...mimic aspects of organ morphology and physiology in vitro and have emerged as a powerful model for understanding...for doing gene targeting, reporter generation, controllable gene knockouts, and inducible gene inactivation...design. B) Strategy for leak-free transcriptional control. C) Iterative transduction workflow overview. Image...SL-insensitive fluorophore (LSSmOrange) as an internal control and a SL-sensitive fluorophore (circularly permuted...the cpGFP without altering that of the internal control (Fig 1). Because this method relies on the fluorescence...-500nM range) and a high specificity for SLs in vitro, and they further demonstrated that when tested ...

...activity in the brain was measured with implanted electrodes. The Archon1 voltage reporter allows a way to...cre-dependent version at Addgene! Fluorescent protein and control AAV These fluorescent protein expression vectors...interneurons. These vectors can also be used as the control vectors for experiments encoding a fluorescent-... Addgene in the next 6 months, pending quality control. For an estimate on availability date or to be ... and serotype you’d like to be notified about. Controls Plasmid Serotype Name 114472  AAV5, AAV8 ...Plasmid Serotype Name 119036  AAV1  pAAV-hsyn-flex-Voltron-ST Chemogenetics Plasmid Serotype Name ...

... Many thanks to our guest blogger Jon Backstrom. Jon Backstrom, Ph.D is a biochemist in the Vanderbilt...This post was contributed by guest blogger Jon Backstrom, a biochemist in the Vanderbilt Eye Institute ...oligomerization of biotinylated proteins on avidin can be controlled easier than formation of GST dimers. Avi-Tag ... of added biotins per molecule cannot be well-controlled. The lack of site selectivity typically diminishes...relatively low efficiency of biotinylation, at least in vitro. Our solution was to exploit the best of both worlds...

...exhibit profound differences in tropism between cell types (astrocytes, microglia, neurons, etc.) and ...can not recombine with each other. When Cre is introduced to the system it binds one pair of lox sites,...your cells.  Some promoters, such as CAG are very strong and may require less viral particles or relatively...or glial fibrillary acidic protein (GFAP) for astrocytes it is important to test it in the brain region... S (2013) Analysis of Transduction Efficiency, Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, ...and 5 Display Differential Efficiency and Cell Tropism after Delivery to Different Regions of the Central...

...plasmid deposit please sign up here.        A retroviral mouse CRISPR knockout library Article contributed...phenotype, and this depends on the method used to introduce the gRNAs into cells. Most CRISPR pooled libraries...issue, Sarah Teichmann and colleagues created a retroviral CRISPR knockout library using gRNAs designed ...designed by the Kosuke Yusa Lab. This retroviral library was used to transduce T helper type 2 (Th2) cells from...differentiation factors as the screen readout. This retroviral mouse CRISPR pooled library can be used for screening...concentration of ApoB-LPs has been shown to have a strong correlation with cardiovascular disease and indirect...

... inserted GFP. After characterizing iGluSnFR in vitro, showing that it is selectively activated by glutamate...higher expression levels in bacteria and produces stronger fluorescent signals upon sensing glutamate in ...variants differing in kinetics, affinity, and ∆F/F0. Introduce the two variants in three different membrane-targeting...iGluSnFR3 have higher expression levels and produce stronger fluorescent signals upon sensing glutamate using...addition to the original PDGFR TMD, the researchers introduce iGluSnFR3 in two additional anchoring domains ...induction to express soluble protein CAG: Generic strong promoter. Formulated as AAV-DJ for neuronal expression...

...CRISPR-Csm: A novel RNA targeting system by: Susanna Stroik The Doudna lab’s recent work characterized a new...heat-inducible lentiviral plasmids here!   Wu Y, et. al. Control of the activity of CAR-T cells within tumours via...chemically stable tool for microscopy by: Susanna Stroik Microscopy techniques have been moving at the speed...microscopy applications such as super resolution, electron, and expansion microscopy. hfYFP is not only brilliantly...denaturing conditions, high temperatures, and other chaotropic conditions better than most FPs. The group also...expansion microscopy as well as correlative light and electron microscopy and observed high fluorescence resilience...

... to neuroscience. In fact, precise light-based control of biological processes is highly useful in other... to be optimized by Tabor’s lab, making light-controlled gene expression in E. coli easier and more robust...engineered to deliver precise, consistent, and controllable pulses of light. Complicated patterns of light...new pathways with sophisticated gene expression controls. Most cell types do not respond to low-intensity...two systems can be coexpressed for sophisticated control of gene expression. An E. coli light-sensitive...from this publication at Addgene. Multichromatic control of gene expression in Escherichia coli. Tabor JJ...

...need to ensure you have a bead-only control alongside your input control. Elute the DNA after washing and..., which uses formaldehyde or UV light to create strong but reversible bonds. This will keep the protein...of histone modifying proteins, which bind quite strongly to DNA, crosslinking may not be necessary. This...Enzymatic digestion, while gentler on the sample, introduces bias due to the selective nature of the cutting...Antibodies 101: Polyclonal Antibodies Antibodies 101: Introduction to Antibodies Resources on Addgene.org ...

...sequence NGG TTTV PAM site preserved? Usually Destroyed Yes, Cpf1 cleaves 5' of the protospacer Multiplexing...Cassettes transfection gRNAs expressed from a polycistronic transcript  Requires co-transfection of Cas9...Cassettes transfection gRNAs expressed from a polycistronic transcript Requires co-transfection of Cas9  ...showing that Cpf1 can cleave an array of 4 crRNAs in vitro and when expressed in 293 cells. This allows for...a GFP tag. Four weeks post-infection, there was strong expression of Cpf1 and GFP in the targeted brain...effective method with multiple applications (in vitro, in vivo, transfection, viral transduction) for ...

...regeneration studies, and cancer biology. I was first introduced to bioelectricity when I attended the Developmental...living cells. Since bioelectrical signals are controlled by the opening and closing of ion channel proteins...genetically encoded, light-gated ion translocators to control tumorigenesis." Oncotarget 7.15 (2016): 19575. ...signaling networks: exploiting voltage gradients for control of growth and form." Annual review of biomedical... neural and gap junction protein-mediated cues control polarity during planarian regeneration." Developmental...Vaibhav P., et al. "Transmembrane voltage potential controls embryonic eye patterning in Xenopus laevis." Development...

...Be prepared to troubleshoot and be sure to include proper positive and negative controls as you get your...someone to turn to for advice if you need to troubleshoot. What do you know about the vendor and the vendor...anti-tag antibody; and immunoprecipitation-mass spectrometry strategies that isolate an antibody’s immune...complexes and confirm interacting partners by mass spectrometry (Uhlen, 2016). Be sure that the antibody you...The Basics of Western blottingAntibodies 101: Introduction to AntibodiesAntibodies 101: Epitope availability...

...Scientific Support Team here at Addgene, so troubleshooting molecular biology experiments was in the job...core part of our academic training.  Developing strong problem solving skills is just a matter of applying...think back to the last experiment you had to troubleshoot; you probably did not just automatically come...As such, scientists are uniquely prepared to be strong problem solvers both in and out of the lab. The...position. Of course, there are limits to how far strong problem solving skills alone can take you - just...effectively does not mean I am qualified to be an astronaut. But, knowing that this skillset is a part of ...

...research that was done and what it means. The introduction should give you just enough information to allow... addressed. In primary research papers, the introductions should be fairly focused. Consult a review article... etc)? Did they perform all of the appropriate controls? Refer back to the methods section for finer points...automate the process as well. Sign up for eTOC (electronic table of contents) alerts from journals you read...useful papers. For many, this collection is purely electronic. Gone are the days of file cabinets filled to...She is a fan of photosynthesis and writes about autotrophs and biochemistry on her blog. Follow her on Twitter...

...activity in HEK293 by reducing editing from ~30% in control samples to 0-10% in Acr samples. Acr proteins can...When added to human cells six hours after the introduction of Cas9 RNPs, AcrIIA4 reduced off-target editing...specific site. 2. Temporal, spatial, or conditional control of CRISPR activity Regulating Acr proteins by inducible...molecules could achieve rapid, dynamic, and spatial control of CRISPR-Cas activity. For example, fusing AcrA4II...LOV2 domain allows anti-CRISPR activity to be controlled by blue light (Bubeck et al., 2018). 3. Reduce...Your Next Experiment Read more about Optogenetic Control of CRISPR   Resources on Addgene.org Find CRISPR...

...approach of CeNGEN, check out their publication.   3’UTRome The 3’UTRs of mRNA are important for post-transcriptional...expression in C.elegans. In this system a promoter controls the expression of a GAL-4 driver that can bind...also find additional SapTrap plasmids used for introducing point mutations that was published in a microPublication...Central PMCID: PMC3531189. Mangone, Marco, et al. "UTRome. org: a platform for 3′ UTR biology in C. elegans... L., & Jorgensen, E. M. "SapTrap vectors for introducing point mutations with unc- 119 + selection." MicroPublication...split intein for refined spatiotemporal transgene control in Caenorhabditis elegans." Proceedings of the ...

...are monoclonal antibodies that are generated in vitro using synthetic genes that are typically expressed... a patient’s blood), scientists can use mass spectrometry to determine the amino acid sequences of the...process of antibody selection and maturation in vitro by selecting for antigen binding from libraries ...or mammalian display (Tsuruta et al., 2017). In vitro selection allows scientists to create antibodies...into an expression plasmid, the plasmid can be introduced into host cells, such as bacterial, yeast, or...on the Addgene blog Read our Antibodies 101: Introduction to Antibodies blog post Learn more about hybridomas...

...response (e.g., graft vs. host) as well as the introduction of the cancer-fighting component, CAR. Clinical... edit with the goal of ‘fixing’ mutations or introducing a desired edit, but this particular class of ...of treatments has quite the opposite aim: to destroy. One treatment currently in clinical trials includes...but are not limited to, ones targeting muscular dystrophy, cardiovascular disease, inflammatory diseases...by Kendall Morgan and updated in 2023 by Susanna Stroik.  References and Resources References Yuxuan Wu...blog CRISPR Antimicrobials Treating Muscular Dystrophy with CRISPR CRISPR 101: Drug Discovery and CRISPR...

... calcium binding proteins (e.g., calmodulin or troponin C). Their fluorescent readouts are used to monitor... is a FRET-based calcium reporter derived from troponin C. It is engineered with a reduced number of calcium...fluorescent state only by simultaneous (1) user-controlled illumination with violet light and (2) exposure...different versions optimized for neuron-specific, astrocyte-specific (GCaMP6 only) or mammalian cell expression...101: Viral Applications Viral Vectors 101: An Introduction to AAV Find AAV Calcium Biosensor Vectors at... Susanna Stroik...

...Tips for Plasmid Quality Control Plasmids 101: In-house NGS Plasmid Quality Control Now at Addgene Bioinformatics... has many points at which small errors can be introduced. Eventually, these errors could cause the plasmid...Figure 1. Potential points where DNA errors can be introduced, indicated by exclamation marks. Error points...Addgene, we put a lot of effort into our quality control (QC) processes to screen for and reduce these errors...non-negotiable. By maintaining a high standard of quality control, we have built a trusted plasmid catalog containing...