...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 ...