...GBPs/GFP. The new system, CRE-DOG (Cre Dependent On GFP), is activated by GFP and derivatives GFP and ...to bind GFP. The idea of using GFP as a scaffold suddenly seemed very realistic. Envisioning GFP as a substrate...presence of GFP. These systems, known as T-DDOG and Cre-DOG, respectively, repurpose popular GFP reporter...lines expressing GFP in specific cell types to do more than just label cells. If GFP could be co-opted...co-opted to control gene expression, he could then selectively manipulate only GFP-labeled cells. Once Tang ...tested pairs of their GFP-binding proteins (GBPs) to find those that could co-occupy GFP. Once they’d found...culture system. In cells cotransfected with GFP and the GBPs, GFP linked the two GBPs together to create a...

...process worked well with the common GFP variants EGFP, mGFP4-T, and S65T GFP, and no red color was observed...repurpose GFP is great news for microscopy enthusiasts! Addgene has empty backbones containing GFP variants...exciting GFP-labeled plant cells at 405 nm instead of 488 nm, Sattarzadeh et al. noticed that GFP had turned...cytosol-targeted GFP. White circles represent the irradiated area. Photoconverted GFP spreads through ...Applications and limitations of GFP photoconversion One concern with photoconverting GFP is the expression level...intensity increases with GFP concentration and saturates around 30 uM. For low-abundance GFP fusions, a photoconversion...have created a transgenic GFP-containing organism,” says Hanson, “using a gene encoding one of the unexpectedly...

...scientists to begin to study GFP and deduce its biochemical properties. Now GFP and its fluorescent derivatives...and scientists employ GFP for a wide number of functions, including: tagging genes for elucidating their...Placing GFP under the control of a promoter of interest can be used to effectively monitor gene expression...type may have the GFP under the control of an additional promoter from that of the gene of interest, or ...expressing GFP from cells that are not. Developmental/transgenic uses: Because of its stability, GFP can be...implantation and generation of transgenic mice. Purification: GFP can be used as a general epitope tag for...scientists began engineering new versions of GFP through mutagenesis in order to improve its physical and biochemical...

...S65T) in the original jellyfish GFP (AvGFP) to create enhanced GFP (EGFP), a variant with improved brightness... There are numerous GFP variants. Here I use the name ‘GFP’, which refers to mEGFP. This variant was made...examined on a case-by-case basis. GFP is sensitive to acid The chromophore of GFP can exist in a protonated and...its acid tolerance is maintained in cells.   GFP is big GFP is a 28 kDa protein that resembles a cylinder...2013; Hilaire et al., 2017). GFP can only be used for tagging proteins GFP fusion proteins are made by ... a fusion with GFP allows for detection of genomic locations (Chen et al., 2013). GFP fluorescence overlaps...necessary to generate the strictly monomeric EGFP variant, mEGFP (Zacharias et al., 2002). GFP needs oxygen...

...laboratory. The discovery of GFP The story of how fluorescent proteins like GFP came to be used as research...He began to study the A. victoria gene responsible for encoding GFP, and, in 1992, he reported its sequence...their work on GFP. This great honor serves to highlight how useful fluorescent proteins like GFP really are...fluorescent ones, like Green Fluorescent Protein (GFP). These molecules occur naturally in bioluminescent... calcium ions, and the second, which we now call GFP, absorbs that blue light and glows green. In the ...new green glowing protein could be used to measure gene transcription, as well as to track protein localization... Prasher’s collaborator Martin Chalfie expressed GFP in exogenous organisms (E. coli, and later C. elegans...

...decrease in red puncta. Find the mRFP-GFP-LC3 plasmid at Addgene! GFP-LC3-RFP-LC3∆G: A fluorescent probe ...flux. mRFP-GFP-LC3 functions as an autophagy bionsensor via the same mechanism as Rosella: GFP is quenched...autophagy biosensor is the GFP-LC3-RFP-LC3ΔG probe. It’s similar to the mRFP-GFP-LC3 Tandem Fluorescent Protein...Autophagic flux is estimated by the GFP/RFP ratio: a decrease in GFP fluorescence relative to RFP fluorescence...the mitochondria-targeted Keima variant at Addgene! mRFP-GFP-LC3 tandem fluorescent protein This autophagy...translation, this fusion protein is cleaved to generate one molecule of GFP-tagged LC3 for every one molecule of...decrease in autophagic flux.   Find the GFP-LC3-RFP-LC3∆G plasmid at Addgene! References Klionsky, Daniel J....

... at Addgene. Additional Resources the Addgene Blog Learn More about SunTag Catch up on Your GFP Background...sites on the nascent polypeptide recruit scFV-GFP, generating green puncta that colocalize with the red reporter...switching, TRICK requires the following components: a GFP protein that binds to the coding region of a reporter...reporter mRNA via a PP7 coat protein (NLS-PCP-GFP) an RFP protein that binds to the 3’ UTR of the reporter...Plasmid 64542 or 64543) Before translation, both GFP and RFP are bound to the reporter, causing it to ...appear yellow. The act of translation pushes NLS-PCP-GFP off of the mRNA, leaving only a red FP bound to the... contains a nuclear localization signal, NLS-PCP-GFP then returns to the nucleus to bind cognate untranslated...

...released Anti-GFP [N86/8R] as another option for targeting GFP. But why might you need a second GFP antibody...on the Anti-GFP [N86/8R] page for more details. Image credit: Addgene.   Find anti-GFP [N86/8R] here...were compared head-to-head for gene expression changes of non-target genes and RNA-seq of a nuclear (MALAT1...PMCID: PMC9015817.   Meet anti-GFP [N86/8R]! by: Ashley Waldron Anti-GFP [N86/38.1R] was among the first...antibody?  Similarities:  Generated against full length GFP and converted to recombinant form by the NeuroMab...system Heat-inducible lentiviral Car constructs Anti-GFP [N86/8R] ready-to-use antibody Hyperfolder YFP ...variable domains that recognize different epitopes on GFP Monoclonal antibodies can yield relatively low signal...

... their hypothesis with GFP fluorescence. To do this they generated a silent GFP construct, that contains...engineered BFP variant that converts to GFP after a base editing event. GFP positive cells can be sorted and ...without relying solely on GFP (Katti et al., 2020). They named this reporter “Gene On” or GO. GO works by...changes the histidine to a tyrosine generating a GFP variant (GFPY66) that has a shifted emission spectra...lab was inspired by work that used an integrated GFP reporter that is converted into BFP (blue fluorescent...engineered a BFP variant that undergoes conversion to GFP after being targeted by a cytidine deaminase base... a successful base editing event would result in GFP fluorescence that can be visualized and sorted via...

...al. observed some GFP aggregation, which they reduced by using superfolder GFP (sfGFP) with the small solubility...scaffold fully occupied with GFP has a molecular weight of 1,400 kDa vs 24 kDa for GFP alone. Although traditional...nomenclature throughout the rest of this blog post, GFP refers to sfGFP-GB1.   Tanenbaum et al. examined the power... copies of the short epitope GCN4. GCN4 recruits GFP fused to the cognate scFV antibody, which is expressed...physiology. Having seen that mitochondrial tracker GFP-mitoNEET can impair mitochondrial function, they ...they examined the effects of mitoNEET-SunTag-GFP. As expected, they obtained bright images of mitochondria...used to improve CRISPR-based activation of target genes, but we’ll save that application for another day...

...the most efficient for gene editing. A virus encoding GFP plus a gRNA targeting GFP (pXPR_011, 59702-LV)...by the virally-delivered GFP gRNA, will edit out the virally-delivered GFP gene. So, cells with no Cas9... BRAF-edited and GFP-edited amplification products (BRAF-GFP duplex) and from GFP-edited amplification... to make my first CRISPR/Cas9 gene edit. I wanted to inactivate the gene BRAF (a kinase implicated in ...I thought I could see GFP expression with a fluorescence microscope, but the GFP expression was so low...expressed GFP) that I couldn’t get a good image. You could probably also do Western blotting against GFP expression...Targeting different regions of the gene is also useful in case the gene has a weird mutation that you didn...

... the GFP-Puro will be inserted just upstream of a stop codon. One potential concern is if the GFP-Puro...ready-made PITCh plasmids available from Addgene are perfect for expressing GFP from a given promoter, and the ...harnessed MMEJ to create a new method for CRISPR gene knock-in, termed PITCh (Precise Integration into Target ...Target Chromosomes). Using their PITCh plasmids, GFP knock-in cell lines can be created in about a month and...than that observed with NHEJ. PITCh: Using MMEJ for gene knock-in Building on the lab’s previous work, Sakuma...detailed protocol for MMEJ-mediated knock-in of a GFP-Puro cassette into a given locus, just upstream of...microhomology to the insertion locus flanking the GFP-Puro cassette. Three double stranded breaks are necessary...

... of GFP, it's variants, and their relevant mutations, check out Marcy's post from last week on GFP. Additionally... a few hours. For example, superfolder GFP (sfGFP) and mNeonGFP can fold in <10min at 37°C, mCherry takes...are PA-GFP, Dendra2, and the mEOS proteins. Some proteins are reversibly switchable (e.g. rsEGFP, Dreiklang...these biosensors can be found at Addgene. Split FPs – some FPs (e.g. GFP, Venus) can be split into two ...affected by the temperature. For instance, enhanced GFP (EGFP) was optimized for 37°C, and is therefore most... the discovery of the Green Fluorescent Protein (GFP), a protein from the jellyfish Aequorea Victoria....below (or emission wavelenght range).  By mutating GFP, the variants blue FP (BFP), cyan FP (CFP), and yellow...

...respectively of a GFP dependent Cre. The two segments come together in the presence of GFP to form a functional...supply a variety of GFP expression patterns, thus allowing you to further restrict gene activation to cells...cells expressing both Cre and GFP. The GFP dependent FLPo, Flp-DOG (75469), is also available in AAV1. ...AAVrg pAAV-hDlx-Flex-GFP-Fishell_6 Gordon Fishell   Optogenetics Optogenetics continues to supply neuroscience...-2 proteins ChR2 for targeted expression DREADDs GFP dependent Cre and FLPo And more!  New lentiviral...inhibition library that inhibits over 18,000 human genes and is used for genome-wide inhibition screening...in new serotypes, as well as a new Cre dependent GFP control plasmid for expression in forebrain GABA-...

...pAAV.hSyn-FLEX.iGABASnFR.F102G pAAV-Syn-Archon1-KGC-GFP-ER2 pAAV-Syn-FLEX-rc [Archon1-KGC-GFP-ER2] pAAV-FLEX-tdTomato pAAV-GFAP104...pAAV-mDlx-NLS-mRuby2 CAG-NLS-GFP pAAV-hSyn-mCherry Read on to learn more!  Calcium sensor AAV The GFP-based calcium...pAAV-Syn-Archon1-KGC-GFP-ER2) and cre-dependent version (pAAV-Syn-FLEX-rc [Archon1-KGC-GFP-ER2]) of this membrane-embedding...mCherry, pAAV-mDlx-NLS-mRuby2, CAG-NLS-GFP,and pAAV-hSyn-mCherry at Addgene. Viral vectors coming soon! These...37825  AAV8*, AAV9*  pAAV-CAG-GFP 50465  AAV8*, AAV9*  pAAV-hSyn-EGFP *Also coming in our new 20 ...variant of jGCaMP7 in AAV1 at Addgene! See all calcium sensor AAV at Addgene. GABA sensor AAV GABA is the...IRES EGFP Chimeric channels for neuronal manipulation 119742  AAV5  AAV SYN PSAM4 GlyR IRES EGFP Chimeric...

... have 10 HA or 10 MYC tags built into a GFP scaffold. Some GFP nanobodies  stain poorly when tagged with...weak signals of fluorescent protein (i.e. GFP, RFP) Detecting GFP reconstitution across synaptic partners...Monster because they recognize this GFP scaffold itself. Fortunately the GFP1 HA Spaghetti Monster RANbody doesn... sequences that targeted the following antigens: GFP, RFP,  the histone protein H2A2B, and the actin-binding...recognize the scaffold and is useful for detecting GFP. Spaghetti Monster tagged RANbodies can be detected...expensive Can only stain with one HRP-RANbody at a time GFP, RFP, H2A2B, Gelsolin Spaghetti Monster: HA (H)...Requires secondary antibody staining HA Tagged GFP, RFP, H2A2B Myc Tagged RFP, H2A2B Chicken IgY ...

...remain unaware that EGFP/GFP is prone to forming noncovalent dimers. This property of EGFP can lead to significant...Green Fluorescent Protein (GFP/EGFP) for a transcriptional reporter or as a general cytoplasmic label of cells...contributed by guest blogger Erik L. Snapp. Stop using EGFP/GFP for fusion proteins! Despite multiple studies ...in the world, is not inert. In all seriousness, EGFP/GFP has a real nontrivial propensity to noncovalently...best solution is to use truly monomeric FPs. EGFP and other GFP family members (superfolder, Emerald, CFP...However, if you fuse your protein of interest (POI) to GFP to study the protein's behavior in cells, in solution...noncovalently dimerize. That means that your POI fused to GFP or another fluorescent protein (FP) could be forming...

... Figure 1: Gene editing is the addition of a useful coding sequence (i.e. GFP) into a gene while leaving...features of the gene intact. Here, we show an example gene targeted for tagging with GFP at the C terminus...allele, or gene copy. The coding sequence for GFP is transcribed and translated along with the gene, producing...tag a wide variety of genes with GFP. These plasmids are available through Addgene, and the stem cell lines..., we inserted a linker and GFP tag at the end of the last exon of the gene to ensure it would be transcribed... kb homology arms matching the gene of interest, a linker, and the GFP tag sequence to be inserted. Created...insertion of the GFP tag precisely where we need it, in this example, at the C-terminus of the gene of interest...

...self-assembling split GFP (GFP1-9 and GFP10-11) by flipping a single β-strand of GFP, hence the name FlipGFP. FlipGFP...apoptosis reporter, ZipGFP, which utilized the self-assembling split GFP (GFP1-10 and GFP11) to increase fluorescence... reporters (Zhang et al., 2019). How does FlipGFP work? GFP contains 11 β-strands and one central ɑ-helix...Lab used FlipGFP to track the spatiotemporal dynamics of apoptosis during embryo development. GFP fluorescence...protein-protein interaction sensor based on tripartite split-GFP association." Scientific reports 3 (2013): 2854. ...PMC2117903. To, Tsz-Leung, et al. "Rational design of a GFP-based fluorogenic caspase reporter for imaging apoptosis...fluorogenic protease reporter by flipping a beta strand of GFP for imaging apoptosis in animals." Journal of the...

... structures GFP is limited in its ability to label subcellular structures in the CNS. GFP and other FPs...probes at Addgene Uses of smFPs 1. Multi-channel connectomic tracers Fluorescent proteins such as GFP have...nucleus AAVs CREATEd for gene delivery to the CNS and PNS Resources on Addgene.org Find the Spaghetti Monster...with the development of methodologies to deliver genes to the CNS have recently made it much easier to ..., and the long-term expression of AAV delivered genes. AAV serotypes have been extensively studied in ...fluorescent signals or using anti-FP antibodies. GFP emits light in only a single channel. In multi-color...these FPs is that they are less photostable than GFP. In addition, antibody-based amplification of their...

...targeted gene will be removed from the chromosome and and replaced by the resistance gene.The gene is thus...strategies to investigate a gene's function is to inactivate, or "knockout", the gene by replacing it or disrupting...endogenous gene. Here, we'll describe the techniques and the plasmids used to inactivate specific genes in mammalian...given gene.   Figure 1: A knockout targeting vector designed to insert a resistance gene. The ...targeted gene have been cloned into the vector on either side of an antibiotic resistance gene. To avoid... resistance gene through the insertion of flanking LoxP sequences, you can remove the gene through the...later remove a resistance gene. In this construct, the neomycin resistance gene is floxed so that it can...

...Check out Addgene’s monoclonal antibodies (mouse, rabbit, and chicken) to this tag! GFP  GFP is more than...If you need monoclonal GFP antibodies or guidance on designing a GFP tag Addgene has you covered!   Potential...amino acid protein that can also be used as a tag! GFP was originally derived from jellyfish (Aequorea victoria...epitope tag as well as a fluorescent marker. The GFP tag is ideal for live cell imaging, FRET, flow cytometry...antibodies also exist for imaging. The obvious con of GFP is its size – which can possibly interfere with protein...nature18308   Additional resources on Addgene.org Addgene’s General Use Antibodies Antibody guide Antibody...applications. You can find FLAG antibodies on Addgene’s catalog including a mouse monoclonal (M2) as well...

...stably integrate a reporter gene that contains a puromycin resistance gene followed by the target site...that contains a puromycin resistance gene linked to an in-frame H2B-GFP. This visual read out allows for ...that targets your desired gene (whose sequence is also found in the reporter gene). A control RFP expression... the supplied donor plasmid will express GFP. If there’s GFP expression, presumably the endogenous target...experiment that targeted the gene NFE2L2, the Madison lab found that H2B-GFP expressing cells exhibited ...RFP to GFP expressing cells provides a real-time read out of Cas9 activity.  By using the RFP:GFP ratio...in the genome) and an out-of-frame H2B (histone)-GFP reporter into the genome of mammalian cells.  ...

...Chang-Deng Hu Green sfGFP Split super-folder GFP, our most-requested GFP(1-10) and GFP(11) Versatile protein...Commun. 2016 Bo Huang spGFP Split superpositive GFP Split-superpositive GFP reassembly is a fast, efficient...base FP (for example, EBFP2(1-10) can be used with GFP(11)). Some of the articles linked below only provide...Blue EBFP2 EBFP2(1-10) and Capri(1-10) for use with GFP(11) Multiplexed labeling of cellular proteins with... Kamiyama Cerulean Cerulean(1-10) for use with GFP(11) Multiplexed labeling of cellular proteins with...background signal (especially for some older split-GFP constructs or certain cell environments). Also consider...resources on the Addgene blog Addgene Blog Biosensors posts Resources on Addgene.org Fluorescent Biosensors...

...live cells are GFP+. In this plot, with adjusted voltage, cells that did not receive the GFP protein are ...-case result. For example, you have introduced a GFP protein into your cells (visualized via the FITC ... to be clustered at signals below 103, while the GFP+ population cluster would be shifted to the right...samples and controls were processed the same way, the GFP- population should overlap with the negative control...control population (Figure 1). Based on where the GFP- population ends, you can draw a gate which selects...selects anything to the right of it, marking it as GFP+. Quantification for such graphs could simply be the... Labeling in Flow Cytometry Resource on addgene.org Addgene's Antibody Guide rAb Affinity Purification...

...Methods for Delivering Multiple Genes to Cells for Gene Therapy 28:40-35:10 : GFP Activatable Tools Being Developed...Strategies to Make Gene Therapies More General (more than correcting a defect in a single gene) 26:00-28:39 ...publication at Addgene. Resources on the Addgene Blog Read our Recent Blog Post on GFP Activable Plasmid...Targeting in Retinal Gene Therapies 10:17-14:34 : Progress in Treating Macular Degeneration and Other Retinopathies...publication at Addgene. 2. Tang, Jonathan C.Y., et al. “Cell type-specific manipulation with GFP-dependent...about Professor Cepko's recent work on developing GFP-activated proteins, read our recent blog post. Check...fluorescent proteins as scaffolds for cell-specific gene manipulation.” Cell 154(4) (2013): 928-939. PubMed...

...reported was PA-GFP. PA-GFP was derived from Jellyfish (Aequorea victoria) GFP (wtGFP) by mutating Threonine...mutation in PA-GFP generates a chromophore population that is more neutral, causing PA-GFP to display almost...example, by engineering PA-GFP tagged to a mitochondrial matrix protein (mito-PAGFP) the Youle Lab was able...Multi-Color Imaging Catch Up on Your GFP Background Resources at Addgene.org Find Photoactivatable FPs in ...the neutral to the anionic form, which allows PA-GFP to exhibit up to a 100-fold increase in fluorescence...when illuminated with blue light (1). Similar to PA-GFP, scientists later developed red fluorescent PA-FPs...in vivo imaging models perfer the PA-RFPs over PA-GFP. Another class of irreversible PA-FPs are the photoconvertible...

... comparison, capsids were packed with a GFP transgene and GFP intensity and transduction efficiency of...of AAV-PHP.B versus AAV-PHP.eB in the CNS. A GFP transgene was packaged into either an AAV-PHP.B or an ...efficiency of AAV.9 versus AAV.PHP.S in the PNS. A GFP transgene was packaged into either an AAV.9 or an AAV....viral (AAV) vectors are the most frequently used gene-transfer tools in the study of the brain and spinal...organs to the CNS, are also an important target for gene delivery, especially for the study of pain. While...all possible combinations into a region of the cap gene. In this special CREATE cap plasmid, loxP sites ...primers to determine which version(s) of the capsid gene successfully infected the cell type of interest....

... popular tool for gene delivery, but it has a drawback: how do you ensure your gene goes where you want... a Cre-dependent fluorescent CatCH-GFP reporter. Expression of GFP will only result in cells where Cre-mCherry...of the same transgene: an optogenetic reporter, channelrhodopsin, fused to a fluorescent GFP marker (CatCH-GFP...cell-type specific genes from mice. Strategy #2 (ProB): Create promoters using phylogenetically conserved sequences...CatCH-GFP). The lab deposited AAV plasmids containing the active promoters at Addgene. Performance of ...projections. Generating animal models: Transgenic animals allow for the expression of genes in particular...Additional resources on the Addgene blog Read more about using AAV for retinal gene therapy Find more about...

...newly formed proteins.  Limitations with GFP in imaging  While GFP has been an immensely powerful tool to...impossible for scientists to use GFP to image nascent proteins. In addition GFP tags are large, dim, and permanently...proteins in vivo, it does have some limitations. GFP has long protein maturation times which limits its...experiment the Stasevich lab observed colocalization of GFP tagged HA frankenbody with an mCherry, 4xHA tagged...lab used an alternative strategy to develop a genetically encoded antibody based probe that detects the...multi-color imaging. Find the HA frankenbody tool-kit at Addgene to get started with your experiments! What else..., T., Sato, Y., … Stasevich, T. J. (2019). A genetically encoded probe for imaging nascent and mature ...

...the limitations is the size of GFP: ~240 amino acids or about 28 kDa. GFP and its homologues have a beta-barrel...fluorescent protein (GFP) from Aequorea victoria and its variants are genetically encoded fluorescent ...interference is known as steric hindrance. For instance, GFP may occlude catalytic sites or obstruct binding sites...post-translational modifications. Despite these potential issues, GFP has been used successfully in countless fusion proteins... an inert light emitting module, any tendency of GFP to homodimerize should be avoided because it will...the linker sequence short In the early days of GFP applications, many were concerned with steric hindrance...fluorescent proteins derived from Aequorea victoria GFP sticks out and can be considered a linker (figure...

...pSpCas9(BB)). The 2A-GFP allows for screening of transfected cells. Find pSpCas9(BB)-2A-GFP (PX458). lentiCRISPR...pSpCas9n(BB)-2A-GFP (PX461) - Similar to pSpCas9(BB)-2A-Puro (PX458), pSpCas9n(BB)-2A-GFP (PX461) expresses...HIV gag, pol, rev, and tet genes. It can be used with 2nd or 3rd generation lentiviral vectors and an ...BB)-2A-GFP (PX458) - This plasmid from the Zhang lab expresses Cas9 from Streptococcus pyogenes with an...3rd generation lentiviral packaging plasmid from the Trono lab that contains the gag and pol genes. It...packaging plasmid from the Trono lab contains the rev gene. It’s meant to be used with pMDLg/pRRE and the envelope...doxycycline to the grown media resulting in target gene knockdown. Note: this plasmid was called pLKO-Tet-On...

...sender” cells express GFP on the surface and “receiver” cells express an anti-GFP nanobody on the cell ... the anti-GFP nanobody cross-reacts with BFP, sender cells expressing surface BFP generated BFP-labeled...to track cell-cell interactions By Jennifer Tsang GFP-based Touching Nexus (G-baToN) is a new tool developed...cell surface. When the cells interact, GFP is transferred to the receiver cell, and the receiver cell turns...from Jiazhang Lian’s lab that enables simultaneous gene activation, repression, and editing with a single...expression of cytoplasmic-tdTomato and Synaptophysin-GFP. Synapse targeting GCaMP6s from the Rylan Larsen ...studies.    Duong et al., Genetics 2021. https://doi.org/10.1093/genetics/iyab006       The CRISPR ...

...Serotype Name 105530  AAV1 pAAV.CMV.PI.EGFP.WPRE.bGH 37825  AAV1 pAAV-CAG-GFP 59462  AAV2 pAAV-CAG-tdTomato... testing) pAAV-CAG-GFP 50465 AAV8, 9 (20 µL size serotype testing) AAV-hSyn-EGFP Biosensor AAV The...vectors New optogenetics inhibitors Plasmid Serotype Name 99039  AAVrg pAAV-CamKII-ArchT-GFP (PV2527)...testing) pAAV-CAG-GFP 50465 AAV1, 2, 5, rg (20 µL size serotype testing) pAAV-hSyn-EGFP Recombinases...various serotypes of two popular control AAV encoding GFP. Check out our inventory of serotype testing AAV ...pAAV-hsyn-flex-Voltron-ST DREADDs / Chemogenetics AAV We have also expanded our inventory of chemogenetic AAV for activating... New chemogenetics AAV Plasmid Serotype Name 119741  AAV5  AAV SYN flex PSAM4 GlyR IRES EGFP Recombinase...

...time-consuming, as it only changes one gene at a time. CRISPR-based systems enable gene knockout in immune cells in... to create mice with gene knockouts in their immune systems. However, many genes have different functions...combination with different genes. Having CRISPR systems for assessing genes in defined contexts would ... C-CHIME Knocks out a pair of genes pXPR_219 Violet-excited GFP (vex) Rosa26-Cas9 I-CHIME ... Inducible knockout of a single gene pXPR_068 Violet-excited GFP (vex) Rosa26-FlpO-ERT2; H11-Cas9... out a single gene in defined cell types (lineage-based) pXPR_070 Violet-excited GFP (vex) Lineage-Cre...S-CHIME Knocks out pairs of genes sequentially pXPR_220 Violet-excited GFP (vex) Rosa26-FlpO-ERT2;...

...line expressing a GFP-tagged version of a protein of interest. The binding of the GFP nanobody to its target... proximity to the GFP-tagged protein (albeit slightly offset due to the nanobody-GFP interaction). The...ligase TurboID fused to a conditionally stabilized GFP-nanobody rather than a specific protein of interest...dCas9 and microbial SSAPs (dCas9-SSAP) which insert genes in a cleavage free manner. The dCas9-SSAP system...Fig. 3: Development of a cleavage-free dCas9-based gene editor using microbial SSAPs. Image from Wang, et...reduces the need to generate “single-use” transgenic lines. Instead, researchers can generate and maintain transgenic...each protein of interest and, for in vivo work, generate a dedicated transgenic line. Using zebrafish as...

...dim GFPs The availability of the three dimensional structure of GFP, its brighter mutant S65T-GFP, and...and GFP shRNA. Mice carrying the R26DsRedR; CRUSH-GFP; and Nestin-Cre alleles showed efficient GFP knockdown...they can successfully create GFP fusion proteins with a variety of genes across the genome. Although this...effectively targets your gene/region of interest is a key step for CRISPR/Cas9 gene editing. Dr. Masahito...sequence or gene of interest into the pCoofy vectors, select the appropriate pair of vector and gene primers...vector and gene primer for SLIC cloning. Amplify the pCoofy vector and the sequence of gene of interest... can be used to generate random mutagenesis libraries or to insert heterologous genes, reporters, or other...

... is time consuming. GFP-tagged Atg8p or LC3: ATG8 is part of a group of genes that affect autophagy ...Resources on the Addgene Blog Beware of Fluorescent Protein Oligomerization Seeing Red: Simple GFP Photoconversion...Alkaline phosphatase activity: In yeast, the PHO8 gene encodes a vacuolar alkaline phosphatase. Normally...with autophagosomal membranes, so tagging it with GFP allows for tracking the localization or accumulation...autophagosomes and autophagic bodies in yeast. A GFP-tagged version of LC3, the mammalian Atg8p homolog...pH-insensitive RFP variant, DsRed.T3 and a pH-sensitive GFP variant, super ecliptic pHluorin (SEP). They are ...fluorescent biosensor that was recently deposited with Addgene by Dr. Mark Prescott. This system was developed...

...sensor was engineered from a GFP variant and a DA receptor such that GFP fluoresces when the receptor ...on a similar strategy: a GFP variant bound to a DA receptor, such that the GFP fluoresces upon DA-receptor...viral vectors 101 eBook Resources on Addgene.org Learn about Addgene's viral service Find viral protocols...associated with the brain’s reward circuits are generally linked to dopamine (DA), a well-known reward chemical...in freely moving mice Record DA dynamics of optogenetically activated and inhibited neurons in live mice...neuronal imaging of dopamine dynamics with designed genetically encoded sensors." Science 360.6396 (2018): eaat4422... PMCID: PMC6287765. Sun, Fangmiao, et al. "A genetically encoded fluorescent sensor enables rapid and ...

...the sgRNAiBAR library covers every annotated human gene, it enables genome-wide screening with a high MOI...a stop cassette upstream of an effector/reporter gene. In cells where the stop cassette is removed, the...fish, and mice, by replacing the effector with a gene of interest in the cells you are studying. Read .... https://doi.org/10.1101/788679 A destabilized GFP reporter for calcineurin activity in yeast Article...calcineurin-dependent response elements (CDREs) and drives gene expression. Because calcineurin is required for ...antifungals that target calcineurin. Current calcineurin gene reporters are based on the bacterial βgal assay,...reporter consisting of a quickly-degraded destabilized GFP variant fused to a 4X CDRE repeat. In addition to...

...effects of GFP fusions to inntags or traditional FLAG, HA, and Myc tags. Impressively, IT6-GFP did not reduce...yeast cell size or viability compared to untagged GFP overexpression, while the rest of the fusions had...: 315-323. Pubmed PMID: 23435261. Resources at Addgene Read about other Protein Tags in our Plasmids 101...

...this general design scheme to generate two more OptoNBs, one against mCherry and one against GFP. Insertion...Lab monitored how AsLOV2 insertion into mCherry or GFP nanobodies affected binding of their target proteins...co-localization with a membrane-localized mCherry or GFP protein. With this system, if the OptoNB can bind....  AsLOV2 Inertion Site mCherry #1 mCherry #2 GFP Loop 1 dark-induced dark-induced dark-induced ...telephone, until the message reaches the nucleus where gene expression changes lead to cell proliferation and... on Addgene.org Find nanobody expression plasmids here Learn more about optogenetics in Addgene's optogenetics...loop 1 always generated OptoNBs with dark-induced binding, but insertion into loop 6 generated both blue ...

...TOPO E17TAG GFP and pGEX-6P-1. The pCRT7 NT TOPO vector contains a mutant pSer-containing GFP that can be...plasmid - Addgene # 68292 C321.ΔA bacterial strain – Addgene #68306 pCRT7 NT TOPO E17TAG GFP plasmid -...during optimization experiments. This version of GFP will contain a phosphoserine at amino acid 17 instead...instead of a stop codon, which will allow full length GFP to be translated), and second, through a gel shift...protein. Up to 80% p-Ser incorporation into E17TAG GFP has been achieved with the Rinehart system (6); however... - Addgene #68295 SupD plasmid – Addgene # 68307 For further technical reference and protocols for use...candidate regulatory kinase is known, it’s possible to generate non-phosphorylated recombinant protein and perform...

...nature of GFP fluorescence to create an improved fluorescent aptamer system. Knowing that GFP’s  structure...Protein Activity with GFP Resources at Addgene.org Find the Spinach Plasmids at Addgene Find Broccoli Plasmids...Resources on the Addgene Blog Tips for Using FRET in Your Experiments Learn more about GFP Controlling Protein...increase its fluorescence, essentially mimicking GFP.  Though the aptamer, called Spinach, required longer...and used in assays for over half a century; the Addgene blog has an excellent primer and tips from experts...can be created in many different ways, but the general premise is that an aptamer in its unbound state...likely to trigger immunoresponses. Nucleotides, in general, are far less immunogenic than proteins. Assays...

...among other things) gene delivery, generating pluripotent stem cells, and developing gene therapies for monogenic...and lentiviral vectors that are used for gene regulation, gene of interest expression, knockout, and screening...gamma-Retroviral Transfer Vectors   Expressing your gene of interest pMKO.1 GFP (Plasmid #10676) Retroviral vector for...for cloning and expressing your gene of interest with GFP expression.  Cloning and expressing RNAi pMKO...technology CAG-GFP-IRES-CRE (Plasmid #48201) This retroviral vector from the Gage lab encodes GFP and Cre-recombinase...Experimental uses include: Cre-dependent gene expression, gene knockout, and selection marker removal. ...Expressing your gene of interest pLenti-puro (Plasmid #39481) You can clone your gene of interest into...

...detect gene copy number based on the number of fluorescent puncta observed. By labeling two genes of a ...Proteins Control Protein Activity with GFP Resources on Asddgene.org Find Fluorescent Protein Plasmids ...vivo. Catalytically dead Cas9 (dCas9) cannot induce gene editing, but it retains its gRNA-directed targeting...signal to noise ratio of the system, they modified GFP-dCas9 and the gRNA scaffold to enhance complex assembly...the amount of background fluorescence from unbound GFP-dCas9. With these improvements, labeling efficiency...Chen et al. successfully labeled protein-coding genes with both intronic and exonic gRNAs. In fact, the...also monitor the spatial relationship of the two genes over time, at a range of distances from 2-75 Mbp...

...array plus a GFP tag. Four weeks post-infection, there was strong expression of Cpf1 and GFP in the targeted...Cpf1 multiplexing method, other multiplex CRISPR gene editing methods relied solely on Cas9. Overall, ...4 targets when an array of 4 crRNAs, Cpf1, and a GFP tag were transfected on a single plasmid. This is...Lastly, an AAV vector was used to multiplex edit 3-4 genes in a primary culture of mouse neurons and in vivo...~75% of neurons were co-transduced with Cpf1 and GFP.  Of these neurons, ~15% had indels at all 3 targeted...summary of multiplexing efficiency in vivo. Multiplex gene editing with CRISPR-Cpf1 is one of the latest developments...transfection, viral transduction) for simultaneous multi-gene editing with a single crRNA array. Are you interested...

...includes a set of plasmids each containing an anti-GFP nanobody fused with a different functional element...using different detection methods. Since the anti-GFP nanobody is able to bind fluorescent proteins derived...here can be used to target any protein fused with GFP, YFP, mCerulean, or other related fluorophores. Find...specifically investigated protein binding at the TERT gene, but this method enables the unbiased discovery ...cDNA sequencing and RNA sequencing of complex heterogeneous RNA samples. Furthermore, such high processivity...

...systems allow researchers to understand bacterial gene clusters that produce useful natural materials, ...capable of robust expression of high G+C content genes, as validated through monitoring the expression ...coding sequence for a green fluorescent protein (GFP). The success of the A-to-G conversion could be seen...through the success of the change in expression of GFP, for the stop codon should have changed from “TGA...cytometry is used to sort populations by mCherry and GFP expression to check for ABE’s. b) Quantification ...human mRNA binding proteins with 10 guide RNAs per gene. The lab modified the lentiviral backbone to encode...pooled library from Junjie Chen's lab targets human genes known or suspected to be involved in DNA damage ...

... to track where GFP labelled B cells were in mounted slices of these organs. These GFP labelled cells ...enabled by fluorescent proteins The optimization of GFP and its derivatives by the late Roger Tsien changed...stimulus. Using an epifluorescence microscope and GFP fusion proteins controlled by promoters specific ...the immune lineage, scientists could easily track GFP expressing cells. For example, by harvesting lymph... the infiltration of neutrophils expressing LysM-GFP (green) into a draining lymph node after infection...molecule (see figure to the right). The excitation generated by these two-photons occurs only at a chosen focal...Laser include: Blue-green fluorescent proteins like EGFP Yellow-orange fluorescent proteins like TagRFP, ...