We narrowed to 741 results for: RAN-1

...effective recovery of knock-ins, but did result in 1-2 kilobases of additional sequence being introduced...facilitating recovery of edited animals. Figure 1: In the background are 504 restriction digests of ...selection for repair of a temperature-sensitive pha-1 mutation significantly enriches for knock-in of 2x... 3xFLAG epitopes into other, non-linked loci; pha-1(e2123) mutant worms are perfectly viable at 15 ºC,... repair pathway. Reagents required to perform pha-1 co-conversion are available through Addgene. In 2019... technologies have transformed the palette of experiments possible in a wide range of organisms and cell...genomic locus (CRISPR-ChAP-MS) could further transform the range of experiments imaginable in C. elegans. ...

... levels. That is, if transcription factor 1 controls expression of transcription factor 2, which directly...by modulating the induction of transcription factors 1 and 2. See Aranda-Díaz et al. for more information...difficult-to-manipulate primary cells, achieving knockdown within 1-2 hr of reagent delivery. TRIM-Away is suitable for...expression, Aranda-Díaz et al transformed yeast with both a GAL4/estradiol controlled transcription factor ... a broad pH range. The four biometric probes successfully targeted endosomes under a range of pH conditions...ligand binding domains and the MSN2 transactivation domain. Transcription factor activation via the appropriate...progesterone controlled transcription factor as well as reporters specific to each transcription factor. They then...

... help improve the transport of protein of interest the membrane.   Figure 1: Electron microscope...proportion were found on the plasma membrane (top panel in Fig. 1). Of course, this was problematic, because...expression of hM4Di in plasma membranes in both species (bottom panel in Fig. 1). Our results indicate that... the transgene of interest. In our studies, mCherry interfered with the transport to the membrane of the...the hM4Di might improve the transport of this DREADD to the plasma membrane. The haemagglutinin (HA) tag...the expression of the transgene of interest and its localization in the membrane. The lesson we learned... the plasma membrane. Note that the use of the HA tag significantly improves the transport of hM4Di to...

...) vector (Figure 1). Everything else outside the ITRs gets left behind.    Figure 1: Recombinant AAV ...reduces rAAV productivity but increases transgene expression. Sci Rep, 7(1), 5432. https://doi.org/10.1038/s41598...containing transcription factor binding sites can increase transgene expression and transduction efficiency...as the gene that they package. In a transfer plasmid, the transgene is flanked by two ITRs, and the ITRs...vectors, modifications can affect gene transfer efficiency and transgene expression (Zhou et al. 2017). For...these mutations can affect transduction efficiency and subsequent transgene expression. Replacing the ...nucleotide sequences that flank the ends of the single stranded DNA genome of AAV in the native virus. Since the...

... Figure 1: Strand separation and target DNA binding to the Cas9 nuclease. Stable strand separation ...by 2 sets of interactions : 1. Interactions between the non-target strand and a positively charged groove... improvements in genome engineering! References 1. Slaymaker, Ian M., et al. "Rationally engineered ...increases when target DNA strand separation is stabilized (2,3). Stable strand separation is maintained...and therefore destabilize basal strand separation. This decreased strand separation combined with the weak...the non-target strand and the HNH/RuvC groove, and interactions between the target strand and the gRNA....nuclease domains 2. Interactions between the target strand and the gRNA (see figure below) Because off-target...

...neuron which generates an action potential (Figure 1). This light-induced excitation can take place in ...channelrhodopsin activation to a small volume of brain tissue (~1 mm3). Current channelrhodopsins also need high-intensity...fiber-optic cables into the brain. Figure 1: With no blue light, the channelrhodopsin remains ... that determine membrane expression and plasma membrane localization of transmembrane proteins, like rhodopsins...capable of broad transduction of the nervous system which eliminates the need for intracranial injections. ... to perform in vivo optogenetic experiments. Intracranial injections of AAV are commonly used to deliver...channelrhodopsin-based optogenetics minimally-invasive, Frances Arnold’s lab and Viviana Gradinaru’s lab used machine...

...Collection Fluorescent tagging strategy Figure 1: Gene editing is the addition of a useful coding sequence...plasmid design strategy. A donor plasmid contains 1 kb homology arms matching the gene of interest, a ...Institute website at alleninstitute.org. References 1. Roberts, Brock, et al. "Systematic gene tagging using... copy. The coding sequence for GFP is transcribed and translated along with the gene, producing both a...exon of the gene to ensure it would be transcribed and translated. For an N-terminally tagged protein, ...destroyed by CRISPR/Cas9 during transfection. Making the double strand break and introducing the tag through...base pairs on each side, in fact. Later, when we transfect the cells, these large regions of homology enable...

...Figure 1: The Addgene Requests Dashboard   The dashboard will display the following items (Fig. 1): ...depositor, but it can also be valuable data to share in grant proposals or to demonstrate impact in annual reports...

...Popular gamma-Retroviral Transfer Vectors   Expressing your gene of interest pMKO.1 GFP (Plasmid #10676) ...GFP expression.  Cloning and expressing RNAi pMKO.1 puro (Plasmid #8452) RNA interference (RNAi) is an...information and a detailed protocol on how to use the pLKO.1 vector can be found here. Cre-Lox technology Puro....split-vector systems’ by separating viral genes and transgene expression across several plasmids. The most commonly...separate envelope and packaging plasmids as well as transfer plasmids. This concept ensures safe handling and...Gamma-retroviral vectors have a relatively high transduction efficiency, but in contrast to lentiviral vectors... are very versatile tools as they are able to transduce dividing and non-dividing cells. More detailed...

... two of the remaining viral proteins. Only Nsp11 ran bigger than expected, and Orf7b gave a lot of background...received so many requests (175 and counting) that we ran out of FedEx supplies at one point and had to go ...faster. Years of science, done in weeks Figure 1: Sending out 80 FedEx envelopes in a single day --...right) from the University of California, San Francisco. Manon Eckhardt and Melanie Brewer are on the ...

... biosensor, so that the (1) binding of calcium, (2) allosteric rearrangements of the sensor, and (3) subsequent... to a red fluorescent state only by simultaneous (1) user-controlled illumination with violet light and...fluorescent indicators. Nature protocols. 2006 Aug;1(3):1057-65. Resources on Addgene.org Addgene’s AAV...your expected range. For example, calcium in the ER lumen reaches the sub-millimolar range, much higher...are used to monitor calcium concentrations and transients in living cells and organisms. Unlike chemical...sensors available that will localize at the plasma membrane. GCaMP-X is a nucleus-targeted GCaMP6 with an ...an extra apoCaM-binding motif to prevent aberrant binding of the biosensor to endogenous calmodulin. Multiplexing...

... activity to cut off-target sites. Figure 1: Rectangles represent the on-target activity of a ...specificity you need for your particular experiment. Step 1: Choose your target well If multiple target sites ...detectable off-targets. Nature Communications, 14(1), 5746. https://doi.org/10.1038/s41467-023-41393-5...with 5′G-extended sgRNAs. Nature Communications, 11(1), 1223. https://doi.org/10.1038/s41467-020-15021-5...IFNs can be arranged along a spectrum of decreasing activity/increasing fidelity (activity rank) and likewise... three selected IFNs that divide the target ranking range into four approximately proportional sections...the lowest-ranking active (green) variant but with higher activity than the highest-ranking inactive (...

...the other (Sandomenico et al., 2020). Figure 1: Comparison between the IgG antibody and scFv. ...intensity of a tagged protein. SunTag has two components: 1) a protein of interest that’s tagged with 10-24 copies...Application. Clinical and Developmental Immunology 2012:1–15 . https://doi.org/10.1155/2012/980250  Bates A,...without disulfide bonds, made by molecular evolution 1 1Edited by I. A. Wilson. Journal of Molecular Biology... the CAR facilitates signal transduction and release of cytotoxic granules from the T cell. When a CAR...., 2012, Bates and Power., 2019): rapid blood clearance, which is useful for imaging applications; better...small size (Bates and Power., 2019). Their rapid clearance from blood can be a drawback for therapeutic applications...

...yourself in a creative rut, read on!   Fig 1: Blugene using creative problem-solving in the lab...Blog Transferable Skills Guide: Problem-Solving Transferable Skills Guide: Teamwork Transferable Skills...feel they lack formal training in a variety of transferable skills. At Addgene we're helping to fill this...both helping researchers recognize their MANY transferable skills learned while working in the lab and ...Today, we’re highlighting an oft-underlooked transferable skill: creativity. I’ve heard quite a few scientists..., it’s clear that creativity is absolutely a transferable skill developed during PhD training. Creativity...problem. In a study outlined in Originals by Adam Grant, a creativity expert at Stanford conducted an experiment...

...domain (Figure 1).  Figure 1: Examples of sites on antibodies used for conjugation. 1) Endogenous ...microscopy, flow cytometry, or spectrophotometry. The range of fluorophores available for antibody conjugation... choice for ELISAs.  Biotin Biotin is used in a range of antibody-based assays. Biotin itself is not visible...Endogenous carbohydrates added to antibodies post-translationally. 4) Modifications made to antibodies, such ...commercially conjugated antibodies available, that never guarantees the antibody you need will be conjugated, or ...Stephenson W, et al (2017) Simultaneous epitope and transcriptome measurement in single cells. Nat Methods 14:...

...current state (Green when off, dark when on). Figure 1: Photoswitchable MEK1 (psMEK1). Is it possible to ...Below are a few examples demonstrated in Zhou et al. 1. Testing kinase inhibitors Zhou et al examined the...the protein and pdDronpa are preserved. References 1. Zhou, Xin X., Linlin Z. Fan, Pengpeng Li, Kang Shen...Signal transduction pathways are a lot like cell phone networks. Protein kinases deliver messages to ..., and how long their signals last all impact transduction of the message. To study signaling pathways,... game of telephone, often the signal must be transmitted by intermediate messengers. Optical control of...signaling by using psMEK1 and an ERK kinase translocation reporter with a mRuby2 fluorescent reporter ...

...glassware should be treated with a DEPC solution for 1 hour, and autoclaved to remove residual DEPC. Alternatively...more large, expensive ultracentrifuge.  Figure 1: An outline of the different steps in RNA extraction...extraction: twenty-something years on. Nature Protocols 1:581–585. https://doi.org/10.1038/nprot.2006.83 Additional...expense, and less of being left with a bunch of random solutions when all the spin columns run out. In... inherently not as stable as DNA- it is single stranded and its ribose groups are susceptible to hydrolysis...effectively isolate intact RNA, a fast, strong protein denaturant would be required- something that broke down ...Chirgwin and colleagues showed that a strong protein denaturant, guanidinium thiocyanate, did just this (Chirgwin...

... of the biotin-avidin interaction   Figure 1: Biotin is enriched in egg yolk while its binding ... substrate (Holden and Cremer, 2003). References 1. Chapman-Smith, A., & Cronan, J. E. “Molecular Biology...matrices.” Journal of Nanobiotechnology 13.1 (2015): 1–14. PubMed PMID: 25980573. PubMed Central PMCID: PMC4461904...detection by northern blotting.” Rice 7.1 (2014): 1–7. PubMed PMID: 26224555. PubMed Central PMCID: PMC4884002...purification of tagged transcription factors in mammalian cells and transgenic mice.” PNAS 100.13 (2003...and decarboxylases. These enzymes catalyze the transfer of carboxyl groups to organic acids to produce...lipogenesis, amino acid metabolism and energy transduction. Biotinylation is the general process by which...

... requires selection. These two steps are (Figure 1): The initial plasmid integration step (the so-called...antibiotic resistance marker from the vector (abR, Figure 1). The resolution step (“double crossover”) which leads... loss of a specific phenotype (using sacB, Figure 1, further discussed below).  Counter-selectable marker...Current protocols in molecular biology 106.1 (2014): 1-16. PubMed PMID: 24733238. Additional resources on...strong transcriptional terminator BBa_B1002 (from IGEM) upstream of the MCS to reduce transcriptional readthrough...rely on exogenous enzymes, require multiple transformation and plasmid curing steps, and can generate ...worked too. Rhamnose induction relies on the transcriptional activator rhaS. To allow manipulation of bacteria...

...! References 1. Sinche, Melanie, et al. "An evidence-based evaluation of transferrable skills and job...Page Read Other Posts in the Transferable Skills Guide Developing Transferable Skills During Science Training...that they lack formal training in a variety of transferable skills. At Addgene we've set out to fill this... highlighting that researchers do learn MANY transferable skills while working in the lab and by offering...where you might need some help. Today in our transferable skills guide: Collaborating with others outside... had to overcome some hesitation to ‘approach strangers’ and learn how to communicate outside my scientific...message and to keep it short. Soon, discussing with strangers stopped feeling weird and started to be fun. I...