... later, Cre-dependent GFP expression was detected with direct fluorescence. GFP was not detected in the...genomes (vg)/cell, pAAV-CAG-FLEX-rc [Jaws-KGC-GFP-ER2] (Addgene 84445-AAVrg) alone at 1.1E6 vg/mL, or both... Karl Deisseroth (Addgene viral prep # 55632-AAVrg). pAAV-CAG- FLEX-rc [Jaws-KGC-GFP-ER2] was a gift from... plasmid encoding rep and serotype-specific cap genes, and a plasmid encoding adenoviral helper sequences...viral vectors are tested in vitro and in vivo for gene expression and/or function. These data are sometimes...copies of the lentiviral Rev responsive element (RRE) gene and the cellular ribonuclease P/MRP 30 kDa subunit...vector genome titers by droplet digital PCR. Human Gene Therapy Methods, 25(2), Article 2. https://doi.org...

...capture module (SpyTag-GFP nanobody) assembled on beads. C) Micrograph of H1.8-GFP nucleosomes collected...editing, this epigenetic strategy doesn’t induce DNA damage or permanently alter a gene product; it simply...In MagIC-cryo-EM, target proteins (expressed as a GFP fusion in this proof-of-concept study) are captured...SpyTag-SpyCatcher linkers and spacer proteins ending with a GFP nanobody (Figure 1). This enrichment streamlines ...) 3D model of the obtained structure of the H1.8-GFP-bound nucleosome. Images adapted from Arimura et ...on Prime-editing-Assisted Site-Specific Integrase Gene Editing (PASSIGE), their previously-reported technique...). Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases...

...lactose released activates expression of GFP from the lac operon. GFP expression can then be used to determine...(including lactose), and a lac operon controlled GFP. When the enzyme breaks down the HMO of interest,...Fatima's work and keep checking back in with the Addgene blog for future award opportunities. Xin Zhou:...when exposed to ultraviolet light. To do so, she generated fusion proteins in which Dronpa domains flanked...Proteins, or FLiPs.  Xin later optimized these first generation FLiPs by mutating Dronpa to lower its aggregation...second sensor is a fusion protein consisting of sfGFP fused to a His-tagged ABC transporter known to bind...Chemical Biology! Additional Resources on the Addgene Blog Learn More about Photosactivatable Fluorescent...

...plasmids Tagging human transcription factors with GFP Kevin White’s lab recently deposited a set of CRISPR...plasmids to tag human transcription factors with GFP. As part of the Encyclopedia of DNA Elements (ENCODE...proteins for optogenetic control of CRISPR-Cas9. By combining AcrIIA4, a Streptococcus pyogenes Cas9 inhibitor... a few of the new CRISPR plasmids available at Addgene. We will still periodically focus on specific CRISPR...adapted for detecting SNPs. Learn more from this Addgene blog post Read the paper in Science Find the...the Cas14 plasmids Anti-CRISPR proteins for optogenetic control of CRISPR-Cas9 Anti-CRISPR proteins that...editing is now possible. Learn more from this Addgene blog post Read the paper in Nature Methods Find...

...Caenorhabditis elegans. Genetics, 228(2), iyae126. https://doi.org/10.1093/genetics/iyae126.   Gene disruption in...with expression levels comparable to GFP during early embryogenesis (Figure 3). mScarlet-I3 also offered...tags for live imaging in C. elegans CRISPR/Cas9 for gene disruption in M. abscessus  Bioluminescent reporter...through the middle of each embryo are shown, comparing GFP, wrmScarlet (mScarlet), and the new mScarlet3 and...developed a novel dual-plasmid CRISPR/Cas9 system for gene disruption in Mycobacterium abscessus, an increasingly...Here, the researchers were able to target multiple genes in M. abscessus either simultaneously or stepwise...system is induced to cause disruption of the target gene. Lastly, curing the mCherry-sgRNA plasmid leaves...

... cassette to the gene of interest. The gene to be expressed is amplified using a gene-specific forward...sgRNA-targeted gene, resulting in increased activation of endogenous transcription of the target gene. The brilliant...of downstream genes. Nihongaki et al describe the targeted activation of endogenous genes and detail the...efficiency and gene expression. The pUCXKT vector ensures that all screened plasmids contain a gene variant*...resistance gene (missing the first two codons). Two stop codons are inserted between the truncated gene and ...both the GFP and the RFP proteins in the nucleus. During the first round of translation, the GFP proteins... inducible, gene knockdown with shRNA or shRNA-miR30, (2) tamoxifen-inducible CreERT2 gene deletion, (...

...deletion of one or several of these genes permits the expression of genes farther downstream in the construct... two tandem sets of genes aligned head-to-head and the deletion of one of the gene pairs. Brainbow 2.0... from the default GFP (green) to RFP, CFP, or YFP fluorescence. In Brainbow 2.1, GFP is tagged with a ...recombination in this mouse generates cells that randomly express either nuclear GFP, membrane CFP, cytoplasmic...Brainbow uses a Cre/lox system to label neurons. Genes encoding the fluorescent proteins are organized ...organized in tandem with a single promoter, but only the gene directly following the promoter is expressed. Cre-mediated... which uses Cre to trigger the inversion of two genes aligned head-to-head. Another variation, Brainbow...

...reporter gene (e.g. GFP) to identify and enrich positive cells or a selection marker to generate stable ...permanently knock out or knock-in a gene? Do you want to activate or repress gene expression? Are you trying to...separate transfer vectors. May contain reporter gene (e.g. GFP) to identify and enrich positive cells. Likely... create a fusion with a reporter protein such as GFP? All of these outcomes can be most effectively achieved...conjunction with activator or repressor domains to control gene expression. Base editors can help you make precise... for each, and potential delivery methods.   General considerations for planning your CRISPR experiment...Wildtype Cas9 or Cas9 nickase are appropriate for generating knockouts or knock-ins, or introducing mutations...

...Joachim Goedhart shares some strategies for generating GFP fusion proteins. From considering protein size... PS: they’re free. 3. Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection by ... biology over the past year: CRISPR, optogenetics, chemogenetics, plasmids, fluorescent proteins, careers...proteins at Addgene. 7. Designing Your Chalk Talk for the Academic Job Interview by Erik Snapp Addgene depositor...2020! Additional resources on the Addgene Blog Read other Addgene news Find the most recent plasmid ...posts Resources at Addgene Browse the repository Find educational resources at Addgene Search for plasmids...discovery. We’ve documented many new tools on the Addgene blog and have created and updated guides on various...

...easily modified to tag hundreds of genes, generating powerful genetic screening libraries. Additional protein... to screen for the genetically modified strains that contain your newly tagged gene. Most insertions can... to generate high-throughput targeting vectors. These vectors can be used to tag endogenous genes and ...-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans.” Genetics. 202(4) (2016):1277-88....Since the discovery of GFP over 50 years ago, the growing spectrum of fluorescent proteins (FPs) has ...protein fusions is to replace the genomic copy of your gene of interest with a fluorescent protein fusion. CRISPR...repair pathway, a new user-defined DNA sequence (like GFP) can be inserted at the DSB. CRISPR has allowed scientists...

...section. The GFP story has its own Franklin in Doug Prasher, who originally isolated the GFP gene but was ...years, but lay readers may be less familiar with gene drives and their ramifications. Readers unfamiliar...the identification of Green Fluorescent Protein (GFP) and its development into an essential tool in biology...time on Addgene’s role in the rapid dissemination of these technologies, proposing that “Addgene, with ...contributed to the steadily increasing distrust the general public has in science and scientists (as discussed...replace scientists themselves.  The sections on optogenetics and CRISPR of course address some of the ethical...has been a key driver of the technology.” As an Addgene vet of 12 years, I’m very familiar with the role...

...expression of Gene X using shRNA expressed from a plasmid   Figure 1: Expression Level of Gene X The results... level of Gene X in cells treated with Plasmid A is similar to the expression level of Gene X in the untreated...backbone itself on gene expression in your target cells. In experiments employing gene targeting or genome...trying to repress a gene, your control might be a system where expression of that gene is knocked out completely... shRNA targeting human Gene X in Plasmid A is the decreased expression of Gene X. Ergo the positive control...targeting human Gene X. The non-targeting control also assesses any effects of the general introduction ... wherein Plasmid A (encoding shRNA against human Gene X in backbone Y) was transfected into human cells...

...viral prep pAAV-CAG-GFP AAV9-X1.1 Controls Edward Boyden New serotype pAAV-hSyn-EGFP AAV PHP.eB Controls...AAV9. You can now target these cells with AAV-CAG-GFP packaged in AAV9-X1.1!  We also have eight new AAV-PHP.eB...preps available in the Addgene viral vector repository! This includes the first Addgene prep packaged in the... AAV1 Optogenetics Karl Deisseroth New serotype pAAV-hSyn-hChR2(H134R)-EYFP AAV2 Optogenetics Karl Deisseroth... Brian O'Neill.  More resources on the Addgene blog Addgene's Data Hub What is a Virus (and a Viral Vector...other new AAV vectors as well — including next-generation biosensors for GABA, dopamine, serotonin, and...Table 1: New ready-to-use viral vectors in the Addgene repository  Name Serotype Category Depositor...

...delivery of genetic cargo  By Alyssa NeuhausTo improve delivery and reduce off-target effects of gene therapy...transcriptional activation events as bands of induced GFP-iPAK4 expressed under promoters like the tet-responsive...fluorescent signal in the ticker tape from cFos::GFP-iPAK4 within an hour. Analyzing the ticker tape images...to red at 12 h, and PMA activation induces cFos::GFP-iPAK4 at 3 or 6 h. Scale bar 5 µm. Images adapted...system to improve on-target delivery of genetic cargo Optogenetic proximity labeling and TransitID Tracking...continuous evolution and protein engineering to generate seven new prime editors, PE6a–g, that overcome...10.1038/s41467-023-40788-8. PMID 37612276.   Optogenetic proximity labeling and TransitID By Mike Lacy...

...exon of a gene, and thus they do not disrupt the gene expression of the gene they monitor. Gene traps Gene...expression of a nearby gene. The reporter gene serves as a proxy for the endogenous gene – its expression levels...predicted genes have unidentified roles as well (Vanni, et al). Is it true that this many genes are truly...modifying the gene of interest in any way. For example, if green fluorescent protein (GFP) was the reporter... infer that the gene which GFP was monitoring was being expressed in the same pattern. These systems suggest...biologically relevant situations. Tissue-specific genes – the genes of interest may not be expressed in the tissue...are working with. Development-specific genes – if a gene functions at multiple stages in development...

...substitutions to LanYFP and the addition of the enhanced GFP (EGFP)-type termini. mNeonGreen was reported as the ... to 3 times brighter than the most commonly used GFPs and YFPs. Its excitation maxima is at 506 nm and...Oregon created mTFP1 by transforming E. coli with a gene library of approximately 484 variants, including... your research Subscribe to the Addgene blog Resources on Addgene.org Learn about the Michael Davidson...Allele Biotechnology that now can be deposited at Addgene. What makes these fluorescent proteins unique and...was then screened through successive rounds of mutagenesis for the brightest teal fluorescent proteins that...scientists from Florida State University, they used a mutagenesis approach and found that specific amino acid changes...

...experiment, consider co-transfecting with GFP. This allows you to sort for GFP-positive cells and to enrich for...further consolidate these FAQs, Le Cong worked with Addgene Senior Scientist, Matt Ferenc, to organize the ...full list of 16 FAQs answered by Le Cong Read Addgene's CRISPR guide for background information on CRISPR...the most frequently requested CRISPR plasmids at Addgene Find protocols and gRNA design tools: List of...scientific community and sharing these resources with Addgene. Thank you, Le!    ...

...since become a gold standard in gene expression analysis and a luciferase gene (one of many available to choose... Proteins in Nano Lanterns Reporter gene assays A reporter gene, such as luciferase, usually serves as.... The effect of promoters or enhancer regions on gene expression can be determined by detection of the...such as excitation of Green Fluorescent Protein (GFP) because of the nature of the chemical reaction compared...to using a high-intensity laser to rapidly excite GFP. As a result of the different mechanisms to produce...effect of mutations of these regulatory elements on gene expression. The regulatory element (wild-type or...or mutant) is cloned upstream of the luciferase gene in the plasmid, which is then introduced into the...

...imaging with this set are mTagBFP2, EGFP or one of the improved GFP variants, mRuby2 or TagRFP-T, and an... / em 527 nm) can be imaged together but CFP and GFP (ex 488 nm / em 507 nm) show some crosstalk between...TagRFP to give a brighter signal than mCherry. In general, filter sets designed for Rhodamine / Cy3 will ...mammalian cells, one of the improved folding variants of EGFP like mEmerald or Clover is probably best; mNeonGreen...and have reported brightness measurements. Here, EGFP outperforms the improved folding variants, presumably...set of proteins the green and red proteins are generally the most detectable and so should be used to tag...

...leader sequence. Using a variety of GFP and RFP fusions as their test genes, the researchers at the BIOFAB ...translational repressor to down-regulate a gene of interest. You can generate PUF proteins with a wide variety ...restriction enzymes and standardized primers to clone your gene of interest into both sequencing (pInitial) and ...abundance (meaning they are unlikely to be found in your gene of interest) and prevent incorporation of extensive...kit contain a variety of promoters to facilitate gene expression in bacteria, yeast, mammalian cells, ...various combinations of protein tags (His, Avi, and GFP) as well as protease sites to facilitate protein ...hope this kit will allow you to quickly study your gene of interest in a variety of model systems, thereby...

...binding scFv that’s fused to GFP. When the multiple copies of the scFv-GFP fusion bind the SunTag scaffold...also allows for screening scFv generated from the B cell antibody genes from diseased or vaccinated humans...   Some phage display libraries screen antibody genes from B cells of immunized animals, but it's possible...scFv that’s fused to a fluorescent protein such as GFP. The HA frankenbody works just like an antibody-based....1791  Deal CE, Balazs AB (2015) Vectored antibody gene delivery for the prevention or treatment of HIV ...also allows for generations of scFvs against two antigens that are impossible to generate scFvs against ...probe, but is easier and cheaper to generate since it’s genetically encoded.   Translational applications...

...each recruits a different fluorescent protein (BFP, GFP, or RFP) to the genomic loci of interest. Pairs of...hairpin sequence (sgRNA-Sirius-8XPP7) to recruit GFP to the loci of interest. To determine how the sgRNA-Sirius... observing low-copy genomic loci or single copy genes. If you want to visualize genomic loci brightly,...PP7, or boxB sequences inserted into the sgRNA generates blue, green, or red colors for labeling. A combination... PP7, or boxB sequences in the same sgRNA will generate additional colors. Image from Ma et al., 2016 ...CRISPR-Sirius a try. Find CRISPR-Sirius plasmids at Addgene. References Ma, Hanhui et al. “Multicolor CRISPR...PubMed PMID: 30377374. Additional resources on the Addgene blog Download our CRISPR eBook Learn about fluorescent...

...regulation. To create iGluSnFR, the Looger lab wedged GFP between two halves of a bacterially derived periplasmic...increase in fluorescence intensity from the inserted GFP. After characterizing iGluSnFR in vitro, showing ...zebrafish, and mice. Second Generation iGluSnFR: SF-iGluSnFR In the second generation of iGluSnFR, the Looger...Looger lab swapped eGFP for sfGFP to create SF-iGluSnFR. This improved variant has higher expression levels...being deposited at Addgene. Other plasmids and pAAVs can be ordered directly from Addgene. The Podgorski ... for Synaptic Imaging However, the first two generations of iGluSnFR had limitations: they were not optimal...extrasynaptic glutamate release. Building on the previous generation of the SF-Venus-iGluSnFR (itself an improvement...

...to me…  A recombinant anti-GFP!    🎶On the third day of Antibody, Addgene gave to me…  An online Antibody...Antibody, Addgene gave to me…  An Antibodies 101 eBook!  🎶On the second day of Antibody, Addgene gave to...Antibody, Addgene gave to me…  A recombinant Anti-Arl13b!    🎶On the fifth day of Antibody, Addgene gave ...gave to me… The Addgene Antibody Data Hub!    🎶On the sixth day of Antibody, Addgene gave to me… A recombinant...Antibody, Addgene gave to me… Antibodies developed by IPI!   🎶On the eighth day of Antibody, Addgene gave ...Antibody, Addgene gave to me…  A recombinant Anti-APP!   🎶On the eleventh day of Antibody, Addgene gave to...Anti-DYKDDDDK!   🎶On the ninth day of Antibody, Addgene gave to me… A neuroscience antibody collection!...

...of where the genes were expressed. What’s the use of stable gene expression? Constant gene expression can...Researchers express genes of interest from plasmids in order to study gene function or to engineer cells...input and gene expression is the output. The input, plasmid copy number, directly influences gene expression... varied in size or carried different genes. In these cases, gene expression also remained stable regardless...media and growth stage can both affect gene copy number. Moreover, gene copy number is affected differently... constitutive promoter upstream of GFP increased expression of GFP six-fold, but a stabilized promoter...the levels of gene expression from stabilized promoters were equivalent whether the genes were expressed...

...anti-FLAG-FB-GFP binding to FLAG-tag H2B for nuclear localization. (c) Applications of anti-FLAG-FB-GFP labeling...between MARCH5 and WSB2 genes using Cas12a’s ability to knock out multiple genes in parallel. Lastly, this...to induce high levels of gene activation for both reporter and endogenous genes. It is highly specific ...cellular targets Optimized genetic screening technique using Cas12a for multiplexed gene perturbation The Penn...10846-1.   Optimized genetic screening technique using Cas12a for multiplexed gene perturbation by: Gabrielle...many CRISPR-based genetic screening methods have primarily used Cas9 to knock out genes, researchers at ...multiple genes within a single cassette provides increased simplicity for combinatorial genetic screens...

...limits the delivery of larger transgenes, such as base editors which have a gene size of ~5.2 kb. To overcome...Article contributed by Jennifer Tsang AAVs are a great gene therapy delivery mechanism, but their small packaging...levels similar to that of a single AAV expressing GFP. The lab tested the base editors in vivo and achieved...dosage comparable to those currently used in human gene therapy trials. They further tested the base editors...voltage indicators, however, fall short compared to GFP-based sensors in terms of response and brightness...expression levels from different promoters, and inhibited gene expression using CRISPRi. The kit functions in the...that increases temporal and spatial precision of gene expression and reduces toxicity as compared to previous...

... and allows translation of the reporter gene resulting in GFP signal. With the addition of standards, ...well as a selectable marker and a desired genetic payload (ex: GFP). Thus, cells can be both sorted using...ribosome binding site (RBS) followed by a reporter gene. The hairpin blocks translation by sequestering ...antibiotic resistance markers, promoters, and inducible gene expression systems in bee gut microbiome species...or preparation, and deliver CRISPR components for gene disruption or CRISPRi. While designed to study the...address these questions, biologists often use next-generation sequencing. Sequencing the 16S rRNA reveals the... also study the microbiome by using engineered genetic tools and reporter microbes. Let’s take a look ...

...interest (analyte), and accumulation of ATP activated GFP expression (receptor), then the transducer could ...plate reader, or any other instrument which measured GFP levels and converted that signal into a readable ... on the Addgene blog An Overview of AAV Encoded Calcium Biosensors Illuminating Epigenetics with a ... systems that measure biological phenomena by generating an output which is directly proportionate to ...antibody, enzyme, DNA, etc. The receptors job is to generate a quantitative signal upon recognizing the analyte... (fluorescence, pH, etc.). The transducer will generate a numerical value which represents the amount ...: Almost all of the biosensors discussed are genetically encoded, and luckily, we have pre-designed plasmids...

...rescue of a gene knockout: To test if the phenotype you are observing is really due to a single gene deleted...sequence (such as GFP) in the sense orientation, followed by coding sequence of the gene you would like ...endogenous gene. Spatio-temporal expression of optogenetic reporters: Since most optogenetic reporters...transduced reporter gene. Generating a FLEx switch to control expression of an optogenetic reporter would ... were designed as a genetic tool for researchers to conditionally manipulate gene expression in vivo using...conditionally turn off one gene and activate another. In the field of mouse genetics, FLEx is currently being... vivo conditional gene rescue experiments, as well as for improvement of optogenetic adeno-associated ...

...Joachim Goedhart’s article on the Addgene blog: Fluorescent Proteins 101: GFP Fusion Proteins – Making the ...protein of interest. The green fluorescent protein (GFP) and mCherry, two of the most common tags currently...References on Addgene.org Check out Addgene's Chemogenetics Guide Find fluorescent proteins at Addgene ...on the Addgene blog Browse Fluorescent 101 blog posts Read about chemogenetics vs. optogenetics Find more...Emory University School of Medicine. Optogenetics and chemogenetics are powerful tools to modulate the ...neurons and other brain cells. Since the opsins or chemogenetic receptors used in these techniques cannot be...versatile method to identify the expression of transgenes of interest. Furthermore, fluorophore variants...

...cell type Gene targeted by guide Editing efficiency hiPSCs  EMX1  67% Mouse BM cells from GFP transgenic...alternative method for generating transgenic mice strains and for non-invasive in vivo gene editing. The bottom...repression, transgenic mouse line creation, and in vivo gene editing of mice, with the potential to be used for... Primary cells are often refractory to gene delivery methods, but the researchers was able to edit hiPSCs...transgenic mouse  GFP  ~60-65% Human hepatocytes  Myd88  ~50% Human HSCs  Myd88  ~50% Table 1: Nanoblades...cells when using a gRNA targeting the Fto gene, while editing was not detected in cells electroporated with... used Nanoblades to disrupt the tyrosinase (Tyr) gene in mouse zygotes, which results in albinism in mice...

...plasmids described in this work including the soCoChR-GFP plasmid as well as several derivatives encoding alternative...ShadowG, a novel dark green fluorescent protein (GFP) with an absorption peak of 486 nm and ShadowY, a... to the Hot Plasmids episode! Expanding the optogenetics toolbox with CRY2clust Article contributed by...Basic Science (IBS) has developed another useful optogenetic tool that enables robust and efficient oligomerization...CRY2clust, when integrated into a Ca2+ -modulating optogenetic system (OptoSTIM1), caused two-fold faster changes...soCoChR: A novel opsin that enables single-cell optogenetics Article contributed by Meghan Rego  Listen ...Listen to the soCoChR podcast segment Optogenetic stimulation is a powerful tool used by neuroscientists to...

...where his group focuses on gene regulation in nematode molting and spermatogenesis. His group also builds ... one of the model organisms which I use to study gene regulation during developmental processes, CRISPR...drug resistance (Chen et al., 2013), fluorescence (GFP; Tzur et al., 2013), or rescue of mutant phenotypes...Craig Mello’s lab – used inactivation of the unc-22 gene as their selection marker (Kim et al., 2014). Meanwhile...homology arms to knock in large epitopes, such as GFP (Paix et al., 2014). A further development of this... I was able to knock-in a 2xFLAG epitope into my gene of interest, I encountered low efficiency (0.13%...efficiencies, obtaining as many as 100 independent GFP knock-ins from a single injected animal.  PAM sequence...

... FRET tools, double dimerization GFPs, and circular permutation GFPs. Hopefully we will have new tools...her work has always been focused on gene transfer, from transgenes to plants, chloroplast to the nucleus...easy to express genes in, which makes it a good laboratory workhorse. Tips & tricks Addgene: Do you have ...The MoClo Plant Kit makes it really easy to clone genes for expression in plant cells. Sylvester and I chose... Now there are many, so if you want to make a 10-gene construct, you don’t have to worry about repeating...constructs to allow people to, for example, knock out a gene with simultaneous complementation, with a knockout...Marillonnet share with the scientific community via Addgene, the struggles of plant scientists and how they...

...library - Ampl133 targets S. cerevisiae genes with 6-12 guides per gene where gRNA expression is induced by... hSyn FLEx mGFP-2A-Synaptophysin-mRuby from Liqun Luo’s lab expresses membrane tethered GFP to enhance...  After validating this method through monitoring GFP degradation, this protein compartment was used to...a high efficiency intron-optimized SpCas9-coding gene. SlugCas9 is a compact Cas9 ortholog from Staphylococcus...targeted synaptophysin tagged eGFP. AAV phSyn1(S)-FLEX-tdTomato-T2A-SypEGFP-WPRE from Hongkui Zeng's lab... Figure 1. (A) SARS-CoV-2 RBD DMS Library generation and sequencing. From Starr et al Cell., 2020....scFV library and characterized them by ELISA, homogeneous time resolved fluorescence, and suspension bead...

...with a fluorescent reporter, such as GFP or mCherry, through genetic modification. As your protein of interest...overlap. Figure 4 compares the spectra of enhanced GFP (eGFP) and FITC, the latter being a commonly used fluorophore... you can also use fluorescent reporters, such as GFP, and fluorophore-conjugated antibodies together in...Resources on Addgene.org Addgene's Antibody Guide Ready-to-use antibodies in the Addgene repository Antibody...vector also encodes for eGFP, which serves as a transduction reporter. Note that eGFP is not fused to CD45...-Cy7. Next comes our transduction reporter, eGFP. As eGFP emits fluorescence by itself and does not need...cytometer which channel to use to detect eGFP. As you saw in Figure 4, eGFP strongly overlaps with FITC, hence...

...red light instead of blue to excite a molecule of GFP. Because red light has approximately half the energy...light, two photons of red light are needed to excite GFP compared to only one of blue. Only at the focal point...infinitely small point of light – think of a single GFP molecule – will appear as a fuzzy 200-300 nm spot...PubMed PMID: 26186186.  Additional Resouces on the Addgene Blog Lightsheet Microscopy Choosing Your Fluorescent...Fluorescent Protein for Your Experiments Resources on Addgene.org Browse Our Fluorescent Protein Resources Find...Fluorescent Protein Backbones Find Plasmids for Optogenetics ...

...overexpressing a gene of interest in the plant. A common method for silencing genes Virus induced gene silencing...by replacing the genes on the T-DNA region with our gene of interest or a reporter gene so that the transformation...transformation. Choose good reporter genes There are many great reporter genes available and which one you choose...transformation success. Some reporter gene examples are below. The GUS gene (β-glucuronidase) with histochemical...Evaluation and comparison of the GUS, LUC and GFP reporter system for gene expression studies in plants." Plant...Hikmet Budak. "Virus-induced gene silencing, a post transcriptional gene silencing method." International...stable cell line or plant is regenerated from the transformed tissue. Gene function is commonly studied...

...Adeno-Associated Viral Vector-Mediated Gene Delivery to the Hypothalamus. Human Gene Therapy, 21(6), 673–682. https...conditions commonly encountered in human gene therapy trials. Human Gene Therapy Methods, 26(2), 71–76. https...neurons and GFP (green) to label neurons transduced by each serotype. Working at Addgene has shown ... cassette with an ubiquitous promoter to express GFP in a group of young rats. We found that, in this ...Grames, M. S., & Klein, R. L. (2018). More expansive gene transfer to the rat CNS: AAV PHP.EB vector dose–...dose–response and comparison to AAV PHP.B. Gene Therapy, 25(5), 392–400. https://doi.org/10.1038/s41434-018...Adeno-Associated Virus Serotype 9 (AAV9) Preparations. Human Gene Therapy Methods, 30(6), 214–225. https://doi.org...

...stem cells By Jennifer Tsang CRISPR gene interference (CRISPRi) and gene activation (CRISPRa) are great ways...GABA-ergic interneurons. These include mDlx-GFP, hDlx-FLEX-GFP, hDlx-Flex-dTomato, mDlx-GCaMP6f, hDlx-GiDREADD-dTomato...the plasmids at Addgene! A new tool for synthetic biology uses CRISPRi to control gene expression. This...nucleus and modulate transcription of the mCitrine gene. mCherry is constitutively expressed. Cells are ...selectivity and found that the best motif was from the Lck gene. For marking microtubules of mammalian cells, the... ways to modulate the expression of nearly any gene via specific guide RNA (gRNA) sequences. But these...-IgG1-Fc, Addgene #140700) pMAK463 (NBαMouse-IgK, Addgene #140701) pMAK464 (NBαEGFP, Addgene #140702) ...

...chemistry! The discovery of green fluorescent protein (GFP) sparked a renaissance in biological imaging. Suddenly...advances lead to an obvious question: In this age of GFP and its ilk, why should cell biologists talk to chemists...from transient overexpression of protein fusions to gene-edited cells can decrease the number of fluorophores...Fluorescent Protein Should I Use? Resources on Addgene.org Generate Fluorescent Protein Fusions Request Plasmids...in-cell labeling strategies have two parts: (1) a genetically encoded “tag” expressed as a fusion with your... dye ligand (e.g., FlAsH, ReAsH) and a short genetically encoded tetracysteine (Cys4) peptide tag could...TMP-tag) – These widely used systems consist of a genetically encoded enzyme variant tag that reacts specifically...

...protease protection. Thus far Plasmids 101 has covered GFP and its related fluorescent proteins, which are sometimes... I Use? Plasmids 101: Green Fluorescent Protein (GFP) Fluorescent Protein Guide Expression Vectors from... overexpress a recombinant protein? It is not generally in a cell’s best interest to overexpress a protein...for affinity and purification An affinity tag, generally a relatively small sequence of amino acids, is...interest would eventually get ridiculous (you generally don’t want more tag than protein), 2-3 tags is...using plasmids found in this paper (available at Addgene).   Table 2: Protease recognition sites commonly...purification is provided by GE Healthcare Life Sciences. Addgene pages and blog posts of interest: Which Fluorescent...

...mouse brains expressing green fluorescent protein (GFP) (6).   The Dodt and Frank Bradke laboratories later...subpopulation of neurons are brightly labeled with GFP (e.g. Thy1-YFP-H mice).  However, we recently developed... based on using a thin plane (sheet) of light generated by sunlight to observe single gold particles with... nerve afferents labelled with a AAV8 expressing eGFP under the human ubiquitin C promoter. Anatomical...).  These viral plasmids are available through Addgene.  The combination of LSFM with multiple tissue ...

...antibodies against popular fluorescent proteins (GFP and YFP) and protein tags (c-MYC and HA). We have...ways of generating antibodies, for example through the use of less toxic adjuvants; 2) generating complex...and 4) generating mAbs against new cancer biomarkers. We believe that repositories, such as Addgene and ...The 10 Most Distributed Plasmid Technologies in Addgene's First 10 Years Data Freedom: The Expansion of...

...Reporter Genes (ARGs)? Fluorescent and bioluminescent reporter genes are widely used to study gene expression...for visualizing gene expression in vivo.  But what ultrasound lacked was reporter genes that could produce...Lab - developed Acoustic Reporter Genes (ARGs), which are sets of genes that, when expressed, enable cells...vesicles gene clusters from a diverse set of gas vesicle-expressing species. The gas vesicle genes from Serratia...structural gene gvpA marked with EBFP2 and the second cassette contains the P2A-linked accessory genes gvpNJKFGWV...first-generation mammalian ARGs (mARGs) enabled ultrasound visualization of in situ mammalian gene expression...hypothetical protein, improving expression, and named this gene set bARGSer, for bacterial ARG from Serratia sp....

...REVeRT, for gene delivery using reconstitution via mRNA trans-splicing. By splitting a large gene into smaller...the introduction of extraneous genetic material to reconstitute the gene of interest, reducing the immunogenicity...hIBA1a, 466 bp) that drives high microglia-specific gene expression when incorporated in an AAV vector (Figure...Figure 5: A) Representative confocal images showing GFP expression from ssAAV5s with hIBA1 or hIBA1a promoters...Quantifications showing high microglia specificity of GFP expression for the indicated ssAAV5s. C) Quantifications...doi.org/10.1101/2023.12.12.571321.  Revolutionizing Gene Therapy: The REVeRT Dual AAV Vector System By Vaibhav...AAVs, and then splicing their mRNAs, the functional gene product is reconstituted within the target cells...

...the puromycin resistance gene through a 2A peptide. The puromycin resistance gene enables selection of cells...to translate Cas9-2A and the Puromycin resistance gene as separate peptides. PX459 This is the vector backbone...had a point mutation in the puromycin resistance gene that made it less effective in certain cell lines... lines. Alternative forms of this vector contain GFP-tagged and un-tagged versions of SpCas9 and further...Resources on the Addgene Blog Browse Our CRISPR Posts Learn about Using CRISPR to Generate Knockouts Read...Purpose  SpCas9 This plasmid expresses Streptococcus pyogenes (Sp) Cas9 from a Cbh promoter. Cbh is a modified... the key component of most CRISPR systems and generates double strange breaks at target sites 5' of the...

... time or location can be critical when, say, the gene is essential during development and a knockout is...activity can be monitored by phosphatidic acid probe GFP-PASS. B) Time series of HEK 293T cells expressing...membrane-targeted SuperPLDx30 (magenta) and PA probe GFP-PASS (green). After 30 minutes of blue light illumination...the Addgene blog Viral Vectors 101: Optogenetics Additional resources on Addgene.org Optogenetics Guide... The Chow Lab has also generated a variety of membrane-recruitment optogenetic tools for manipulating ...a full toolbox and workflow for generating new Rho GTPase optogenetic systems (Berlew et al., 2022). Stopping...LOV, CRY2-CIB, and other optogenetic modules? Or want to find more optogenetic tools in our collection?...

...translational. Ex: the discovery of GFP. Chapter 4: Goals of the Center for Regenerative Meidince - Understanding...Additional Resources from Addgene Learn about Retinal Gene Therapy from Connie Cepko Find Plasmids from the...treat disease through tissue repair, stem cells, gene therapy, and tissue engineering. How the multidisciplinary...Production Specialist at Addgene. He is the audio engineer and producer for the Addgene Podcast.     Additional...Darrell Kotton is the director of the Center for Regenerative Medicine at Boston University Medical School...source biology in his own lab, at the Center for Regenerative Medicine, and within the biomedical research...Along these lines, Darrell recently joined the Addgene Board of Directors where he hopes to help keep ...