...101 blog series Learn more about CRISPR and genome editing in our CRISPR 101 blog series Resources on Addgene.org... in gene modification tools and methods such as CRISPR have vastly improved scientists’ ability to use...use in the axolotl including plasmids for use in CRISPR and Tol2 transposase induced transgenesis.  Xenopus...Xie, Z., Zhang, C., & Xing, X.-H. (2018). Pooled CRISPR interference screening enables genome-scale functional...regulate regeneration and wound healing are conserved across species, studying the axolotl may reveal the potential...10.1111/j.1365-313x.2009.04086.x Krämer, U. (2015). Planting molecular functions in an ecological context with...

.... The continued development of more and smaller CRISPR tools is an exciting area for future AAV application...- a primer for beginners Adenoviral delivery of CRISPR/Cas9 Tips for a first time AAV user Resources ...and somewhat more reliable quantitation. Differences in sample treatment across methods can affect reported...advise that users keep titering method consistent across samples within a project. Targeting Your AAV Q4...e.g. first at 2 months, then at 4 months? Or do antibodies become a prohibitive problem? A: Re-administration...for AAV due to the development of neutralizing antibodies in animal models. All of our engineered AAV are...

...Educational Resource Top Five by Views Guides CRISPR Guide Lentiviral Guide AAV Guide Optogenetics Guide... What is a Plasmid? How to Design Your gRNA for CRISPR Editing The Promoter Region - Let's Go! How to ...primers to help users get started with tools like CRISPR, lentivirus, and optogenetics — and shared the ...to identify and reduce barriers for scientists wanting to share their tools with Addgene.  In our next...materials both available and accessible to scientists across the globe?” By asking this question, and by listening...Addgene blog Twenty Years of Addgene Sharing: CRISPRTwenty Years of Sharing: Addgene's Viral Vector Service...

...applicable across many areas of research. Beyond fluorescence, SunTag can also be used to improve CRISPR-based... how its localization and expression may change across various conditions. However, this system is far...GCN4. GCN4 recruits GFP fused to the cognate scFV antibody, which is expressed from a separate plasmid. This... track run lengths of the motor protein kinesin across microtubules. Tanenbaum et al. then tested their...

... first ever CRISPR-based clinical trial in 18 lung cancer patients. This study used CRISPR gene editing...are a few of the reasons antibodies may result in a lack of response. Antibodies targeting the tumor-augmenting...effectively in mice. Chimeric antigen receptors T cells producing chimeric antigen receptors (CARs) on their...acquire genetic alterations and alter cell-surface antigen production to evade the immune system. 3. Escape...sidestep immune surveillance: Suppressing Function Antigen presenting cells (APCs) that would normally alert...regulates the expression of the tolerance-producing antigens enunciated above. Finally, signals released from... produces modified dendritic cells (DCs) and macrophages (M1 and M2) that help create welcoming environments...

... Addgene Blog Learn How CRISPR Can Be Combined with Phage for Antimicrobial Purposes Use Yeast Two Hybrid...Systems to Probe Protein-Protein Interactions Use CRISPR For Genome-Wide Screens Resources on Addgene.org...characterization, these proteins could function as surrogate antibodies for identifying and characterizing sugars and...transformants for isolated colonies (150-200 per plate) across 20 fresh selective plates. Step 2: Express random... good start if you’re using a pET vector) and antibiotic for plasmid selection. Remove air bubbles between...temperature. If you’re lucky enough to have an antibody or other protein on hand that will specifically...

...interactions with Alzheimer's, I found others using CRISPR to generate new knockout mice, using adenoviral...by how neuroscientists are using techniques from across different disciplines to better understand neurobiology...There were, of course, many companies selling antibodies, microscopes, and dissection tools, but there...brain function. These scientists pulled tools from across the biological spectrum to learn more about the...

...or preparation, and deliver CRISPR components for gene disruption or CRISPRi. While designed to study the...can be both sorted using FACS and isolated with antibiotic selection. The team tested this system in vitro...and in vivo. They identified cells from 19 genera across four phyla that have taken up the plasmid through...the addition of standards, the assay is semi-quantitative and can approximate mRNA concentration within...host-range of the plasmids and suitability of various antibiotic resistance markers, promoters, and inducible ...microbiome responds to infection, dysbiosis, and antibiotic treatment. To facilitate these studies, Pamela...

... interactions Learn how biotin can be used with CRISPR Additional resources Idtdna educational pages... and antibody labelling. In these experiments, biotin is added to a primary or secondary antibody that...your macromolecule of interest To utilize the natural binding of biotin and avidin, macromolecules of interest...process by which biotin is added to a protein or macromolecule. Biotin can be added enzymatically or chemically...soluble molecule that does not interfere with the macromolecules it is added to. Biotin also has an exposed ...that targets your protein of interest or primary antibody respectively. Avidin bound to a reporter is then... on this complex can bind to your biotinylated antibody. This creates a large meshwork of biotin-avidin...

...for transfer plasmids with small inserts (such as CRISPR guides) while a single 72h harvest is best for ...the transfer gene. By dividing the viral elements across multiple plasmids, the risk of recombination and...generally a good place to start. If you don’t anticipate that you will need large volumes of lentivirus...

... of other technologies such as optogenetics and CRISPR/Cas9 will help immunologists create even better...with the engineering of fluorescently labelled antibodies made it possible to track cells from one organ...harvesting lymph nodes at different times after antigen challenge, scientists were able to track where ...diverse tissue environments. These studies provide quantitative measurements of cellular motility, interactions...By specifically labeling T cells, B cells, and antigen presenting cells, scientists have been able to ...the bloodstream to invade lymph nodes and scan antigen presenting cells, but two-photon microscopy enabled...scanning was and how fast T cells could jump from one antigen presenting cell to another. Indeed, we now know...

...vectors fantastic options for delivering shRNA, CRISPR/Cas9 components, and fluorescent sensors. Retroviral...quiescent or terminally differentiated cells such as macrophages and microglia. This property makes the lentivirus... method detects the expression of viral capsid antigen p24, the FACS dosing method correlates viral titre...reverse transcriptase activity assay uses qPCR to quantify viral RNA. All of these methods have been described...

...delivered: fluorescent proteins, optogenetic sensors, CRISPR elements, etc... *These are only starting recommendations...capsids, when writing this post, we didn’t come across a broadly used working definition of systemic capsids...concurrently to minimize the impact of neutralizing antibodies developed in response to the first exposure to...AAV-PHP.eB. For this reason, the Gradinaru lab anticipates that high-dose AAV-PHP.AX will be the capsid ...expression (EGFP, in the depositing manuscript) across the brain. AAV-BI-28 has been tested in C57BL/6J...brain cells, with a higher efficiency (82 — 85% across the brain in mice) and greater specificity than...efficiency, and is not neutralized by pre-existing antibodies against AAV9-based vectors (and vice versa), ...

...characteristics like pH, redox state, and voltage across membranes. These sensors generally follow the same...Experiments Experiment  Detection Method Notes Quantification of a process Plate-based assays (plate reader...reader) Detect the average amount of fluorescence across a population of cells (i.e. in a plate well). Use... HPLC (proteins or other molecules) Isolate or quantify cells, proteins or other molecules that have been...Autophagy Sensor Read about Aptamer Fluorophores Use CRISPRainbow for Genome Visualization Resources on Addgene.org...