...vitro and in vivo for gene expression and/or function. These data are sometimes reported or posted on the ... 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...recipient's initial thaw will be accounted for in our reported titers. Lentiviral vectors are titered using a...copies of the lentiviral Rev responsive element (RRE) gene and the cellular ribonuclease P/MRP 30 kDa subunit...

...sequence (UAS) of its target gene, in this case a reporter gene (e.g. luciferase or GFP), regardless of whether... promoter of the reporter gene to activate expression (Figure 2c). The reporter gene then allows detection...rescue genes as reporters and some that repress instead of activate transcription. The HIS3 reporter system...co-expressed in a yeast strain that also contains a reporter gene cloned downstream of a promoter containing the...to as the “bait”, Figure 2b) cannot activate reporter gene expression on its own, but, if the two POI’s... activator, bacterial LexA DBD and the lacZ reporter gene (3, 4), there are now many variations, including... to reduce the likelihood of indiscriminate reporter gene activation. Including a prey-only control also...

...targeting a gene associated with diphtheria toxin lethality and were sorted into single wells to generate clonal...PubMed. Find the Plasmids @Addgene: iTOP-tested proteins and reporters Previous Protein Delivery ...instance, it’s much more difficult to introduce a gene of interest into primary cells, so most primary ... cells and activate expression of a luciferase reporter. CPP was not required for protein entry into cells...primary cell types using Cre and various loxP reporters. In both mouse and human embryonic stem cells,...Neuronal stem cells transduced with iTOP resulting in GFP expression. Image courtesy of Diego D'Astolfo. ... risk of viral integration causing mutations and gene expression changes elsewhere in the genome. Second...

...promoter for transgene and CMV drives expression of RFP as a reporter. See plasmid 17618 for GFP plasmid. ...viral particle) and toxicity of the gene product (i.e., if the gene encoded by the transfer plasmid is ...hUbC-driven EGFP; can be used for cDNA expression Baltimore 12247 pLVTHM 2nd EF-1a-driven GFP and shRNA ...Expresses shRNA under mouse U6 promoter; CMV-EGFP reporter cassette is included to monitor expression Parijs...Expresses shRNA under mouse U6 promoter; CMV-EGFP reporter cassette is included to monitor expression Kissler...24129 pULTRA 3rd bi-cistronic expression of EGFP and the gene of interest, also can express shRNA (cloning... which can be modified to encode a wide range of gene products. The main restrictions on a lentiviral ...

...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 ...It is a curated set of technical reports on AAV vectors from Addgene and the CLOVER Center, each one submitted...submitted by researchers like you. Reports are reviewed by Addgene scientists for clarity and accuracy... 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–...

...imaging with this set are mTagBFP2, EGFP or one of the improved GFP variants, mRuby2 or TagRFP-T, and an...with this filter set and have reported brightness measurements. Here, EGFP outperforms the improved folding... / 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...set of proteins the green and red proteins are generally the most detectable and so should be used to tag...

...lines and are not used for general plasmid selection. Instead, reporter genes are often employed to easily...test for your gene of interest may be another method to assess transfection success. GFP is often used...expression levels in the cells. Ideally, the chosen reporter is unique to the cell, is expressed from the plasmid...used as a reporter and we will be covering this ever-versatile fluorophore in a later post, so stay tuned...conjunction with positive selection to ensure your gene gets targeted to a specific location within the ...used selection markers are listed below: Name Gene Conferring Resistance Cell Types* Mode of Action...use G418. This can be confusing since the neo/kan gene confers G418 resistance; however, like gentamicin...

...fluorescent protein reporter under the control of promoters that control cell cycle gene expression.  Additional...Resources on the Addgene Blog Visit the fluorescent protein topics page Get the basics on GFP Find microbiology..., Bernach plans to use a suite of fluorescent reporters. Bacteria containing unstable fluorescent proteins...colonizer, labelled with a constitutively fluorescent reporter can also be used to track how one population responds...work in Lin Tian’s lab at UC Davis is to generate a genetically encoded fluorescent sensor for serotonin... the TIMER growth rate bioreporter. During protein maturation, the bioreporter spontaneously changes from...sequence an individual’s genome. But when they find genetic variants, what does that mean? Previous research...

...both introduce genes into an organism’s genome (see figure) and to disrupt endogenous genes at the site ...puromycin, hygromycin) and fluorescent protein reporters (GFP, BFP, RFP, no FP). The vectors each contain... fused to a region of the RPL41 gene, to drive the selection/reporter parts of each construct. This new...first-generation transposase (as determined by transposition assays of antibiotic-resistance genes conducted...). In their Nature Genetics paper, the lab showed that efficiencies of stable gene transfer using SB100x... the rapid generation of stable transgenic cell lines which can robustly express your gene-of-interest... exciting tool for producing specific genetic mutations and gene disruptions without the use of viral ...

...and a mCherry reporter downstream of the PBAD promoter. To use this plasmid with your gene of interest,...the DNA-binding domain of AraC using a mCherry reporter downstream of a PBAD promoter. While none of the...interest, you would replace mCherry with your gene of interest. pBLADEONLY: This plasmid contains only BLADE ...already have a pBAD33-based plasmid expressing your gene of interest, all you have to do is co-transform ...co-transform pBLADEONLY. That way, a previously cloned gene of interest, under the pBAD promoter, can be controlled...strain transformed with pBLADE expressing superfolder GFP instead of mCherry under the PBAD promoter. They ... Resources on Addgene.org Check out the Molecular Biology Reference Download Addgene's Plasmids 101 eBook...

...popular studies involve using a reporter vector to tag YGOI with lacZ and/or GFP (e.g. pPD80_08) and following...applications used to study gene function. From making transgenic animals, gene targeting and genome modifications...modifications to reporter vectors (e.g. pBV-Luc) and gene silencing (e.g. pLKO.1 - TRC cloning vector). ...These vectors allow you to test the function of Your Gene Of Interest (YGOI) in a controlled environment under...design. You can create a new strain with a disrupted gene by transforming wild-type yeast with an integrating...visualize YGOI in vivo (try pcDNA3-EGFP). If you can get away with generating transiently transfected cells... More Plasmid Resources from Addgene: Plasmids 101 blog series Addgene's Molecular Cloning Guide & Other...

...capture module (SpyTag-GFP nanobody) assembled on beads. C) Micrograph of H1.8-GFP nucleosomes collected...Prime-editing-Assisted Site-Specific Integrase Gene Editing (PASSIGE), their previously-reported technique for precisely integrating...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 ...). Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases...

...GCaMP6 family of sensors are commonly used calcium reporters with different kinetic parameters. There are three...ones? Many of the available biosensors use a single GFP or CFP/YFP-derived FRET pair. If you need to free...also available. Detection methods Many calcium reporters use a single fluorescent protein (FP), requiring...unwanted variables. Twitch is a FRET-based calcium reporter derived from troponin C. It is engineered with...for in vivo studies) There are several calcium reporters specially designed for the aims and requirements...Resources on Addgene.org Addgene’s AAV Guide Fluorescent Proteins: Biosensors Addgene’s Viral Services...calcium sensors. Selecting a calcium sensor Genetically encoded calcium indicators (GECIs) are fluorescent...

...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...buffered against the effects of copy number on reporter gene expression. On the other hand, using a constitutive... 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...

...to induce high levels of gene activation for both reporter and endogenous genes. It is highly specific ...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...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...Ace-mScarlet voltage indicator plasmids at Addgene! Beck et al., Sci Report 2019. PubMed PMID: 31685893. A MoClo...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...

...has dubbed these fusions LumiFluor Reporters. The LumiFluor reporter constructs were designed to be packaged... bright enough to detect endogenous tagged genes generated through CRISPR/Cas9 knock-in. Finally, NLuc...imaging tools generate cyan, yellow, green, orange and red fluorescence. The paper reports a wide variety...photoactivatable, optogenetic accuators to depolarize or hyperpolarize membranes. Traditional FRET reporters could...would also activate the optogenetic accuators. The researchers took a FRET reporter and adapted it into a...Fusion Protein, codon-optimized Firefly luciferase genes (e.g., luc2), and NanoLuc® Luciferase are available...couples NLuc to fluorescent proteins to make better reporters and the second couples NLuc to fluorescent proteins...

...Serotype Testing AAV These AAV encode fluorescent reporters and can be used to compare the tropism of different...AAV1). AAV Vectors for Serotype Testing pAAV-CAG-GFP (Plasmid #37825) Description : Ready-to-use AAV in...plasmid 37825 (deposited by Edward Boyden ) and direct GFP expression from the CAG promoter. For information...Ready-to-use AAV available from Addgene's viral service. Control EGFP vectors in various serotypes for... 37825-AAVrg.T 20 µL $ 140 Add to Cart pAAV-hSyn-EGFP (Plasmid #50465) Description : Ready-to-use AAV ...plasmid 50465 (deposited by Bryan Roth ) and direct EGFP expression from the human synpasin promoter. For...

...Control Protein Activity with GFP Resources at Addgene.org Find KTR reporter plasmids Find photoactivatable... an ERK kinase translocation reporter with a mRuby2 fluorescent reporter (ERK KTR-mRuby2). The ERK KTR...spatiotemporal resolution than pharmacological or genetic approaches, but only a few such methods exist and...Solution: In Zhou et al, the Lin lab present a generic design for creating photoswitchable kinases and...possible to create other photoswitchable kinases? The general design of psMEK1 also worked for creating three...PMCID: PMC4097317. Additional Resources on the Addgene Blog Learn More about Photoactivatable Fluorescent...

...is typically used for gRNA May contain reporter gene (e.g. GFP) to identify and enrich positive cells,... separate transfer vectors May contain reporter gene (e.g. GFP) or selection marker to identify and enrich...knockin. Select gene and genetic element to be manipulated In order to manipulate a given gene using CRISPR...mutant allele of a gene ( point mutant )? Increase or decrease expression of a target gene? Once you have ...sequence for the gene you are trying to target. However, the exact region of the gene you target will ...want to: Generate complete and permanent loss of gene expression or function ( knockout )? Generate a specific... Edit Generate a specific user-defined sequence change in a particular gene, such as generating a point...