...most cases, interested in transferring genes to mammalian cells (i.e. transferring DNA), yet retroviruses...producer cell membrane, and acquired as an envelope during exocytotic budding, as the viral particle emerges...transfer of RT, which would actually occur in step 2 during uncoating. After uncoating, reverse transcription... integrated by the viral integrase protein (IN) during step 3 (King et al., 2008) (Hu & Temin, 1990). ...

... the Addgene blog Learn more about plasmid sharing during COVID-19 Resources on Addgene.org Find plasmids...False, type='scriptV4', hide_playlist=True, viral_sharing=False, embed_button=False, autoplay=False, hidden_controls...to follow an exponential decay model, with dips during weekends and significant holidays. Figure...likelihood by the corresponding factor 𝜙z if t is during event z. The "events" we considered for our model...

...suppose you have a piece string that’s 10 meters long. You twist and roll the string into a tight ball. Now...in a way that brings linearly distant regions close together. gRNA Selection and Filtering for Off-target...Now the two ends of the string, instead of being 10 meters apart, are mere millimeters apart. Similarly...

...bacteria and prevents sensitive bacteria from acquiring resistance genes. Yosef et al. suggest that it...not previously infected by the lysogenic phage, rendering the population sensitive to antibiotics. Created... Lemire, Diana P. Pires, and Timothy K. Lu. “Engineering Modular Viral Scaffolds for Targeted Bacterial...Addgene Blog CRISPR Methods for Bacterial Genome Engineering Treating Muscular Dystrophy with CRISPR Gene ... Mary Gearing...

...presence of antigens (such as viral vectors) without triggering a destructive inflammatory immune response. At...after the cloning of the RPE65 gene, a naturally occurring animal model, the RPE65−/− dog, was characterized...retinal neurons. Nowadays a toolbox of naturally occurring and engineered AAV serotypes that further expands...being done to further improve the technology: engineering of new capsid variants, designing expression ... optimize transgene levels, and large scale manufacturing of viral vectors to support trials for prevalent...

...If you're interested in sharing your science, one of the big questions you'll be faced with is "how?"...for longer educational videos, and Twitter, for sharing updates, new deposits, and new blog posts!  ...  of proficiency.  What kind of content are you sharing?  Sometimes your subject matter will drive the ...fair, or a semester-long after school program? Comparing your desired outcome to the limitations and potential...response. High connection levels and interaction. Can bring in things to be physically interacted with. Public...

...studying chemical engineering. Mengtong "Tom" Duan is a PhD student studying bioengineering. They are both...smaller genetic footprint, and nonlinear acoustic scattering. The best candidate for this was the Anabaena...was sufficient to saturate expression. When engineering a cell line, cotransfect the mARG cassettes 2...acoustic reporter genes for real-time in vivo monitoring of tumors and tumor-homing bacteria. Nat Biotechnol...

...expand and exploit CRISPR, both of which we're featuring in today's blog. Cas7-11 Development and Discovery...alongside some creative (and significant!) protein engineering. They combined Prime editing with an integrase...potential applications. But they weren’t done engineering quite yet – surely, they thought, the integration...optimized the specific attachment sites via sequence engineering, enabling even more efficient integration activity...: Adding Precision and Flexibility to CRISPR Engineering CRISPR 101: RNA Editing With Cas13  CRISPR Cheat...

...editing reporters that can help you do just that. Monitoring APOBEC and Cas9-mediated editing in real time...results in two DSBs that can be repaired by NHEJ restoring mCherry expression. Cells can then be sorted using...editing efficiency can be easily determined by comparing cells expressing only eGFP expression to cells...real-time, it relies on DSBs which is not ideal for monitoring single base edits (St. Martin et al., 2018). ...the Harris Lab circumvents the need for DSBs in monitoring on-target DNA editing efficiency of APOBEC-Cas9...

...viral genome). Empty viral capsids can be formed during viral production depending on the  experimental...usually report? Addgene and other viral vector manufacturing facilities report the physical titer of a viral...to two-fold. How can we get more reliable AAV titering? For our viral service, we currently titrate AAV...different sources in the same experiment, consider retitering both AAV in your lab. The absolute titer values...Addgene, we don’t reserve lots, but if you’re reordering the same virus feel free to email us and we can...

...pyogenes (SpCas9), is truly an exceptional genome engineering tool. It is easy to use, functional in most species... cell stress, and RNA size distribution. When comparing LwaCas13a to shRNA, they found that LwaCas13a ...vivo RNA imaging. In short, the Cas13a molecule brings a much anticipated element of diversity to the ... the CRISPR field. We feel that the culture of sharing within the CRISPR community is an essential part...scientific community. Note: Cary Valley and Mary Gearing contributed to the update of this post. References...

... data. Although it may be useful to selectively bring particular items into an ELN – e.g. an image that...the integration with protocols.io, the protocol sharing repository. As illustrated in this video the integration...RSpace becomes part of a workflow that enhances the sharing and reproducibility ecosystem protocols.io makes...help with searchability, record-keeping, and data sharing, not all of them support reproducibility in the...Reproducibility Read all blog posts about scientific sharing Resources on Addgene.org Find protocols of molecular...

...software. When you are happy with your selection, ordering the sgRNA is simple and easy. Several companies...but fret not! The key to saving time and energy during your in vivo work is to screen you gRNA in vitro...in mice to the day you have your F0 generation (barring life’s little accidents and stuff ups). This is...multiple genes by CRISPR/Cas-mediated genome engineering." Cell 153.4 (2013): 910-918. PubMed PMID: 23643243...Addgene Blog Get the Basics of Mouse Genome Engineering with CRISPR Validate Your Genome Edit Learn about...

...resulted from excision by uracil N-glycosylase (UNG) during base excision repair. Adding a second copy of the...off-target mutations than those derived from naturally occurring cytidine deaminases. Adenine base editors The ...compared to ABEmax. In an alternative approach, engineering and directed evolution of the TadA domain has...) This post was originally written by Mary Gearing in August 2016 and was updated by Jennifer Tsang...Y., Guan, Y., Siwko, S., … Li, D. (2023). Re-engineering the adenine deaminase TadA-8e for efficient and...applicability of base editing through protein engineering and protein delivery. Nature Communications, ...

...unfamiliar, scFvs are often the building blocks for engineering proteins. So let’s get acquainted with scFvs....CD3 on T cells and a tumor-specific antigen, it brings T cells to a tumor site. ScFvs can also be fused...viral vectors like AAV, which could be useful for delivering scFvs that inhibit HIV.   References and Resources...Bruccoleri RE, Haber E, Crea R (1988) Protein engineering of antibody binding sites: recovery of specific...more about antibodies  Read about another reagent sharing platform for antibodies, the Developmental Studies...

...Proximity labelling relies on the labelling of neighboring proteins within a cell with biotin. Biotin, is...promiscuous biotin ligase that can efficiently label neighboring proteins The first biotin based proximity labelling...from the ligase and bind to exposed lysines on neighboring proteins. Thus to capture protein complexes, ...electron-rich amino acids such as tyrosine on neighboring proteins resulting in their biotinylation. The...The advantage of APEX is its speed, labeling neighboring proteins in minutes rather than hours like BioID...

...proven useful in the engineering of riboswitches. Riboswitches are naturally occurring regions in the 5’ ...into the content of Addgene’s aptamer soup by exploring the development and applications of fluorescent...Warner et al., 2014); its length reduces artefacts during live cell imaging. Bunch of baby spinach, or BoBS...transcribed by RNA Polymerase III (5S, tRNA and U6) and measuring Corn fluorescence by flow cytometry, the Jaffrey...

...also ideal for signal amplification during these experiments. During detection, a biotinylated reporter... biotin. Thus something in the egg white was sequestering biotin from being absorbed. In 1940 Esmond E...can be added to either terminus of a DNA sequence during a Polymerase Chain Reaction (PCR) by using a biotinylated.... Biotin can also be enzymatically incorporated during PCR using a biotin labelled dUTP. There are multiple...

...then used to measure the frequency of each variant during the selection process. Each variant is assigned...of MnCl2. Disadvantages of this method include favoring of certain types of mutations, difficulty controlling...then both strands of the plasmid will be nicked during the first BbvCI digest and the whole plasmid will...direction, both strands of the plasmid will be nicked during the first BbvCI digest and the whole plasmid will...Douglas M., Jason J. Stephany, and Stanley Fields. “Measuring the Activity of Protein Variants on a Large Scale...

... nonprofit institution, you can order plasmids covering a wide range of fields from Addgene. What you ...4740. You can find more info on pCri in Mary Gearing’s blog post on the system Easing the Protein Purification...Seq kit has been used in microbiology studies monitoring sequence dynamics under various selective pressures... can lead to surprisingly large fitness effects during adaptive evolution of antibiotic resistance." Proceedings...2016): 895-899. Choudhury, Rajarshi, et al. "Engineering RNA endonucleases with customized sequence specificities...

...post-translational modifications, identifies a staggering ~250,000 phosphorylation events in the proteomes... led to the eventual discovery of a naturally occurring phosphoserine-tRNA synthetase that serves as an...elongation factor that accepts Sep-charged tRNAs during translation. Many ‘flavors’ of Sep-OTS plasmids...pSer-containing GFP that can be used as a positive control during optimization experiments. This version of GFP will...dephosphorylate the pSer-incorporated protein, ideally bringing the phenotype assayed closer to that of the wild...

... require anaerobic conditions. In experiments requiring anaerobic conditions, proteins that bind endogenous...bilirubin (Kumagai, 2013). Next to these naturally occurring co-factor binding proteins, systems wherein a ...proteins can be accurately determined in vitro by measuring the fluorescence intensity of purified protein...usually dim relative to fluorescent probes, recent engineering efforts have increased the brightness of these..., Marieke, et al. "A FRET-based biosensor for measuring Gα13 activation in single cells." PloS one 13.3...

...How much do you need [for your lab start up]?” A hiring chair or dean will often ask a faculty candidate...community is the ultimate value proposition. By preparing a budget before hand, you’re saving the institute...turned down a faculty position because none of the offering institutes were able to provide the resources ...If the resources aren’t available, then you’re entering a win-lose situation that can ultimately set you... be afraid to walk away from a negotiation. If securing a faculty position is your goal, then a resource...

..., and as of today, will stop posting, monitoring, and resharing content on X.  Addgene deeply values our..., TikTok, Facebook, and LinkedIn. We are also exploring other social media platforms with active scientific...in the scientific community. Addgene has been considering a gradual move off X as a response to major platform...

...the recent Keystone Meeting "Precision Genome Engineering and Synthetic Biology". His reflections on the...scientific community. Data on CRISPR and synBio plasmid sharing In a recent review on CRISPR technology, for example...online user forums such as http://www.genome-engineering.org and http://www.egenome.org." At the Keystone...Keystone Meeting Eric presented a poster with data comparing Addgene global deposit and distribution trends...

...accosted by dozens of tiny black dots during our stay there. Yet capturing the flies is nearly impossible, ..., due to their small size and inconspicuous coloring.  FLIS plasmid Recently, the Phuels lab deposited...everywhere find and re-contain any specimens that escape during routine lab activities.  Here, we demonstrate the...

...treated identically, used to assess naturally occurring random biological variability. If one were studying...species-on-species staining can be partially addressed with stringent staining protocols and the use of isotype-specific...standard protocol, but the primary antibody is omitted during the incubation step. Samples are incubated with...will briefly touch on.  Western blotting When comparing samples in different lanes in a western blot, ...controls address the spectral overlap that occurs during multiplexing. Compensation controls are single ...

...cell-type specific. If it is something you’re considering for your experiment, note that you cannot extrapolate...hardware types, construction, and testing used for delivering your light stimulus. This can make or break your...Photoswitching Proteins (Vol. 2173, pp. 21–51). Springer US. https://doi.org/10.1007/978-1-0716-0755-8_...2019). Cortical layer–specific critical dynamics triggering perception. Science, 365(6453), eaaw5202. https...

...bound repressor then prevents transcription from occurring, which means that transcription is now OFF.   ... ADH1 promoter pADH1m, which maintains activity during the late ethanol consumption phase of yeast culture... al. Using Binary Systems Together You may be wondering, “why is it necessary to have all 3 of the different...experiments! This post was originally written by Mary Gearing in 2018, and updated by Hannah Dotson in Dec. 2022...Introduction and expression of genes for metabolic engineering applications in Saccharomyces cerevisiae.” FEMS...

...impossible. With the genome engineering revolution came epigenome-engineering tools - zinc finger nucleases...enhancer regions (Mendenhall et al., 2013). By comparing gene activation when enhancers were active or ...Sauka-Spengler T (2017). Genome and epigenome engineering CRISPR toolkit for probing in vivo cis-regulatory... Mary Gearing...

...Series. We’ll be covering the basics of science communication, tips and tricks for sharing your science with...Addgene TikTok at the end of this month! We’ll be sharing our educational resources and tips and tricks for... now! (Bonus: Tim kindly offered to finishing answering all submitted questions via blog post, which we...

...CRISPR during his postdoc 12:01 - 13:21 : The Wyss Institute for Biologically Inspired Engineering and why...the Wyss institute for Biologically Inspired Engineering. Alex is the first MD PhD we've had on the podcast... of medical school. 7:05 - 10:55 : Alex's focus during graduate school - DNA damage and longevity. 10:...

...I can bring to Addgene -- that comfort with saying, “if we do this, then what?” I hope to bring that business...my personality. Another big part of my role is bringing experience and judgement to a wide variety of ...business world from the mindset of a lawyer can bring a new perspective to an organization. I'm used to...

...successful and the importance of lentiviral vectors in ensuring that success Chapter 6 - Developing Addgene’s ...Future, the research team, and the importance of sharing information/knowledge Outro Many thanks to our...received his Bachelor of Science degree in Computer Engineering from Tufts University and his Master of Arts ...Bioworks. He also holds a SB and a Masters in Engineering from MIT, both in Computer Science.   Special...

... grown steadily during 2014. We were excited to have more and more scientists offering to share their ...Will You Be My Mentor? Finding and Asking for Mentoring Support By Joanne Kamens Plasmids 101: Antibiotic...Margo R. Monroe Best wishes for your research during this holiday season and we look forward to seeing...

...protein can also influence antibody binding by altering the relative concentration of potential antibody...to validate that your antibody is specifically capturing your RNA-binding protein of interest. So you ...antibody’s trustworthiness. You may also consider sharing your validation attempts by other means, such as.... These outlets can be particularly useful for sharing negative data. Negative data often goes overlooked...antibody (perhaps one from Addgene!), consider sharing your results so that others in the community can...

...are thought to interrogate dsDNA for proper base pairing through minor grove contacts, and thus do not read... do with the slight melting of the 5 ́-terminus during the reaction. This “peeling back” of the 5 ́-phosphorylated...upstream probes and four down-stream probes, each differing only in the base at the ligation junction. Thus...prepared, each with a different complement strand covering all 16 possible NN pairs in the template. The ...The probes were designed such that each possible pairing resulted in a product of a different length, with...

...on how they've managed to keep themselves happy during their time in grad school. As you'll learn, grad... hard about picking a good mentor, and begin considering career options early. Tune in for great advice... in grad school? As part of this discussion, we bring up a performance Ben and Niroshi organized for Tyler's...Depace's Talk on "Precision and Plasticity in Mentoring" We'd like to thank Niroshi and Ben for taking...

...can help with plasmid sharing! Judging by how many prizes were given out during the lunchtime pizza and...see a real working lab full of busy Addgenies preparing plasmid samples for shipping. This was the favorite... that are offered with a biology major.” After hearing about many Addgenies’ career paths, Michelle Healy...

...articles appearing in lower-prestige journals has risen steadily from 1995 to 2013. Considering citations...publications alone aren’t enough. Scientists are sharing what they call “raw science” – data sets, code,...researchers to track and share their own libraries and offering another way to consider and quantify the impact...

...exciting developments surrounding CRISPR genome engineering in the past year, we're excited to announce that...haven’t already used it yourself, you’re probably wondering: What does it do and how does it work? LentiCRISPR...work probing mTOR signaling in cancer cells, deciphering the mechanism of matrix metalloproteinase-9 secretion...Addgene and look forward to another great year of sharing!   References 1. Shalem, Ophir, et al. "Genome-scale...

...focused on our core mission to promote scientific sharing. Listen to learn more about Darrell, his research...repair, stem cells, gene therapy, and tissue engineering. How the multidisciplinary nature of the Center...practices and promotes open science in the lab - sharing reagents, cell lines, data, and knowledge without...Addgene board of directors - increasing open source sharing and helping us stick to our core mission. Chapter...

...cells using flow cytometry, you are typically measuring the presence or absence of certain markers on ...photon is emitted. This step is called emission. During excitation, some of the photon's energy is lost... red laser excites at 640 nm (Table 1), while comparing the two fluorophore's excitation spectra around...you can see in Figure 1, emission spectra of neighboring colors can and do overlap. For example, APC overlaps...hypothetical experiment in the lab. Let's say I am culturing HEK 293T cells in vitro. I transduced the cultured...

...in order to directly show that, for example, the firing of a specific type of neuron in a specific brain...whether you wish to stimulate or inhibit neuronal firing, the cell-type specificity, the kinetics of the... which results in depolarization and subsequent firing of an action potential. Halorhodopsins (HR) are...may more effectively silence neurons than HR. Delivering your opsin  The standard opsin delivery method...science writing. Derek is also a pianist and an aspiring composer. View his blog drsimonsaysscience.org...

...Addgenies Brook Pyhtila and Nicole Waxmonsky Resource sharing shortens the time needed to go from planning an... experiments. Addgene’s dedication to resource sharing has led us to develop a searchable and sortable...can search by any part of the data. For example, entering ‘C. elegans’, or ‘elegans’ will filter the table...CRISPR Technologies Available Through Resource Sharing. 2016. In press. Additional Resources on the Addgene...

...unmatched customer support, as this is vital to ensuring that our services help accelerate your research...support team that can help you through any search, ordering, technical, or quality control issues. Improving...within the widget, you’ll navigate to articles answering your questions making it easier than ever to get...describe your issue and press send. Doing so will bring your concern directly to our support team’s attention...

..., Flp, KD, R, B2 and B3, approximately 50% of offspring carried both the recombinase and GAL4, indicating...normally and thus nontoxic. In contrast, 0/79 offspring carried both Cre and GAL4, further emphasizing...and relevance for the efficiency of BAC/PAC recombineering techniques. Semprini S, Troup TJ, Kotelevtseva... Mary Gearing...

...success.” 2. “Enjoy Teamwork.” Be friendly, open, sharing, rather than intensely competitive. Be curious ...society. We need much, much more science and engineering (STEM) expertise in law, business, journalism...connected to scientists and use the skills they learned during their studies. They suggest that you make time ...Connie Cepko, Tom Ellis, and George Church for answering our burning questions about how they manage and...

...stage you could infer that the gene which GFP was monitoring was being expressed in the same pattern. These...result but will still allow for expression pattern monitoring. Enhancer traps Like promoter traps, enhancer...unambiguously identify the gene that they are monitoring as they are directly integrated into it. Enhancer...e67667. DOI: https://doi.org/10.7554/eLife.67667 Springer, S. P. Gene Traps. Plant Cell. (2000) 12(7): 1007...

...can remain present until cleared by the body. Administering the drug once can result in several hours of... Controlling Stimulation Some studies rely on delivering reversible or varied amounts of stimulation to...injection. The main challenge in using ligands is ensuring the agonist is able to cross the blood brain barrier...Bryan Roth lab for feedback on this article and sharing their expertise with us. References Urban DJ, Roth...