We narrowed to 835 results for: Eras
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TypeBlog PostPublishedJuly 14, 2022, 1:15 p.m....including ours at Addgene, the overall purpose of a data pipeline is to perform several sequential computational...enabled us to rapidly iterate and optimize our pipeline. After reviewing and testing several platforms for engineering...parallels between Seqera’s mission and our own, as both address the need to accelerate science using different...sequencing at our facility using Illumina MiSeq Next Generation Sequencing (NGS) technology. As our repository...search for solutions led us to a partnership with Seqera Labs. DNA sequencing drives our bioinformatics...sequences. More than half of these sequences were generated by Addgene as part of our QC process. Automation...other programs running on a computer, so it safely operates in isolation, and can be easily shared with others...
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When Fidelity Matters: A frank discussion about ligase fidelity
TypeBlog PostPublishedMarch 6, 2018, 1:49 p.m....survive high temperatures (up to 95 °C) and is active over a range of elevated temperatures (37–75 °C)....any fragments end-to-end without the exo/polymerase generation of annealed complementary regions. Why ...elevated temperatures, allowing further discrimination by incubating the ligation at a temperature near the...Reaction Temperature Calculator can be used to estimate an appropriate incubation temperature when using...restriction cloning), adaptor ligation for next generation sequencing (NGS) library construction, and circularization... does fidelity mean for a ligase? DNA ligases generally prefer fully Watson-Crick base-paired dsDNA substrates...as a correct base pair. DNA ligases have been generally found to have a higher discrimination at the upstream... -
Antibodies 101: Beyond Surface Labeling
TypeBlog PostPublishedJune 27, 2024, 1:15 p.m....through the biotin-streptavidin interaction (Figure 1a). This interaction brings the fluorophore in proximity...divisions). Figure 4: Proliferation dyes can visualize proliferative capacity and speed of cells...research question and downstream application, one or several of those techniques can be used alongside standard...biotin-streptavidin binding and (b) antibody host species interactions. Created using biorender.com. Another way...perk is that one primary antibody can be bound by several secondary antibodies, increasing the fluorescence...are now stable for much longer, even at room temperature, allowing for a flow analysis to be shifted to...applications of dye stains in flow cytometry are cell proliferation and cell viability. Unlike intracellular labeling... -
Hot Plasmids: Spring 2025
TypeBlog PostPublishedJune 3, 2025, 1:15 p.m....important to know what proteins interact with each other and where those interactions occur in the genome. This...iGluSnFR4f, and iGluSnFR4s. C) Spike-triggered averages for single action potential-evoked axonal glutamate... and provide step-by-step protocols for viral generation and stable cell line creation (Figure 2). Looking...Lenti CMV HA-EGFP Puro (Addgene #236081), then generated lentiviral particles in HEK293T cells with pMD2...much to learn about their myriad functions and therapeutic potential. Our partners at the Institute for ...IPI Glypican Collection here! Figure 3: General glypican domain structure. Glypicans are proteoglycans...Abudayyeh-Gootenberg Lab developed this tool based on several new site-specific retrotransposon elements they... -
A Control for All Seasons
TypeBlog PostPublishedOct. 5, 2023, 1:15 p.m....to detect this undesirable interaction. When non-specific interactions are suspected, use a preadsorption...controls for biological experiments, starting with general controls and then moving on to controls for antibody-related...interpretation of results. A positive control is generally a sample or group that will have a desired response...experiment above, you could repeat the tau western blot several times with the same samples and compare the results...results across runs. If the results vary considerably, then the high degree of variability in the process...antibody-only staining will allow you to see nonspecific interactions between the sample and the secondary antibody...-specifically to other molecules through the interactions of other antibody structures, such as the Fc... -
Antibodies 101: Multiplex Immunofluorescence
TypeBlog PostPublishedMay 23, 2023, 1:15 p.m....The general process is similar to standard immunofluorescence, but with some extra considerations (Im ... same considerations apply as for standard IF. However, there are a few specific considerations to keep...there is (quantification). But what if you’ve got several interesting protein targets and you want to know... in the cell? Or you know that the presence of several biomarkers together in the same tissue sample predicts...just what you need! How does it work? There are several ways to achieve multiplex IF. One of the most common...a good place to get started. One important consideration when deciding whether you can do simultaneous...or conjugate with an enzymatic label such as horseradish peroxidase (HRP). Experiments using enzymatic... -
Truncated gRNAs for Regulating Gene Expression
TypeBlog PostPublishedJan. 10, 2017, 3:37 p.m.... these minor gRNA alterations can be exploited to quickly and inexpensively generate a potent Cas9-based...inactivate a gene or introduce a desired genetic alteration. Listen to Our Podcast Interview with Alex Chavez...Schematic of native (nuclease-competent) Cas9 interacting with either a full-length gRNA (20nt complementarity...from different bacterial species, each of which interacts with a distinct gRNA that allows users to decide...Although this is possible, this strategy suffers from several limitations. Perhaps most importantly, most Cas9...instance, when native (nuclease-competent) Cas9 interacts with a ≤15nt gRNA that contains an RNA adapter...induces potent gene expression with no genetic alterations observed at the target site. Figure ... -
CRISPR 101: Mammalian Expression Systems and Delivery Methods
TypeBlog PostPublishedSept. 24, 2015, 2:30 p.m....each, and potential delivery methods. General considerations for planning your CRISPR experiment As ...Wildtype Cas9 or Cas9 nickase are appropriate for generating knockouts or knock-ins, or introducing mutations...PCR followed by gel electrophoresis and/or next-generation sequencing more appropriate? If you already...have an end product in mind, steps 1 and 2 will generally be straightforward. Likewise for step 4, as this...system, a good first step would be to consult the literature to see if anyone has published a protocol that...enrich positive cells or a selection marker to generate stable cell lines. Transient or stable expression...positive cells. Likely contains a selection marker to generate stable cell lines. Packaging and Envelope plasmids... -
The CRISPR Software Matchmaker: A New Tool for Choosing the Best CRISPR Software for Your Needs
TypeBlog PostPublishedNov. 3, 2015, 3:30 p.m....this reason I generally recommend that laboratories that have already attempted several CRISPR experiments... a part of a group (Biology of Host-parasite Interactions, Institut Pasteur, Paris) that changed genome... in highly similar content and the absence of iterative design; it is caused by not having the time to...to select a tool based on your project and not general opinion. I could spend a lot of time running through...functionality as well as how the user is expected to interact with the tool (a screen shot and overview of the...primer design are more and more common. User interaction: Software design elements that fall into this... category describe how the user is expected to interact with the software. This category is important ... -
AAV Titers: Where Do They Come From And What Do They Mean?
TypeBlog PostPublishedNov. 15, 2017, 3 p.m.... by the temperature parameters. For example, we found that changing the annealing temperature of the PCR...the composition of a viral vector solution, and generally these methods measure different characteristics...depending on the experimental conditions used to generate AAV. One study showed that approximately 50% ...preparations. Addgene has used this method to generally validate our viral vector preparation protocol...: Absolute quantification by qPCR requires generating a standard curve of known concentration. We regularly...qPCR assays, we ensure that the standard curve generated is reliable, and can thus be used to accurately...Paolo, et al. "Systemic errors in quantitative polymerase chain reaction titration of self-complementary... -
New Acoustic Reporter Genes: Ultrasound Imaging of Gene Expression
TypeBlog PostPublishedJan. 24, 2023, 2:15 p.m....first-generation bacterial ARGs (bARGs), used in bacteria, expressed poorly at body temperature (37oC)... transposase and outgrow the cells for a several generations to dilute the unintegrated plasmids (Fig....imaging was used. We set out to develop next-generation bARGs that could scatter ultrasound nonlinearly...Serratia sp. 39006. We next tested bARGSer with several inducible expression systems in E. coli. The arabinose-inducible...L-arabinose at 37oC for at least 12 hours, or at lower temperatures for longer. Cultures induced with arabinose ... ARG expression in mammalian cells The first-generation mammalian ARGs (mARGs) enabled ultrasound visualization...in deep tissue. However, not unlike the first-generation bARGs, poor expression and acoustic properties... -
Antibodies 101: Immunoprecipitation
TypeBlog PostPublishedDec. 7, 2021, 2:15 p.m.... modifications, or study how a target protein interacts with other proteins or nucleic acids. Read on ...affinity for immunoglobulins, the strength of the interaction varies between antibody host species and isotypes...subsequent Western blot. When choosing beads, there are several factors to consider. Agarose beads are sponge-like... can cause loss of beads, break weak protein interactions, and limit high throughput procedures. However...cost-effective option. Target protein/antibody interactions To prepare for an IP, samples are typically ...to preserve native protein conformations and interactions. For difficult to release proteins, including...preclear the sample to remove any proteins that may interact nonspecifically with the capture antibody or Protein... -
Healthcare Consulting: A Door to the Business of Life Sciences
TypeBlog PostPublishedOct. 13, 2016, 2:30 p.m....companies about the pricing and coverage of drugs, and developing internal operational capabilities. In just ...of combining targeted molecular therapies for cancer with chemotherapies, or developing innovative mobile...experts to work on “cases”. Cases present specific operational or commercial challenges or questions that clients...clinical research to discover and test the best new therapeutics. Consultants often work with such teams to help...human resources towards the development of a new therapy based on its market need and anticipated commercial...trained on how to work best with diverse teams, to leverage the preferences of different personality types... scientific background, training in reviewing literature, and critical analyses skills were applicable... -
Tips for CRISPR Gene Editing in Mice
TypeBlog PostPublishedJune 28, 2016, 10:59 a.m....Wang et al., 2013, Cho et al., 2014). There are several variants of the CRISPR/Cas9 system floating around...efficacy in vitro, and using it in mouse embryos to generate gene modified mouse lines. Hopefully this post...to target that region. Or you may even wish to generate a point mutation and need to focus your attention...yourself for potential gRNA sites, or you can utilise several online tools that do it for you. One such option...selection, ordering the sgRNA is simple and easy. Several companies supply gRNA sequences, such as GenScript...validating your edit in mice once they have been generated (more on that later). Find Validated gRNAs to ... of EGFP. Using this technique, you can assess several sgRNA’s at the same time, and choose the most effective... -
"Build Your Plasmid": The Game - Play to Teach
TypeBlog PostPublishedJuly 17, 2018, 12:23 p.m....more hands on While teaching courses about gene therapy and genetic manipulation, I and other professors...absorbed if they don’t get a chance apply it in an interactive setting. The problem is…how can you make a “...similar types of thinking go into developing gene therapy strategies for various diseases. That’s when I ...-on”, but as much as I try being compelling, interactive, and application-driven to boost curiosity, I...skills to apply those concepts and understand how therapeutic strategies work. Find Addgene Blog posts about...pick the best strategy and propose a final gene therapy approach for the disease to be openly discussed... this way, the students also had to practice transferable skills like team building, collaborative critical... -
Viral Vectors 101: Viral Applications
TypeBlog PostPublishedApril 13, 2023, 1:15 p.m....gene therapy have also been in the limelight. The FDA approved the first oncolytic viral therapy in 2015... science research all the way to clinical gene therapy. But how can you use these amazing tools in your... how we understand what genes do! Viruses have several options to allow you to do exactly that: shRNAs...integration of a plasmid. Viral transduction is also generally less toxic to cells than transfections, preserving...libraries makes them the ideal vector for the job. Therapeutic applications Vaccine development Attenuated and...nucleic acid-based vaccines. These vectors provide several advantages over traditional vaccines, including...being exploited as delivery mechanisms for gene therapy. The tractability of AAV, low immunogenicity, and... -
March for Science
TypeBlog PostPublishedApril 21, 2017, 2:30 p.m....17). Federal funding for science has been decreasing for the last five years and the first federal budget...passion for photosynthetic communities, microbial interactions, and science education. Disclaimer: The views... in education, government, and civilization in general (1). Science and politics? Science is an apolitical... human consumption in comparison to 37% of the general public (5, 6). Similarly 90% of active research...mostly due to human activity while much less of the general public agrees (5, 6). Many of the new administration...only be by advocating for science education and literacy that this changes. 2) Lumping all scientists ...marching to advocate for science education and literacy as I believe people that can see and understand... -
Gendered Innovations: Why Does Sex of the Cell Matter?
TypeBlog PostPublishedJuly 21, 2016, 2:30 p.m....recipient interaction should be tested before being ruled out; there are many factors that interact with sex...strategic approaches to gender equality over the past several decades: “Fix the Numbers of Women” focuses on...wrong costs lives and money. According to the US General Accounting Office, between 1997 and 2000, ten drugs...analysis of the US Women’s Health Initiative Hormone Therapy Trial, for example, found that the study saved ...Reduced use of estrogen plus progestin hormone therapy resulted in 76,000 fewer cases of cardiovascular... find 263,000 more osteoporotic fractures. The overall economic savings to the US healthcare system was... cells and tissues, specifically in stem cell therapies. Let’s go back to why ten drugs were withdrawn... -
Hot Plasmids - October 2020
TypeBlog PostPublishedOct. 13, 2020, 1:15 p.m....researchers tested several scenarios from embryo development to myeloid proliferation in acute myeloid ... IL-3 to proliferate. The cells cultured with a low dose of IL-3 showed reduced proliferation and had ...particularly challenging as they are great in number, generally very small, and can contain different neurotransmitters...proteins coupled with Fibronectin intrabodies generated with mRNA display (FingRs), Xue Han’s lab has ...cell cycle attempt to understand development, regeneration, and certain disease states. However, it’s difficult...proportion of blue fluorescence, indicating cell proliferation. BaF3 cells maintained in media without IL-3...withdrawn became entirely red as they could not proliferate. Eastman et al. Cell Reports 2020. https://doi.org... -
A Look Back at One Year of Plasmid Sharing for COVID-19 Research
TypeBlog PostPublishedJuly 12, 2021, 1:15 p.m....year and how these plasmids have been used to accelerate COVID-19 research in other labs. Spike protein... protein has had many opportunities to evolve. Several variants are now circulating and plasmids containing...from Alejandro Balazs’s lab includes those for generating pseudovirus and those encoding different spike...used these pseudoviruses to determine how effective sera from vaccinated individuals are at neutralizing ... the reverse transcriptase, protease, and Bst polymerase constructs from their assay to Addgene. Drew...expressing MMLV reverse transcriptase and Taq polymerase that can be used in a one-step RT-qPCR reaction...SARS-CoV-2 researchers to investigate how the virus interacts with human cells. Addgene’s mammalian target plasmids... -
CRISPR 101: Epigenetics and Editing the Epigenome
TypeBlog PostPublishedJune 24, 2020, 5:45 p.m....efficient. In general, epigenetic chromatin modifications are correlated with alterations in gene expression...activation p300 acetyltransferase dCas9 fused to the catalytic domain of p300 acetyltransferase increases levels...transcription, also includes light-regulated histone methyltransferases and deacetylases. CRISPR and epigenetics ...initiates cytosine demethylation of DNA. However, several proteins in the DNA oxidation and repair pathways...delivery systems. Transcriptional repression DNA Methyltransferase 3 Alpha (DNMT3A) Vlatka Zoldoš’ lab has deposited...repression. Created with BioRender.com. DNA Methyltransferase MQ1 Margaret Goodell's lab has deposited pcDNA3.1...Q147L)-EGFP, a fusion of dCas9 to a small DNA methyltransferase from the prokaryote Mollicutes spiroplasma... -
Viral Vectors 101: Producing Your rAAV
TypeBlog PostPublishedJuly 16, 2024, 1:15 p.m....There are several facilities nationwide where you can obtain excellent quality, high titer rAAV (including...including Addgene!), but you can also generate rAAVs in your own lab with standard molecular biology tools...our ddPCR for AAV quantitation blog post. We generally do not recommend using ELISA-based titering assays...inverted terminal repeat sequences. Human Gene Therapy Methods, 23(1), Article 1. https://doi.org/10.1089... of gene expression in the CNS. Molecular Neurodegeneration, 15(1), Article 1. https://doi.org/10.1186...can limit production of recombinant AAV-2. Gene Therapy, 6(7), Article 7. https://doi.org/10.1038/sj.gt...Inactivation of AAV Serotype 1 Vectors. Human Gene Therapy Methods, 28(1), Article 1. https://doi.org/10.1089... -
MXS Chaining
TypeBlog PostPublishedFeb. 7, 2017, 3:30 p.m....the functions of different genetic elements to generate non-natural tools such as novel biological circuits...and more. By combining these components, one can generate constructs to visualize cell cycle dynamics, titrate...applications. In one example, modular constructs generated with MXS-chaining were used to label subcellular..., MXS Chaining gave researchers the ability to generate the four initial unique constructs and later combine...ligation, the original pattern of the MCS is regenerated at the flanks of each ligation product, which...that the restriction sites of the MCS are only regenerated at the 5’ and 3’ termini of the ligation product...added to the 5’ or 3’ terminus using the newly generated MCS. MXS-chaining also has the advantage of being... -
Negative Can Be Positive: Open AAV Data with Addgene
TypeBlog PostPublishedSept. 6, 2022, 1:15 p.m....providing many of the details not generally captured in the literature, scientists can use the Data Hub...commonly encountered in human gene therapy trials. Human Gene Therapy Methods, 26(2), 71–76. https://doi.org...Working with AAV vectors in the brain and peripheral nervous system presents a special challenge to scientists... process is often not detailed in the primary literature, and as a result can leave scientists under-informed...time and effort to test and optimize our vector - overall, I spent two years testing and reformulating our...dose–response and comparison to AAV PHP.B. Gene Therapy, 25(5), 392–400. https://doi.org/10.1038/s41434... Gene Delivery to the Hypothalamus. Human Gene Therapy, 21(6), 673–682. https://doi.org/10.1089/hum.2009.169... -
A Practical Guide to Optimizing AAV DIO and FLEx Vector Expression
TypeBlog PostPublishedNov. 2, 2021, 1:15 p.m....FLEx cassette using next-generation sequencing (NGS) (Guerin, K. 2020). In general, we observe that 0.1 -... in the Central and Peripheral Nervous System and Relevance to Human Gene Therapy. Front Mol Neurosci ...FLEx Vectors Important Considerations When Using AAVs Three Key Considerations For Precise Neuronal Targeting...Vectors). While this strategy greatly reduced the overall level of off-target transgene expression, low levels...greater off-target expression just due to more overall viral particles getting into those cells. Therefore...transduces your cells of interest. We recommend testing several different serotypes to determine which one is the...course experiment to check expression levels over several weeks allows the researcher to determine when the... -
Sequencing Options for CRISPR Genotyping
TypeBlog PostPublishedOct. 4, 2016, 2:30 p.m....begin to use CRISPR as a therapy. Low frequency off-target effects may generate irreproducible data in ... CRISPR experiments, many are turning to next-generation sequencing options over older assays. Biased... search criteria are not exhaustive. Mismatch tolerance settings are often limited to off-target sites...sites of <4 bp. The off-target list is also generally weighted by the position of the mismatch along the ...CRISPR editing in the model cell line or organism. Several options exist for unbiased off-target detection...labeling, enrichment on streptavidin and next-generation sequencing (BLESS) (Crosetto et al. 2013). GUIDE-seq...consistent set of standards as the field seeks to generate reproducible, quality data on the role of genetic... -
Plasmids 101: Origin of Replication
TypeBlog PostPublishedNov. 11, 2020, 2:23 p.m....plasmids. Growth conditions The amount of aeration, temperature, culture volume, antibiotic, and medium ...survive within cells. The replicons of plasmids are generally different from the those used to replicate the... to make additional copies. ori sequences are generally high in As and Ts. Why, you ask? Well, A-T base...pairs can be separated more readily at lower temperatures and allows the replication machinery room to...few copies depending on how they are regulated. Generally, control of replication is referred to as "relaxed...exclusively in E. coli? Gram negative bacteria in general? Both Gram negatives and Gram positives? Will you...compatibility with one or more other plasmids. Generally speaking, plasmids with the same ori are incompatible... -
Hot Plasmids: Fall 2024
TypeBlog PostPublishedNov. 21, 2024, 1:30 p.m....structural studies of E3 ligase cereblon By Mike Lacy Several types of drugs target the ubiquitin E3 ligase cereblon...Lon and TBD domains, part of the HB domain, and several mutations to improve solubility and stability (...and mScarlet-I3, Oliver Hobert’s lab recently generated new constructs specifically for C. elegans (Cao...showed greater photostability and also matured considerably faster, with expression levels comparable to...notorious for its high resistance to antibiotics. Several CRISPR/Cas-based systems have been developed for...mechanosensor called PILATeS, Pulling-Induced Luciferase Association Tension Sensor (Zhong et al., 2024...sensor STReTCh, the 11-amino acid BiT fragment of luciferase is embedded in the I10 domain of the human titin... -
Hot Plasmids Spring 2024
TypeBlog PostPublishedMay 23, 2024, 1:15 p.m....microglia The REVeRT Dual AAV Vector System Neurodegeneration Collection updates Multiplexed perturbation...2024 under a CC-BY-NC-ND license. In this iteration, the size of the lentiviral insert was minimized...ensure this tRNA processing only occurs on the RNA polymerase III transcript, the sgRNAs were encoded on the...recombinant versions of mouse monoclonal antibodies generated and characterized by the Geiss Lab in their effort...A122D mutation in the rhodopsin domain conferred several new improvements. The new variant has increased...valuable tool for microglia research and broader therapeutic applications. Find microglia-targeting AAV plasmids...10.1101/2023.12.12.571321. Revolutionizing Gene Therapy: The REVeRT Dual AAV Vector System By Vaibhav Kawde... -
Beyond the Blood Brain Barrier: PHP.B Enters the Central Nervous System Via the LY6A Receptor
TypeBlog PostPublishedJuly 23, 2019, 12:46 p.m....One of the biggest barriers to gene therapy treatment of central nervous system (CNS) diseases is the...because in vivo transduction of AAV serotypes generally tracks between strains of mice. This suggests ... engineered PHP.B. PHP.B was created by first generating a diverse library of AAV9 mutants that had a ...inheritance pattern of PHP.B permissiveness. The first generation of outbred mice (F1) had half the level of CNS...transduction as their C57BL/6J parent. The second generation (F2) of outbred mice also showed a Mendelian ...galactose and AAVR to transduce cells. The Wilson Lab generated results similar to the Deverman Lab when they ...Transport across the Blood-Brain Barrier." Molecular Therapy 27.5 (2019): 912-921. PubMed PMID: 30819613. Hordeaux...