...constructs 69570 and 69571 together encode the N and C terminal segments respectively of a GFP dependent ...-N-CretrcintG Connie Cepko 69571 AAV1 pAAV-EF1a-C-CreintG Connie Cepko 75469 AAV1 pAAV-EF1a-Flp-DOG-NW.... The advantages of optogenetics lie in the spatiotemporal control offered by the system that allows for... expression of ChR2(H134R) bound to an mCherry reporter. We now also offer AAVrg for plasmid 83898, which...advantage of the extensive supply of transgenic reporter lines that supply a variety of GFP expression ...

...Center (www.taleffectors.com) . Whether you work in a C. elegans lab and have been struggling to mutate a ..., Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove AJ, Voytas DF. Nucleic...using engineered TALENs. Sander JD, Cade L, Khayter C, Reyon D, Peterson RT, Joung JK, Yeh JR. Nat Biotechnol...every 35bp. Researchers are still determining the importance of context for each TAL effector within an array...plasmid toolbox. Addgene has rapidly become an important resource for this new technology, and we hope ...

...80 °C. Titer All titering is performed on lentiviral preparations that have been stored at -80 °C and ...Preparations are then aliquoted and stored at -80 °C. Titer Titering is either performed by Addgene or ...expression and/or function. These data are sometimes reported or posted on the material page for the corresponding...recipient's initial thaw will be accounted for in our reported titers. Lentiviral vectors are titered using a...This DNA is then used as a template to amplify a portion of the intended lentiviral insert. During this ...

...replicates such as multiple mice in each group and C, technical replicates or repeated measurements will...interactions between the sample and the secondary antibody. C, a label control treats the sample to the full protocol...PubMed Central PMCID: PMC5773976. Waters, Jennifer C. “Accuracy and Precision in Quantitative Fluorescence...binding  Primary antibody specificity is further supported by a pre-immune serum or isotype-specific control...

...come out! References Noren, C. J., Anthony-Cahill, S. J., Griffith, M. C., & Schultz, P. G. (1989). A...describing a technique that allowed for intentional incorporation of an unnatural amino acid in translation (Noren...natural translation machinery; these are often incorporated into the cells via plasmid. Third, a second ...containing the chosen codon that will result in the incorporation of the non-canonical amino acid at the site ...integrated into the cell’s genome, instead of incorporation via plasmid or media. Theoretically, if you ...will produce a protein with a site specifically incorporated ncAA, ready for you do to any number of experiments... They provide resources, knowledge, and most importantly, validated protocols (using readily available...

...Pewe L, Wohlford-Lenane C, Hickey M, Manzel L, Shi L, Netland J, Jia HP, Halabi C, Sigmund CD, Meyerholz...SARS-CoV-2 were developed for broad use and are now important for accelerating research into this virus. One...during the pandemic and in the future, depends on support from the scientific community. Deposits of new ... be deposited into repositories, and financial support to maintain repositories’ capacity for storing,...all ways in which the scientific community can support repositories. Repositories are essential for the...

... a mutation that causes Niemann-Pick disease type C, directly demonstrating the therapeutic potential ...toxicity in long-term experiments.  Figure 4: The C-terminus of Cre (CreC) and the N-terminus of Cre (...rhodopsin Ace2N and mScarlet. Ace2N quenches a proportion of mScarlet's emission via FRET. Image from Beck...indicator plasmids at Addgene! Beck et al., Sci Report 2019. PubMed PMID: 31685893. A MoClo toolkit for... Eisenhoffer lab, is a new tool that increases temporal and spatial precision of gene expression and reduces...

...disorder of glycosylation, and Niemann-Pick disease type C. Although the three DMD studies referenced here represent... 2015 Dec 31. doi:10.1126/science.aae0169 2. Long C, Amoasii L, Mireault AA, McAnally JR, Li H, Sanchez-Ortiz...K, Cheng JK, Chew WL, Widrick JJ, Yan WX, Maesner C, Wu EY, Xiao R, Ran FA, Cong L, Zhang F, Vandenberghe...optimized, it’s important to ascertain how long the rescue phenotype will last, and more importantly, if it does...genetic disease in a postnatal mouse model, an important proof of concept for future preclinical and clinical...does not cause obvious germline modification, an important finding given the controversy associated with ...great step forward for CRISPR gene therapy, it’s important to realize that DMD is a simpler case than other...

...also shown that the Q system is also functional in C. elegans. Subsequent work by the Luo and Potter labs...23995928; PMCID: PMC3811519.Seo SO, Schmidt-Dannert C. Development of a synthetic cumate-inducible gene ...; PMCID: PMC5270762. Wendler, F., Park, S., Hill, C. et al. A LexAop > UAS > QUAS trimeric plasmid to ...stage-specific promoter and design a UAS reporter construct, the reporter will only be expressed where and/or...UAS to examine the expression of reporter genes, or to combine reporter expression from one promoter with...the Q system from a gene cluster in the fungus Neurospora crassa. The QUAS promoter is less leaky than ... transcription from the UAS promoter. This is important because it allows them to be used together for...

...short term storage (think days to weeks) in the 4°C. If you’ll only be using it every few month or if ...be going through small aliquots at a time, the -20°C is your best option, in aliquots no smaller than 10uL...whenever you use the antibody. This information is important, and you’ll need to track it throughout your experiments...

...10.1016/j.gendis.2017.04.001 Maetzig T, Galla M, Baum C, Schambach A. Gammaretroviral vectors: biology, technology...2011 Jun;3(6):677-713. DOI: 10.3390/v3060677 Varanda C, Félix MDR, Campos MD, Materatski P. An Overview of...Lenti particles enter the nucleus through nuclear pores and thus do not require breakdown of the nuclear...

... of cytidine and adenine base editors that enable C-to-T and A-to-G base substitutions respectively. Substitutions...:723–728 . https://doi.org/10.1021/sb500351f Dong C, Fontana J, Patel A, Carothers JM, Zalatan JG (2018... https://doi.org/10.1016/j.cell.2013.02.022 Ronda C, Pedersen LE, Sommer MOA, Nielsen AT (2016) CRMAGE...Vridhambal GS, Rivera AJ, Montgomery SA, Fortier L-C, Barrangou R, Theriot CM, Ousterout DG (2020) In Vivo...the phage recombineering machinery are first electroporated with pCas9. Then, pCRISPR is introduced along... a mammalian cell line (Qi et al., 2013). Two important features of this early system are its regulatable...Euler CW, Jiang W, Nussenzweig PM, Goldberg GW, Duportet X, Fischetti VA, Marraffini LA (2014) Exploiting...

...variables. Twitch is a FRET-based calcium reporter derived from troponin C. It is engineered with a reduced number...calcium binding proteins (e.g., calmodulin or troponin C). Their fluorescent readouts are used to monitor calcium...Calcium is arguably one of the most important metal ions in biology (sodium and potassium are up there..., and an allosteric modulator. Because of its importance, scientists have developed calcium sensors to...GCaMP6 family of sensors are commonly used calcium reporters with different kinetic parameters. There are three...also available. Detection methods Many calcium reporters use a single fluorescent protein (FP), requiring...for in vivo studies) There are several calcium reporters specially designed for the aims and requirements...

...connected by a 9 amino acid linker that’s fused to the C-terminus of DsRed.T3. See Table 1 for a summary of...are available from Addgene. References 1. Rosado, C., Mijaljica, D., Hatzinisiriou, I., Prescott, M., ...spectra. DsRed is the permanent fluorescent tag portion of Rosella: it will emit red fluorescence regardless...mechanisms behind autophagy by tracking what’s being transported to the yeast vacuole (i.e. cytosol, mitochondria...2008). Rosella: A fluorescent pH-biosensor for reporting vacuolar turnover of cytosol and organelles in...using a single fluorescent biosensor. Scientific Reports,5(1). doi:10.1038/srep12397. PubMed PMID: 26215030...

...from an additional 5–10 minutes of incubation at 37 °C. Wash the membrane for 5–10 minutes 2–3x in fresh ...stripping solution and agitate for 30–45 minutes at 50 °C. Wash the membrane for 5–10 minutes 2–6x in fresh ...directly to buffer without a re-wetting step. Important: do not dry your membrane with antibodies on it...the ones shared by Bio-Techne, ThermoFisher, or Millipore Sigma. We’ll share the basic composition of these...Alternatively, companies including ThermoFisher, Millipore Sigma, and Azure Biosystems offer pre-made stripping...your western blot several times — some researchers report up to ten cycles! Just remember that a bit of sample...

...blog post for more details. Figure 2: Dense (c), intermediate (d), and sparse (e) labeling of the...remove a stop cassette upstream of an effector/reporter gene. In cells where the stop cassette is removed...https://doi.org/10.1101/788679 A destabilized GFP reporter for calcineurin activity in yeast Article contributed... it is an attractive drug target. Calcineurin reporters allow scientists to measure calcineurin dynamics... target calcineurin. Current calcineurin gene reporters are based on the bacterial βgal assay, and are...Büttner’s lab designed a calcineurin transcriptional reporter consisting of a quickly-degraded destabilized ...panels) equipped with destabilized GFP calcineurin reporter (blue) and stained with PI (pink) to indicate ...

... its preferred target integration site: TNA(C/G)TTATAA(G/C)TNA. Tol2 can deliver 10-11 kB to mammalian...   References and Resources References Feschotte C, Pritham EJ (2007) DNA Transposons and the Evolution...piggyBac plasmids    Tol2  Tol2 was the first reported active DNA transposon in vertebrates. It was discovered...mutations. In these screens, the transposons encode reporter genes, mutagenic cassettes, or barcodes. When ...2021). Target site preference and host range are important to consider when selecting a transposon for a ...pronuclei of fertilized eggs, which leads to random incorporation of that sequence into the zygote’s genome, a...unpredictable. Transposons, however, efficiently incorporate into the zygote’s genome after injection into...

...application of blue light, Vvd dimerizes bringing the C-terminal and N-terminal Cre fragments together and...the PAM sequence is 5’-TBN-3’ (where B is G, T, or C). The authors demonstrate that CRISPR-Casɸ is active...in a protein.    Cells expressing an RFP reporter plasmid for recombinase activity were exposed ...interest. However, FACS is unable to spatially and temporally target single cells. Now, Francois St-Pierre’...way to isolate individual cells with unique spatiotemporal phenotypes from large heterogeneous cultures...introduction & resolution of two DSBs in a fluorescent reporter. Find the plasmids here Yongsub Kim’s lab engineered...

...These plasmids enable lentiviral expression of N- or C-terminally FKBP12F36V-tagged fusion chimeras or locus-specific... rapid means of comparing functionality of N- and C-terminal fusions and degradation in a cell line of...transcription factors with important roles in Multiple Myeloma), it is important to ensure lack of degradation... chimeras For each protein-of-interest, it is important to determine which terminus can accommodate the...

...easy to switch out reporters, Cas proteins, guide cassettes, promoters, N’ and/or C’ terminus, and selection...Griffith, A. L., Szegletes, Z. M., Wen, B., Kraus, C., Miller, N. W., Steger, R. J., Escude Velasco, B....New to the bench? Fragmid depositor John Doench reports even the greenest members of his lab, high school...vectors are available? What you’re building is important — but so is where you’re building it! Table 1 ...

...doi.org/10.1128/AEM.67.8.3440-3444.2001 Fogarty, E. C., Schechter, M. S., Lolans, K., Sheahan, M. L., Veseli....102684 Shareck, J., Choi, Y., Lee, B., & Miguez, C. B. (2004). Cloning Vectors Based on Cryptic Plasmids...plasmids — more on that in a minute! Fun fact! Electroporation is one popular method scientists use to promote...

... was created by adding a 9-residue peptide to the C-terminus of human codon-optimized  cryptochrome 2 ...a fragment of the kainate receptor subunit to the C-terminus of the protein. Unlike the diffuse neuronal...while also capturing information about spatial and temporal organization. The design use to detect PKA activity...

...mutations in the common cytokine receptor gamma chain (c) and is characterized by a lack of immune cell development...1172-1179. PubMed PMID: 28671695. 6. Münch, Robert C., et al. "Off-target-free gene delivery by affinity-purified...basic research and other ongoing clinical trials support the utility of AAV as a broadly applicable gene...

...in mice. Scientific Reports 9: https://doi.org/10.1038/s41598-019-41088-2 Magnus C, Lee P, Bonaventura...10.1021/cn500325v Goutaudier, R., Coizet, V., Carcenac, C., & Carnicella, S. (2019). DREADDs: The Power of the...Targeted and specific delivery becomes especially important for studies in neuroscience where specific tissues...neuron need to be targeted. Targeting is also important in the development of therapies when expression...Picking Ligands Picking the right ligand is also important for building a chemogenetic system. Your experiment...cellular response you aim to manipulate. It is important to consider that the same receptor can elicit ...drug that only binds to one receptor, so it is important to fully characterize the compound, or compounds...

...Most FP fusions for FRET studies are at the N- or C-terminus of your target protein, but even these modifications...can insert your protein of interest, resulting in a C-terminal fusion of a tetracysteine tag. In addition...for observed FRET efficiencies between 80% and 10%. C Cy3NTA and Cy5NTA bind His tags similarly but with...nonspecifically soak up your fluorophore package. One important concept in FRET experimental design is that the...donor first where labeling specificity is most important. Donor labeling and the need for specificity Donor...protein molecules contain the donor and, more importantly there is no fluorescence background resulting...in the ribosome, resulting in site-specific incorporation of the attached fluorophore. However, since ...

....2307/1542146 Lazarus, J. E., Warr, A. R., Kuehl, C. J., Giorgio, R. T., Davis, B. M., & Waldor, M. K..... D., Jamal, S., Wistrand-Yuen, P., … Forster, A. C. (2018). Engineering a palette of eukaryotic chromoproteins...Characterization of the Pink and Blue Pigments of Pocilloporid and Acroporid Corals. The Biological Bulletin 189:288...chromoproteins and fluorescent proteins, it’s important to note that despite their distinction by name...interactions with cytosolic proteins. This step is important as non-specific binding to proteins can inhibit...improved expression in living cells. Scientific Reports, 9(1). https://doi.org/10.1038/s41598-019-48604...

... CRISPR plasmids deposited with Addgene by year. (C) Cumulative number of laboratories that have deposited...smoothed using locally estimated scatterplot smoothing. (C) Number of requests per year for pooled libraries,...plasmids/year from 2017 to 2023. This growth is supported by new labs joining the Addgene CRISPR community...year of deposit. Outlines show a representative proportion of CRISPR plasmids requested, categorized by ...plasmids deposited by country; square size is proportional to number of plasmids deposited by each country...plasmids requested by country; square size is proportional to the number of plasmids requested by each ...Barrangou for his authorial contributions to, and support for, this and previous Addgene CRISPR publications...

...these methods a try.  References Højland Knudsen C, Ásgrímsdóttir ES, Rahimi K, Gill KP, Frandsen S, ... CRISPR reporters in the near future.  SRIRACCHA (stable, but reversible, integrated reporter for assaying...histone)-GFP reporter into the genome of mammalian cells.  Figure 1: The SRIRACCHA reporter construct...Find GEmCherry2 reporter plasmids at Addgene Overall SRIRACHA and GEmCherry2 reporter systems provide ...Recently scientists have created CRISPR fluorescent reporter assays to quantify targeted Cas9 cleavage, and...and non-homologous end joining (NHEJ). In these reporters, editing typically results in a change in the ...this blog we will focus on two fluorescent CRISPR reporter methods, SRIRACCHA and GEMCherry2. We will cover...

...3rd 4 78,637 Broad GPP Humagne Set C and Humagne Set D 172650 (Set C) 172651 (Set D) Knockout Human Doench...gRNAs By Species Mammalian Bacteria Drosophila Plant C. elegans Yeast Zebrafish Xenopus CRISPR Resources ...pneumoniae M. tuberculosis M. smegmatis Green monkey ( C. sabaeus ) Kaposi's Sarcoma-associated Herpes Virus...collection differ based on some important criteria, and it is important to know which library best...mind: CRISPR screening experiments require electroporation to amplify the pooled library and the use of...best suits your particular experiment. Some important variables include: Type of genetic modification - ...Human Bonifacino 3rd 6-25 11,108 Borodina Yeast Transporter Libraries 153101-153106 Knockout Yeast Borodina...

...Maddalo D, Manchado E, Concepcion CP, Bonetti C, Vidigal JA, Han Y-C, Ogrodowski P, Crippa A, Rekhtman N, de...Scientific Reports paper introducing the delivery method in 2014, Manuel Goncalves lab report that AdV-...the likelihood that donor DNA will interact with sporadic double-stranded chromosomal DNA breaks “that always...