...techniques?  Let us know in the comments! References 1. Bacteriophage P1 site-specific recombination. I. ...using a resistance marker; however, it is often desirable to remove the marker after the initial selection... Combining this with some of the loxP methods described above, a genetic modification can be restricted...

...internalization of two different receptors- angiotensin II type 1 receptor and the β2 adrenoceptor. GPCRs in chemogenetics...new receptors were termed Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) (Roth, 2016...What do smell, taste and sight have in common, besides being one of the five senses? GPCRs or G-protein-coupled...photons or odorants, to elicit downstream signaling cascades.  So let’s break down the name "G-protein coupled...then induce or inhibit intracellular signaling cascades as a response to the extracellular stimuli. Ligand...

... Science Find the SEND plasmids    Figure 1: SEND combines an endogenous Gag homolog, cargo RNA...different labs have recently published work that provides a solution to this problem. A miniature CRISPR-Cas...miniature CRISPR-Cas12f nuclease Quanjiang Ji’s lab describes an effective miniature Cas system that utilizes...Nature Biotechnology paper showcases an improved design of the guide RNA that caps the 3’ end with a structured...that mismatch repair (MMR) interfered with the desired resolution of prime editing intermediates. Building...sequence. As with other prime editing tools, the desired edits are templated by the 3′ extension of the ...

...incorporated at the termini of the crRNA molecule (usually 1-3 of the first 3 bps are modified) for optimal stability...to simply swap out several of the ribonucleotides for deoxynucleotides. A partially DNA gRNA is surprisingly...use in applications like siRNA and antisense oligonucleotides. Some of these synthetic modifications, specifically...photoactivatable and photocleavable gRNAs. Photocleavable guides are generated by introduction of a single photocleavable...Additional resources on the Addgene Blog  How to design your CRISPR gRNA CRISPR 101: Ribonucleoprotein ...

... an entirely new affinity reagent.    Fig. 1: A comparison of an antibody (left) to several alternative... as the OG (as the kids say) affinity reagents deserved to be celebrated, there are many other amazing...its thorns and every affinity reagent has its downsides. These reduced-in-size options often have reduced...°C and can refold correctly after denaturing.  Designed ankyrin repeat proteins, or DARPins, are genetically...a wide variety of targets, including proteins, peptides, amino acids, drugs, metal ions, and cells. They...

...many of which are available in rAAVs!   Figure 1: Diagram of AAV components      Cargo capacity...serotypes The names of AAVs contain two numerical designations, separated by a slash, that indicate the ITR...interested in expressing multiple small genes, you can design single rAAV vectors with multiple genes packaged... an option (McCarty et al., 2001). This AAV is designed so that the single stranded DNA folds back on ...

...see how you use them in your research!  References 1.Tang, Jonathan C.Y., et al. “A nanobody-based system...GFP-labeled cells. Once Tang and Cepko found a description of GFP-binding nanobodies, the project really...stable inside cells. Nanobodies binding GFP were designed in 2009. Subsequent work showed that nanobodies...green-red Cre-lox systems. T-DDOGs can also be designed with other DNA-binding domains, including the ...

...efficiently with adenovirus and AAV.   Table 1: Types of viruses and their features Choosing ...about a specific virus? We have you covered with guides on lentivirus, retrovirus, adenovirus, and AAV....

...with each protein of interest.    Figure 1: The direct approach (left) versus the indirect approach...affinity, many indirect antibody protocols are designed to take place over multiple days. Increased run...

... trafficking of these biosensors.   Figure 1: Schematic of iGluSnFR3 (left). GluSnFR3 in pMinDisplay...sulfurtransferase (MPST) which catalyzes the desulfuration of 3-mercaptopyruvate and generates an enzyme-bound...transferred to thiol-containing acceptors. These hydropersulfides modulate protein activity and have important...

...manipulation, there are two main decisions to make: 1. Which modification? and 2. Which viral vector should...behavioral neuroscience, and address experimental design considerations used in all behavioral studies. ...ion channels. Similarly, chemogenetic tools are designed channels that are chemically activated. Both are...

... – a mechanical device that could be used to bend 1/8th inch steel wire into a backbone structure of a...BioMolecular Modeling and the CEO of 3-D Molecular Designs. Have you ever held your favorite protein in the...favorite protein in your hand? At 3D Molecular Designs, we create physical models of proteins using 3D...off a small, family-run business – 3D Molecular Designs – to commercialize some of the best instructional...reaction to this model on his blog. Or, for a description of how effective this model was in explaining... up close and personal, visit the 3D Molecular Designs web site. Or, if you are visiting a local classroom...check out our hands-on teaching tools that are designed to make the molecular world real for students....

...cereblon, leading to rapid target degradation (Figure 1). Plasmids: We generated a series of plasmids that... molecule degraders” recruit the proteasome to destroy targeted proteins. We previously developed small...locus-specific knock-in of FKBP12F36V. Detailed protocols describing the use of these plasmids are available here....Nabet et al., Nature Chemical Biology additionally provides detailed information on cloning procedures used...degradation, and lead author on the recent paper describing the dTAG technology. Follow him on twitter @behnamnabet... 29581585. This reference contains the full description of the dTAG system. The dTAG system was employed...Conditional Mouse Models CRISPR 101: Cas9 Nickase Design and Homology Directed Repair Plasmids for endogenous...

...your finished constructs at Addgene!). References 1. Li, Chaokun, et al. "FastCloning: a highly simplified...amazing project. You’ve written your protocols, designed your experiments, and prepared your reagents. ...following cloning methodologies have been extensively described in other articles; so if you are interested in...homology. You might also consider FastCloning, first described by Chaokun Li and colleagues. Here you simply ...-generated insert. When mixed, topoisomerase I provides both the endonuclease and ligase function while...restriction enzymes. In Golden Gate cloning, the desired end construct does not contain the type IIS recognition...This feature biases the reaction in favor of the desired assembly to make the process close to 100% efficient...

...significant progress in clinical trials.     Figure 1: Types of delivery and editing strategies for CRISPR...introducing a desired edit, but this particular class of treatments has quite the opposite aim: to destroy. One...potential for treatment is no longer an accurate descriptor; progress in clinical therapy is more fitting...One treatment currently in clinical trials includes Cas3 (the shredder member of the Cas family) as part...CRISPR therapies have made significant clinical strides since we first wrote this blog post. The future...

... CRISPR guides) while a single 72h harvest is best for larger genes such as Cas9. See figure 1 for an ...newer generation lentiviral packaging systems were designed to prevent the formation of replication competent...use 3 plasmids; an envelope plasmid that usually encodes VSV-G, a packaging plasmid containing structural...the best titer possible. While this information provides a great starting point, it may not translate to...

... little signal detected along the neurite (Figure 1.) Moreover, the group was able to demonstrate that...soCoChR-expressing system. Taken together, Shemesh et. al. describe a novel system for the precise mapping of neuronal...the single-cell level. Various soCoChR plasmids described in this work including the soCoChR-GFP plasmid...information about spatial and temporal organization. The design use to detect PKA activity can be generalized to...choice for FRET assays where a dark acceptor is desired due to its very low quantum efficiency. ShadowY...

.... Follow David on Twitter @driglar.   References: 1. Alberts, Bruce, et al. "Rescuing US biomedical research...for affecting change. On Friday, the symposium includes a panel discussion and keynote by former congressman...towards solutions. The 2015 Boston FoR meeting was designed to explore areas where further data is necessary...for students interested in leading the process despite the recent advance. Similarly, grad students throughout...development and implementation of new strategies designed to cope with current increases. These two rulings...

...most comprehensive for searching. Timesaving Tip #1: If you know the species that your sequencing result...terrific getting-started guide for BLAST, which includes a simple explanation of the different BLAST programs...matrix BLOSUM62, but you may want to check the description of the other matrices to see if another would...alternatives Depending on your sequencing result and desired analysis, BLAST may not always be your optimal ...

...base editors with editing windows as small as 1-2 nucleotides! To reduce off-target effects associated with...Lower on-target activity than BE3; editing window ~1-2 nt  87438  pET42b-HF-BE3  HF-Cas9 (NGG)  Very ...cassette via Cre recombination. Each knockout includes unique barcodes and universal priming sites to ensure ... the Chi-Bin Chien lab (Kwan et al., 2007) and includes four promoters, six fluorophores with nonoverlapping...The set of CyRFP constructs available at Addgene includes bacterial and mammalian mCyRFP1 or CyRFP1 expression...codons) with kanamycin cassettes and were carefully designed to allow for easy removal of the resistance cassette...essential in B. subtilis 168. Each individual strain includes a xylose-inducible dCas9 paired with a single,...