... developed four acoustic reporter gene (ARG) plasmids to better monitor gene expression in vivo using ...plasmids at Addgene. Liu Q et al., Neuron. 2019. PubMed PMID: 30704911. Acoustic reporter genes for noninvasive...contributed by Lukas Morgan  Listen to the acoustic reporter gene podcast segment In recent years, scientists ...rely heavily on fluorescent protein genes. These optical reporters have limited deep-tissue performance...demonstrates engineered gas vesicles as the first reporter genes using ultrasound and provide a major benefit...29300010. miRNA reporter vectors shed new light on mechanisms of miRNA-mediated gene regulation Article...collection of reporter plasmids is amenable to Golden Gate assembly for turning the single-input reporters into...

...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...plasmid that constitutively expresses a reporter protein (e.g., GFP or luciferase) that is either co-transfected...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 ...

...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...

... 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...transgenic mice expressing fluorescent protein reporter constructs in the immune lineage. By specifically... the infiltration of neutrophils expressing LysM-GFP (green) into a draining lymph node after infection...using 2-photon microscopy as this hasn't been well reported for many fluorescent proteins. Additional fluorescent...

...molecule it detects. This change then generates a reporter signal. Reporter signals may be electrochemical or...biosensors To focus on a particular gene or protein, check out our gene-specific biosensors. These constructs...light-based, with luminescent and fluorescent reporters being especially popular. We’ll give you an introduction...biosensors as an example. Many calcium biosensors include GFP fused to calmodulin (CaM) and the chicken myosin ...glutamate sensor pCMV(MinDis).iGluSnFR. Picking your reporter Don’t forget about the other end of the biosensor...Visualization Resources on Addgene.org Find Biosensor Plasmids Find Optogenetics Plasmids Visit the Fluorescent... OR enzyme activity. Small molecule biosensors Addgene has many sensors that measure the concentration...

...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 ...plasmids use “Cas9-mediated adenosine transient reporter for editing enrichment”, or XMAS-TREE, to identify...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...

...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...

...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...

...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...into the hPSC genome using AAV1 and contain an EGFP reporter. Expression of the dCas9-KRAB activator or dCas9...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...

...epitopes, such as GFP (Paix et al., 2014). A further development of this approach reported in 2017 uses the... Mello lab also reported that modifying the 5’ modification of oligos used to generate linear dsDNA repair...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...approaches may become even more powerful following a report that one can use linear repair templates (ie. PCR-derived...

...viral replication and transcription as well as reporter genes. To learn how you can use minigenomes in your...repression of your target gene, activation and repression of different genes in a single cell, and localization...Favorite Fluorescent Protein Oligomerizes in Cells GFP oligomerization can lead to misleading artefacts ...Lab at MIT developed plasmid based systems that report the order in which specific chemical inputs are...recombinase-based state machines could potentially be used to report upon and/or engineer “temporally ordered biochemical...derived from Zika virus and, upon binding, produce reporter proteins. These toehold switches can be used to... them in action. AAV: A Versatile Viral Tool for Gene Expression in Mammals Adeno-associated viruses (...

...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...highly customizable in terms of how they actually “report” as biosensors. Making aptamers glow The flexibility...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...

...viral prep pAAV-CAG-GFP AAV9-X1.1 Controls Edward Boyden New serotype pAAV-hSyn-EGFP AAV PHP.eB Controls...services collection or check out Addgene's Data Hub to see viral vector reports from other users. And don't...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...

...irreversible PA-FP reported was PA-GFP. PA-GFP was derived from Jellyfish (Aequorea victoria) GFP (wtGFP) by mutating...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...

...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...

...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...