...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...

... 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 ...

...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 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...

...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...

...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 ...

... 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...

...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...

...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...

... 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...

...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...

...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...

...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...

...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...

...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...

...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...

...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...

...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...

...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...

...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...