...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...

... 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....

...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...

... 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...