.... Two days later, Cre-dependent GFP expression was detected with direct fluorescence. GFP was not detected... the absence of Cre. mCherry expression alone was detected. pAAV-Ef1a-mCherry-IRES-Cre was a gift from...expression and/or function. These data are sometimes reported or posted on the material page for the corresponding...were transduced with either pAAV-Ef1a-mCherry-IRES-Cre (55634-AAVrg) alone at 1.7E6 viral genomes (vg)/cell...recipient’s initial thaw will be accounted for in our reported titers. Lentiviral vectors are titered using a...

...following sucrose treatment. Moreover, several reports have shown that cells treated with sucrose or other...assays designed to test the endocytosis of a fluorescent dye and transduction of GFP-expressing AAV particles...Vandenberghe, Luk H., et al. "Efficient serotype-dependent release of functional vector into the culture ... but did you know that Addgene also performs independent research? Since launching the viral service in...1989; Ozturk and Palsson, 1991; Oh, 1993) and increased yields in adenoviral (Coroadinha, 2006) and retroviral...AAV production yields had yet to be examined. Increased yields with sucrose supplementation is serotype...

... Posts on Fluorescent Proteins Browse Our Fluorescent Protein Pages Find the mOX Fluorescent Proteins ...protein non-fluorescent. Unfortunately, accumulated misfolded and nonfunctional (dark) fluorescent proteins...others have reported that FP cysteine residues form inappropriate disulfide bonds in the secretory pathway...publication illustrated the quantitative increase in fluorescent signal achieved when utilizing an optimized...et al. "Oligomerization of green fluorescent protein in the secretory pathway of endocrine cells." Biochem... monomeric blue fluorescent protein, secBFP2, for studies in the eukaryotic secretory pathway."Biochemical...applicable to many microscopy experiments with fluorescent proteins (FPs) localized to compartments other...

...cassette into the Rosa26 locus, allowing for Cre-dependent expression of a GMAP inserted gene. The applications...with Rosella, a pH-sensitive fluorescent biosensor A pH-sensitive fluorescent biosensor-based assay system...come with mCherry2 or mClover fluorescent proteins allowing you to screen for fusions. This combination...-ome using a quick, affordable, and ubiquitous reporter assay. In their recent publication, Kroeze et ...library In order to identify both core and context-dependent human fitness genes, the laboratory of Jason Moffat...DsRed is relatively pH-insensitive, while SEP fluoresces at cellular pH (7.0) but not at vacuolar pH (...environments that inactivate SEP and leave only DsRed to fluoresce (1). With variants that can be targeted to specific...

... pFUS array vectors, destination vectors and a reporter vector for mammalian cell-based validation assay...N- and C-terminal domains (N153AA, C47AA). The reporter vector pGL4-SSA can be used for single-strand ...plasmids were designed for mammalian expression of fluorescent TAL effectors to visualize subcellular positioning...TAL Effector Assembly Kit Assembly by ligation-independent cloning (LIC). Validated in human cells. Veit...next generation architecture has been shown to increase mutation induction when using TALENs. The FokI...Gersbach These new destination vectors can be used to create TALE-transcription factors and are used at the ...TALEN kit, (ii) a lacZ fragment for blue/white-screening in E.coli , (iii) CAG promoter and Kozak sequence...

...CRISPR-Cas9) or deletion (Cre), together with expression of fluorescent or enzymatic reporters for cellular assays... Mammalian shRNA Tools for RNAi! Cre-Lox technology CAG-GFP-IRES-CRE (Plasmid #48201) This retroviral ...vector from the Gage lab encodes GFP and Cre-recombinase. The Cre-lox system is a technology that can be...mouse transgenics. Experimental uses include: Cre-dependent gene expression, gene knockout, and selection...the pLKO.1 vector can be found here. Cre-Lox technology Puro.Cre empty vector (Plasmid #17408)   This ... vector deposited by Dr. Tyler Jacks. It carries CRE recombinase and a puromycin resistance cassette. ...CRISPR pooled libraries that allow functional screening of thousands of genes in genome-wide experiments...

...oligomerization of fluorescent proteins is a concern. The propensity of fluorescent proteins to oligomerize...of the fluorescent protein are relatively close together (figure 1). Where should the fluorescent protein...length, the specific fluorescent protein variant, and where to add the fluorescent tag. These choices will...vivo characterisation of fluorescent proteins in budding yeast." Scientific reports 9.1 (2019): 2234. PubMed...dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins." Proceedings...Addgene blog Read our fluorescent proteins blog posts Download the fluorescent proteins 101 eBook Read....org Browse the fluorescent protein collection Find empty backbones for fluorescent protein fusions Browse...

...CRISPRing typical mouse models, including creating Cre-dependent conditional alleles, has remained a challenge...used cassettes, such as fluorescent proteins (EGFP, mCherry etc.), recombinases (Cre, Flp etc.), and tetracycline... make. For example, large insertions, like a fluorescent protein knock-in, suffer from low efficiency ... your desired insertion feature (e.g., a fluorescent protein), you can amplify the desired region using..., et al. "One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated...plasmid pmScarlet_C1! After creating the T7-DNA template, you'd use ivTRT to create the ssDNA construct as ...CRISPR genome editing has made it easier to create knockout alleles in a variety of species, including...

...developed a way to rapidly screen thousands of proteins for one that can report electrical activity via ...minutes of inactivating the CASANOVA complex, fluorescent CRISPR complexes were observed binding telomeric...30377362. Studying neuronal activity using the fluorescent voltage indicator Archon Article contributed ...team identified Archon1, a novel opsin-based fluorescent voltage indicator. Archon1 can embed itself into...Heckler et al., 2015) or ratiometric Bimolecular Fluorescence Complementation (rBiFC, Grefen and Blatt, 2012...adapted cells expressing these plasmids, tgRFP fluorescence localizes to the mitochondria, because cPYP ...simultaneously clone two genes-of-interest (GOI) into two independent expression cassettes on a single plasmid (Grefen...

...et al., 2023). Additionally, their performance depends on the specific protocol used and the abundance...fits all. The approaches used will be different depending on the sample and target type. To start the validation...methanol, we found that the optimal perm/fix solution depended on the specific antibody:target combination of... cells were labelled with a green or violet, fluorescent dye, respectively. WT and KO cells were mixed...the optimal knockdown of the protein target will depend on the protein turnover rate. If you need a more...through an epitope tag such as FLAG/His/GFP. Fluorescent protein tags, which correlate closely with target... expression. Its reliability will be entirely dependent on the selectivity of the antibody used in the...

... the expression of a Cre recombinase contained within the knock-in. Including Cre-mediated marker excision...enough, depending on perspective – to work with inefficient sgRNAs in my initial direct screening efforts...such as drug resistance (Chen et al., 2013), fluorescence (GFP; Tzur et al., 2013), or rescue of mutant...kilobases of additional sequence being introduced. Cre-mediated excision of the selective cassette minimizes...experimental manipulation. In some cases, injection of Cre is required, however the development of the self-...approaches may become even more powerful following a report that one can use linear repair templates (ie. PCR-derived... 2014). A further development of this approach reported in 2017 uses the direct injection of a CRISPR/...

...sigh. The problem is that many of these studies report correlations without clearly testing whether a ...enzymatic activity cause demethylation?  Dividing cells depend on DNA methyltransferase 1 (DNMT1) to copy methylation... floxed dCas9 so that it may later be excised by Cre recombinase. Addgene has several great options: pLV...T2A-GFP for removable dCas9 expression and pLM-CMV-R-Cre. Selection for cells stably and highly expressing...expressing these constructs can be achieved by fluorescence-activated cell sorting, and stable gRNA expression...are dioxygenases that oxidize 5-methylcytosine to create rare DNA bases that are recognized by distinct ...domains tethered to dCas9 often interact with DNA independently of dCas9 targeting (Galonska 2018). This means...

...protein coding genes in the human genome with fluorescent proteins. For each gene, we needed to design ...While CrispyCrunch may evolve over time, you can depend on it to stick to a few guiding principles that...analysis because of its ease-of-use and comprehensive reports. With CrispyCrunch, you get the all information...the sequence inserted by HDR may inadvertently re-create a match for the active guide. With these constraints.... To use this tool, simply follow these steps: Create a new experiment in the program Design gRNAs by...

...transfection conditions for each cell line with a reporter construct to ensure robust plasmid delivery before...editing efficiency, but 37 °C is acceptable. Fluorescence Activated Cell Sorting (FACS) of Transfected...incubate at 37 °C for 7 - 14 days. Vary these times depending on the doubling time of the cell line used. NOTE... Bauer's labs details the use of CRISPR/Cas9 to create genomic deletions in mammalian cell lines. Along... pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order...strategies for genes and non-coding elements. For creating a gene knockout, two sgRNA located within exons...strand ( Figure 2A ). However, DSB will occur independent of orientation of the protospacer sequence/PAM...

...sequences using a fluorescent reporter. In SHERLOCK, a quenched ssRNA fluorescent reporter is added to the...viral genome, and a ssDNA-fluorescently quenched reporter is added. As this reporter is degraded by Cas12a...inactive dCas9 to a fluorescent marker like green fluorescent protein (GFP), creating a customizable DNA...have also led to reduced dependency on specific PAM sequences and the creation of new tools. Figure 3: ...binding signal for Cas9, but the exact sequence depends on which Cas protein you use, and there are several...homology arms). The length of each homology arm depends on the size of the change being introduced; larger...larger insertions require longer homology arms. Depending on the application, the donor DNA may be a single-stranded...

...recognizes the different fluorescent labels. The machine detects which fluorescently labeled nucleotide is...antibiotic resistance. This benefit can be context-dependent, and thus the plasmid exists in a symbiotic relationship...the control of a non-bacterial promoter) or a fluorescent protein (that can be used to select or sort the...cell types (mammalian, yeast, bacterial, etc.), depending largely on which promoter is used to initiate ...information on CRISPR check out Addgene’s CRISPR guide . Reporter Plasmids - Used for studying the function of genetic... genetic elements. These plasmids contain a reporter gene (for example, luciferase or GFP) that offers...relies on the random incorporation of modified, fluorescently tagged bases onto the growing DNA strand in ...

...might be later). Find scFv plasmids at Addgene Depending on how an antibody is broken up, there are a few...by reducing the distance between the target and reporter. They have fewer non-specific binding opportunities...it either needs to be directly conjugated to a reporter or you need a light-chain specific conjugated ...fairly easy to find these in a Fab fragment format. Reporter-conjugated anti-IgG Fab fragments make good secondaries...Light chain. C = Constant domain. H = Heavy chain. Created with biorender.com     The modularity of antibodies...hinge region disulfide bonds, resulting in two independent Fab fragments with small tails of the hinge region...above the hinge-region, which results in two independent Fab fragments. Size: ~50 kDa. Fv - These fragments...

...expression plasmids, gene knock-down plasmids, reporter plasmids, viral plasmids, and genome engineering...quantity inside a cell Plasmids 101: Green Fluorescent Protein (GFP) Plasmids 101: Luciferase Monitor...small circular pieces of DNA that replicate independently from the host's chromosomal DNA. They are mainly... foreign DNA into another cell. Minimally, lab-created plasmids have an origin of replication, selection...cloning methods (restriction enzyme, ligation independent, Gateway, Gibson, etc). The cloning method is...

...chemogenetic reporter ensures that the reporter remains silent until a cell or tissue-specific Cre is provided...luciferase-fluorescent protein fusions, to more-sensitive opsins has led to improved LMOs with increased efficiencies...excite or inhibit the neurons expressing the LMO, depending on its biophysical properties. While LMOs provide...efficiencies. Depending on the type of luciferase used, the luciferin substrate may be the native coelenterazine...turn ON or turn OFF neurons in your experiment? Depending on your answer, you’d pick a chemogenetic activator..., CAG General expression Targeted expression. Depending on the experiment, an AAV-encoded chemogenetic... FLEx vectors block expression in the absence of Cre and are especially useful in AAV experiments where...

...optical switches Sensors are genetically-encoded reporters of molecular signals; e.g., calcium indicators...turn ON or turn OFF neurons in your experiment? Depending on your answer, you’d pick an excitatory or inhibitory...results in more localized expression of the opsin. Depending on the virus and promoter system used, there is...combined with site specific recombinase technology like Cre-lox, which allows you to express an opsin ONLY in...using a viral vector with a floxed opsin in a VGAT-cre animal will result in expression of the opsin only...Alternatively, when one half of a given protein, such as the Cre recombinase, is fused to Cry2 and the other half ...activator FixJ in the absence of blue light. 470 Fluorescent Protein domains Dronpa based Light-inducible ...