...detectable by two different tags: a reporter and an HA tag. The reporter comes in three varieties: HRP, Spaghetti...independent of the reporter. The only caveat is that HA staining is weaker than reporter staining. Picking...Picking the right reporter for your RANBody While all three reporters stain cultured cells and tissue sections...Monster, and Chicken IgY Domain RANbody Reporters. Reporter Detection Method Uses Pros Cons Antigens...genetically tagged and instead are conjugated to reporters (i.e. fluorescent dyes, HRP, etc. ) after purification... is a RANbody? A RANbody is a fusion between a reporter and a nanobody that’s encoded on a plasmid. A ...sections, some reporters are better suited for particular applications. Read on to learn more about each...

... CRISPR reporters in the near future.  SRIRACCHA (stable, but reversible, integrated reporter for assaying...histone)-GFP reporter into the genome of mammalian cells.  Figure 1: The SRIRACCHA reporter construct...Find GEmCherry2 reporter plasmids at Addgene Overall SRIRACHA and GEmCherry2 reporter systems provide ...Recently scientists have created CRISPR fluorescent reporter assays to quantify targeted Cas9 cleavage, and...and non-homologous end joining (NHEJ). In these reporters, editing typically results in a change in the ...this blog we will focus on two fluorescent CRISPR reporter methods, SRIRACCHA and GEMCherry2. We will cover... the Piggybac transposon to stably integrate a reporter gene that contains a puromycin resistance gene...

...fluorescent executioner caspase reporters. Several apoptosis reporters have been developed, such as the...transfer (FRET)-based executioner caspase reporters. In these reporters, the FRET pair is linked by a caspase...FlipCherry: Red fluorescence protease reporter To add to the fluorogenic reporter toolkit, the Shu Lab also designed...novel fluorogenic reporter that has a large dynamic range and high brightness. This reporter achieves approximately...fluorescence emission. FRET and fluorescence based reporters however have several limitations. First, FRET ...study apoptosis in vivo scientists need a protease reporter that has a large dynamic range and high brightness...Shu Lab from UCSF designed an improved apoptosis reporter, ZipGFP, which utilized the self-assembling split...

...What are Acoustic Reporter Genes (ARGs)? Fluorescent and bioluminescent reporter genes are widely used... to study gene expression in cells. But these reporters have limited use in vivo because they are based...expression in vivo.  But what ultrasound lacked was reporter genes that could produce ultrasound contrast. ...our lab - the Shapiro Lab - developed Acoustic Reporter Genes (ARGs), which are sets of genes that, when...5202–5209 (2017). Bourdeau, R. W. et al. Acoustic reporter genes for noninvasive imaging of microorganisms... Hurt, R. C. et al. Genomically mined acoustic reporter genes for real-time in vivo monitoring of tumors...

...real time, but each of these reporters has its drawbacks. Acoustic reporter genes (ARGs) overcome these...existing bacterial reporters Limited Depth of Signal/Imaging: Fluorescent reporters are not as well suited...fluorescent or bioluminescent reporters usually only detects the signal of the reporter and fail to pinpoint what...signal is coming from.   Acoustic reporter genes (ARGs) Acoustic Reporter Genes (ARGs) are engineered gene...imaging tools The luciferase reporter gene The green fluorescent protein reporter gene Resources on Addgene.org... researchers have developed in vivo bacterial reporters that use fluorophores and luciferases to track...these limitations by using gas vesicle reporters that are detectable by an inexpensive and widely available...

... Figure 4: Reporters in Gene On include a mScarlet reporter, luciferase reporter, and neomycin resistance... editing. eGFP reporters for single base editing by APOBEC-Cas9 Though the ACE reporter quantifies APOBEC-Cas9...DSB in their previous reporter, the Harris Lab created a panel of eGFP reporters to quantify the on-target...editing reporters. Image from St. Martin et al., 2019. The Harris lab used these eGFP reporters to analyze...Transient reporter for editing enrichment (TREE) The Wang and Brafman lab developed a transient reporter for...cells segregated solely by a reporter of transfection, which only report the efficiency of plasmid delivery... functional reporter system to identify and enrich for base-editing activity The reporters mentioned above...

...Problems with FRET kinase reporters Difficult to Multiplex: FRET reporters are comprised of two fluorescent...phosphatase-accessible. Kinase Translocation Reporters (KTRs) Kinase Translocation Reporters (KTRs) are genetically encoded...single-cell kinase activity is FRET, but FRET reporters are challenging to design and difficult to multiplex...alternative tool with their Kinase Translocation Reporters (KTRs) whose cellular localization serves as a...multiplexed together. Challenging  to Design: FRET reporter activity depends on both the distance and the .... Slower Dephosphorylation Rates: FRET-based reporters can have slower dephosphorylation rates than KTRs...KTRs which is hypothesized to be caused by FRET reporters having phosphorylated conformations that are less...

...collection of reporter plasmids is amenable to Golden Gate assembly for turning the single-input reporters into...Neuron. 2019. PubMed PMID: 30704911. Acoustic reporter genes for noninvasive imaging of microbes in mammalian...contributed by Lukas Morgan  Listen to the acoustic reporter gene podcast segment In recent years, scientists...heavily on fluorescent protein genes. These optical reporters have limited deep-tissue performance, and sometimes...Mikhail Shapiro’s lab has developed four acoustic reporter gene (ARG) plasmids to better monitor gene expression...with a resolution of less than 100 µm, the ARG reporters could be successfully visualized in vivo using...demonstrates engineered gas vesicles as the first reporter genes using ultrasound and provide a major benefit...

...remove a stop cassette upstream of an effector/reporter gene. In cells where the stop cassette is removed...https://doi.org/10.1101/788679 A destabilized GFP reporter for calcineurin activity in yeast Article contributed... it is an attractive drug target. Calcineurin reporters allow scientists to measure calcineurin dynamics... target calcineurin. Current calcineurin gene reporters are based on the bacterial βgal assay, and are...Büttner’s lab designed a calcineurin transcriptional reporter consisting of a quickly-degraded destabilized ...panels) equipped with destabilized GFP calcineurin reporter (blue) and stained with PI (pink) to indicate ...Image from Diessl et al., 2020. Find the GFP reporter calcineurin activity here Diessl et al., Microbial...

... the 3’ UTR of the reporter mRNA via an MS2 coat protein (NLS-MS2-RFP) the reporter mRNA, which contains...their reporter mRNAs. Using the TRICK plasmids, Halstead et al demonstrated that 91% of reporter mRNAs...customized fluorescent reporters can be challenging enough, but incorporating multiple reporters into a single ...pyralis) reporter plasmids for use in high-throughput screening of pre-mRNA splicing. This reporter system...widely used luciferase reporter gene plasmid pGL3 pGL3 luciferase reporter gene plasmids have been...protein function, thereby creating a single-molecule reporter of intracellular processes. Multimerization via...the cognate att sites are supplied as individual reporter constructs or as the fully assembled pMemoryArray...

... the 3’ UTR of the reporter mRNA via an MS2 coat protein (NLS-MS2-RFP) the reporter mRNA, which contains...proteins to allow translation to occur. For a TRICK reporter mRNA this displacement leads to a change in the...GFP protein that binds to the coding region of a reporter mRNA via a PP7 coat protein (NLS-PCP-GFP) an RFP...translation, both GFP and RFP are bound to the reporter, causing it to appear yellow. The act of translation...of the mRNA, leaving only a red FP bound to the reporter. Because it contains a nuclear localization signal...copies of the FP-coat protein fusions to their reporter mRNAs. Using translation inhibitors cycloheximide...is necessary for removal of GFP from the TRICK reporter. Delayed translation is known to be important ...

...the promoter of the reporter gene to activate expression (Figure 2c). The reporter gene then allows detection...genes as reporters and some that repress instead of activate transcription. The HIS3 reporter system (...sequence (UAS) of its target gene, in this case a reporter gene (e.g. luciferase or GFP), regardless of whether...co-expressed in a yeast strain that also contains a reporter gene cloned downstream of a promoter containing...referred to as the “bait”, Figure 2b) cannot activate reporter gene expression on its own, but, if the two POI...Gal4 activator, bacterial LexA DBD and the lacZ reporter gene (3, 4), there are now many variations, including...to have Y2H systems that avoid transcriptional reporters altogether; In the split-luciferase system, intein-mediated...

... to cleave and activate a reporter. The detectable signal from this reporter can be measured and quantified...and a Csm6 specific reporter (in the same fluorescent channel as the Cas13 reporter), they could significantly...method termed Specific High Sensitivity Enzymatic Reporter UnLOCKING (SHERLOCK and SHERLOCKv2) (Gootenberg... termed DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) (Chen et al., 2018). Both SHERLOCK and... SHERLOCK- Specific High Sensitivity Enzymatic Reporter UnLOCKING Cas13 (C2c2) was first identified in...molecules. In SHERLOCK a quenched fluorescent ssRNA reporter is added to the reaction. Cleavage of the quenchable...amplification step in combination with the ssRNA reporter enables SHERLOCK to detect DNA or RNA with atomolar...

...has dubbed these fusions LumiFluor Reporters. The LumiFluor reporter constructs were designed to be packaged... The researchers took a FRET reporter and adapted it into a BRET reporter by using NanoLuc® Luciferase...couples NLuc to fluorescent proteins to make better reporters and the second couples NLuc to fluorescent proteins...host cells. The fluorescent moiety of the BRET reporter was used to sort successfully infected/expressing...NanoLuc luciferase. Both GpNLuc and OgNLuc BRET reporters produced bright signals without exogenous excitation...near 600 nm – a clear demonstration of why a BRET reporter can improve deep tissue imaging. The constructs...NLuc) and set about to create an in vivo imaging reporter by linking two CyOFP1 molecules to NLuc (CyOFP1...

...Fluorecent Proteins in Nano Lanterns Reporter gene assays A reporter gene, such as luciferase, usually serves... is also the most commonly used bioluminescent reporter. This beetle emits a yellow-green light with a...several studies utilized luciferase as a genetic reporter in plant and mammalian cells. Luciferase assays...available to choose from) is now a common feature in reporter plasmids.  Learn How Luciferase Can Be Used In...transcription within cells, where detection of the reporter protein or its enzymatic activity is measured....expression can be determined by detection of the reporter in a specific assay, which ideally would have .... Production of photons by this bioluminescent reporter occurs slower than fluorescent-based methods, ...

... registered reports, the gold standard for testing hypotheses Finally, Registered Reports combine all ...Reproducibility Project: Cancer Biology, where reported methods may not include how many tests were conducted...attention on the results instead of the methods. Unreported flexibility and questionable research practices...analysis code, and other materials. The use of reporting guidelines can also help remind us to include ...important times to measure that growth rate, you report the results of all four of those tests and not ...fifth time has unpredictably high rates, should be reported as a new discovery that deserves confirmation ...best practices of Open Science. With a Registered Report, peer review occurs before results are known. The...

...invert the reporter and GeneR fragment. Then the second recombination event would excise the reporter from ...Spatio-temporal expression of optogenetic reporters: Since most optogenetic reporters are delivered using AAV injection...transduced reporter gene. Generating a FLEx switch to control expression of an optogenetic reporter would ...Cre-dependent reporter expression, the potential for background expression of the reporter will be greatly...shown in Figure 2, the cassette should contain a reporter DNA sequence (such as GFP) in the sense orientation...the two pairs of target sites. By flanking the reporter with one loxP site and one lox511 site in the ...would ensure that the reporter remains silent until a cell or tissue-specific Cre is provided. Since the FLEx...

...fundamentally the reporter construct monitors the expression of a nearby gene. The reporter gene serves as... screens to introduce reporters throughout an organism’s genome. These reporters have historically been...stimulate the expression of the reporter. The system is made up of a reporter gene flanked by a minimal promoter...promoter – all rely on the random integration of a reporter construct in close proximity to an endogenous ...example, if green fluorescent protein (GFP) was the reporter gene and green cells were observed in the kidneys... for with an uncharacterized gene. The actual reporter construct can be something phenotypic (such as...promoter. The system is composed of a promoter-less reporter which needs to integrate within an exon of a gene...

... transformation. Choose good reporter genes There are many great reporter genes available and which one...the T-DNA region with our gene of interest or a reporter gene so that the transformation and gene expression...different treatments on transformation success. Some reporter gene examples are below. The GUS gene (β-glucuronidase...histochemical GUS-staining is a very useful visual reporter (Figure 2). The GUS enzyme cleaves a substrate...transformation success. Luciferases are great quantitative reporters and use a substrate to produce bioluminescence...half-lives then GUS and are therefore good temporal reporters to measure promoter activity, for example. Useful...Agrobacterium. This is especially problematic with reporter genes as you might observe expression from Agrobacterium...

... versions of this reporter to target the K9 (pcDNA3-K9 histone methylation reporter) and K27 (pcDNA3-K27...The K9 reporter uses the HP1 chromodomain to recognise lysine 9 methylation, while the K27 reporter uses... methylation in living cells. Essentially this reporter construct consists of one substrate (the histone-derived...-K27 histone methylation reporter) positions on histone H3, which are known to be involved in repression...methylation of these sites was controlled, the reporter consistently showed significantly different FRET...states. Thus, this system is a reliable tool in reporting methylation status of these sites. This new technology...Alice Y. Ting. "Genetically encoded fluorescent reporters of histone methylation in living cells." Journal...