. pyogenes Cas9, dCas9-effector fusions, and xCas9, and requires no additional...coordinate emotions, thoughts, and behavior. Historically, scientists study the electrical activity in...

...B978-0-409-90042-2.50021-0  Toh, W. K., Teo, Y. L., Tor, X. Y., Loh, P. C., & Wong, H. L. (2023). Development...Cloning Vectors for Gram-Negative Bacteria. In R. L. Rodriguez & D. T. Denhardt (Eds.), Vectors (pp. 287... inform us on how to create better recombinant vectors. In this blog post, we’ll define plasmid host range...features to create broad host range recombinant vectors.  A plasmid’s host range is determined by the ...groups (Jain and Srivastava 2013).  Recombinant vectors that are made from broad host range plasmids are...different bacterial hosts (Doran et al. 1998). Regulatory elements within the ori also control copy number...The plasmid has its own replication initiation factors and proteins independent of the host. Host range...

...your experiment using the lentiviral vector system. Viral vectors have been increasingly popular in fundamental...primary cells. Amongst the different vectors used, lentiviral vector constructs have proven very useful ...fluorescent sensors. Retroviral vector background Until the development of viral vectors, gene transfer into mammalian...un-transfectable cells, labs use viral vectors and particularly retroviral vectors because they target a broader...attractive choice for a gene transfer vector. Lentiviral vectors Although retroviruses are still in use...generation of lentiviral vectors consisted of three plasmids encoding 1) the lentiviral vector genome which was...lentiviral vector system offers the best safety profile in terms of RCL generation because this vector requires...

...First, some history. In 2008, the Auricchio lab decided to solve the issue of “tiny vector, big transgene... overviews of three split vector strategies plus one additional split vector strategy for increasing the...homology directed repairs (HDR). Split AAV vector approaches Split vector approaches increase the size of the...copies of both vectors. Examples of use: Halbert et al., 2002 used an overlapping vector with 440 or 1000bp...splice site donor and acceptor sequences and the homologous region of the two vectors can be from one of ... hybrid vector, as measured by β-galactosidase activity, was 81% of that from a single vector, but significantly...ideal split vector system. Overall the efficiency of transgene expression from split vectors has been inconsistent...