...AAVs) to treat an inherited retinal degenerative disease called Retinitis Pigmentosa. I quickly became...has limited tropism in the retina, primarily infecting cells of the retinal pigmented epithelium (RPE)...field of gene therapy, more specifically AAV-based retinal gene therapy. The year was 2001 and I started a...delivery, and they had been shown to to transduce the retina but only transiently. However new viral vectors...capable of providing long-term gene expression in retinal cells - AAV - were becoming available. At the same...AdV) and lentivirus (LV) are known to transduce retinal cells, AAV quickly became the vector of choice ...Luxturna™, can efficiently transduce various types of retinal neurons. Nowadays a toolbox of naturally occurring...

...fluorescent units of the FRET pair together and causes an increase in the FRET signal. Subsequent demethylation...progression of cancer through different mechanisms. New FRET based tools to study epigenetics Epigenetic research...lysine-methylated peptides. This construct is in the middle of a FRET pair, which is used to visualise the results. When...separates the fluorescent units and so lowers the FRET signal back again. The Ting lab constructed 2 versions...reporter consistently showed significantly different FRET levels between unmethylated and methylated states...Fluorescent Protein Biosensors Read Our Tips for Using FRET in Your Experiments Post Read Other Blog Posts about...

...offense to BRET, CRET and other RET techniques which have never outclassed the Master. Note: BRET= Bioluminescence... a guitar hero (that I’m not), FRET loves playing “live.” Indeed, FRET was one of the first techniques...each other. FRET reduced the scale of our perception about molecular interactions. What is FRET? In their...Since then FRET has also been used to measure conformational changes, cleavage activity using FRET-based biosensors...its best. What are the Parameters that Affect FRET? FRET occurs when the two fluorophores used are in ...find a balance between FRET efficiency and cross-talk. Methods to Measure FRET for Cell Biology Studies...measure FRET and there is not one that is better than another. It is often recommended by FRET experts...

...orientation of the FRET pair. Since many biological processes occur within the typical FRET range, FRET efficiency...undetectable FRET, well-characterized FRET reference standards can be used to validate FRET measurements... for using FRET in Your Experiments Learn about Fluorophore Targeting for FRET Studies FRET Based Biosensors... further tips for using FRET in your experiments here.     What is this FRET you speak of? Förster Resonance...efficiency of FRET can be measured for a given donor-acceptor pair and a change in FRET efficiency correlates...for conventional light microscopy. Don't FRET - Use these FRET resources instead! As the choice of fluorophore...protein) may be able to boost FRET efficiency. Before constructing your own FRET probe, try searching PubMed...

...The Retroviridae (commonly called retrovirus) family — of which HIV is a member — may seem like an unlikely...molecular biology. “Retroviruses” is an umbrella term which encompasses 1.) the gamma-retroviruses — that have...have been developed into retroviral vectors and 2.) a subclass of retroviruses called the lentiviruses (...foundation for producing and using retroviral vectors effectively. Retroviral structure   Figure 2:..., as the viral particle emerges. Retrovirus life cycle Retroviruses go against the grain of the Central...unlikely candidate to use as a viral vector, but retroviruses have been developed into some of the most widely...Lentiviruses are sometimes referred to as “complex retroviruses ” (Cullen, 1992) because of their more complicated...

...series of fluorescence resonance energy transfer (FRET)-based calcium biosensors. Perhaps most notably,... sensitivity. As reported in Nature Methods, the FRET changes of these Twitch sensor variants were optimized... can now be applied to dozens of other published FRET-based sensors. For example, his team is now applying...Technische Universiteit Eindhoven has similar interest in FRET-based sensor proteins, particularly for the intracellular... them. Merkx says, while in principle adapting a FRET sensor for monitoring calcium versus zinc should...not the other). “It’s quite a robust way to make a FRET sensor with a large dynamic range,” he says. Merkx...in the construction of redCALWY-1, a red-shifted FRET sensor for zinc using variants of mOrange2 and mCherry...

...laboratory, then FRET experiments are a viable biophysical path to answers. What is FRET and what's it ...undergo FRET when they are near each other. How do you label your biomolecule of interest with FRET fluorophores...setting up a FRET-based assay or screen (the Addgene blog has an excellent primer on FRET and how to measure...the donor), then FRET measurements are possible. In fact, even if the majority of FRET acceptors are delivered...-targeted donors never “see” FRET acceptors, and thus never undergo FRET. If this nonspecific labeling... it's okay to be wrong In contrast to FRET donors, absolute FRET acceptor labeling specificity is not ...your FRET assay (Figure 2). These labeling methods are freely interchangeable and new uses for FRET are...

... Viruses of the Retroviridae or Retrovirus family, which includes the gamma-retrovirus and lentivirus ...using retroviral vectors in your experiments. Gamma-retroviral vectors Popular gamma-Retroviral Transfer...genome-wide experiments. Gamma-retroviral vs. lentiviral vector systems Gamma-retroviral vectors have a relatively...cells. Find tips on using gamma-retroviral vectors in our Gamma-retrovirus guide and find popular packaging...plasmids for transcriptional inhibition. Retroviral Plasmid Kit Retroviral Barcoding Library (Kit # 1000000097...viral vectors to choose from, for example gamma-retrovirus, lentivirus, Adenovirus, and Adeno-associated...enable long-term stable gene expression. Gamma-retroviral vectors are derived from the Moloney Murine Leukemia...

...rabies) naturally exhibit retrograde transport and can therefore perform retrograde tracing, but often at ...synapses.)  In this post we’ll describe the AAV retrograde serotype (AAVrg) and how it enables scientists...visualize neurons that send information to a region (retrograde tracer, Fig. 1A), and another to visualize neurons...upstream from the axon terminals to the soma (retrograde tracer) (Fig. 2). Viruses as neuronal scalpels...tracers, but finding a virus that works well for retrograde tracing can be difficult. The in vivo workhorse...adeno associated virus (AAV) shows relatively low retrograde transport efficiency in most serotypes. Some ...directs high-level transgene expression, and has retrograde functionality to monitor and map neurons (Tervo...

...MECP2 in Rett Syndrome Rett Syndrome was first described in 1966 by Austrian physician, Andreas Rett, who ...The Rett Syndrome Research Trust (RSRT) has an ambitious goal of curing Rett Syndrome and Addgene is ...with them to launch a Rett Syndrome Plasmid Collection and Resource Center for Rett Syndrome research. With...veteran and new Rett researchers can find the reagents and resources they need to cure Rett Syndrome.  If...If you’re new to Rett Syndrome, this blog post describes the history of Rett Syndrome research, the role...What is Rett Syndrome? Caused by a mutation in the methyl-CpG binding protein 2 (MECP2) gene, Rett Syndrome...neurological defects of Rett syndrome (Tillotson et al., 2017). Advancing gene therapies for Rett Syndrome MECP2...

...specificity in retinal cells. To do this, the group developed a new strategy for finding retinal cell-type ... and designed using stretches of DNA that were transcriptionally active in retinal cell types in mice....monkey, and human retinas Next, the 113 AAV promoters that were active in mouse retinas were injected into...gene expression in retinal cell types and classes in mice, primate, and human retinas. These synthetic ...types, such as inhibitory neurons, rods, cones, or retinal ganglion. Rather than using naturally occurring...they drove cell type specific expression in the retina.  Four approaches for designing synthetic promoters... with sequences upstream of the start codon of retinal cell-type specific genes from mice. Strategy #2...

...Transfer (FRET). Through the non-radiative transfer for energy between neighboring fluorophores, FRET can be...function. However, FRET experiments can be difficult to implement in practice because FRET depends on a number...number of hard-to-achieve factors. For example, FRET requires that fusion proteins be in close proximity...useable data. What’s more, measuring and quantifying FRET is often easier said than done. Fortunately, a complementary...to potential protein interaction partners. Unlike FRET, BiFC only uses a single fluorescent protein to ...et al., 2002). This would be more difficult with FRET, as the overexpression of fusion proteins it requires...Addgene! How to Design a BiFC Experiment Much like FRET, BiFC experiments must be carefully optimized to...

...powerful the FRET signal. In well-designed FRET-based biosensors, the intensity of the FRET signal can ...packaged into a retrovirus for infection of host cells. The fluorescent moiety of the BRET reporter was ...proteins together thereby increasing their FRET signal. FRET is a non-radiative transfer of energy from...accuators. The researchers took a FRET reporter and adapted it into a BRET reporter by using NanoLuc® Luciferase... allows researchers to convert FRET biosensors into more versatile BRET biosensors that can even be paired...PMC5171807. 6. Machleidt, T., et al. (2015) NanoBRET-A novel BRET platform for the analysis of protein-protein... on the Addgene Blog Learn more about FRET Tips for Using FRET in Your Experiments Learn more about Fluorescent...

...developed a new AAV variant, rAAV2-retro, that migrates retrogradely at higher rates than any other AAV... AAV2-retro vectors with a variety of transgene/promoter combinations. Check out our retrograde AAV blog...Selective retrograde transsynaptic transfer of a protein, tetanus toxin, subsequent to its retrograde axonal...strengthened during memory formation and memory retrieval. In neuronal tracing experiments, brain tissue...bodies to the terminals of their axons; while retrograde tracing outlines neurons in the opposite direction...axons to their cell bodies. Anterograde and retrograde tracing take advantage of existing transport ...myosin, and enzymes for transmitter synthesis. Retrograde transport is used for transport of endocytosed...

...viruses: gamma-retrovirus, lentivirus, adenovirus, and adeno-associated virus. Gamma-retrovirus Retroviruses...type of gamma-retrovirus is Murine Leukemia virus (MLV). Naturally isolated gamma-retroviruses have been ...Lentivirus is a type of retrovirus and shares many common features with gamma-retrovirus including genome architecture... nucleus as gamma-retroviruses do. Fun fact: Since lentivirus and gamma-retrovirus are so closely related...expression (Robinson, 1982). To add insult to injury, retroviruses generally have moderate to high immunogenicity...immune response in the host. So why use a gamma-retrovirus in the lab? Well, they are essentially evolutionarily... types with a high efficiency. However, gamma-retrovirus infection is restricted to dividing cells only...

...compose the secretory pathway, endocytic vesicles, mitochondria, lysosomes or they are secreted into the ...blocked from proceeding through the secretory pathway, and retained in the ER. FPs have been undeniably...include the quality control of secretory proteins in the Endoplasmic Reticulum and live cell imaging approaches... the power of the Dark Side." --Darth Vader in "Return of the Jedi" While Vader was referring to the evil...what if an FP fusion with a resident endoplasmic reticulum (ER) protein instead localizes to the cytoplasm...position requirements. For example, a KDEL ER retrieval sequence only functions at the extreme C-terminus...residues form inappropriate disulfide bonds in the secretory pathway (Figure 1) [4,6–8]. Additionally, FPs ...

.../gamma-retrovirus Lentiviruses are a class of retrovirus (so all lentiviruses are retroviruses, but not...not all retroviruses are lentiviruses.) This means that lentivirus and gamma-retrovirus share a lot of...as tools in the lab. We will discuss the gamma-retrovirus, lentivirus, adenovirus, and adeno-associated...the common components outlined above for gamma-retroviruses. While not required for lentiviral production...   Fig. 1 - Viral vector components of retrovirus  Adeno-associated virus AAV’s elements are...are quite different from gamma-retrovirus, but they perform very similar functions to infiltrate the host...in common with AAV, just like lenti and gamma-retroviruses have a lot in common with each other. Below ...

...monitoring live single-cell kinase activity is FRET, but FRET reporters are challenging to design and difficult...multiplex. Problems with FRET kinase reporters Difficult to Multiplex: FRET reporters are comprised of...be multiplexed together. Challenging  to Design: FRET reporter activity depends on both the distance and...difficult to predict. Slower Dephosphorylation Rates: FRET-based reporters can have slower dephosphorylation... than KTRs which is hypothesized to be caused by FRET reporters having phosphorylated conformations that... activity.  Advantages of KTRs When compared to FRET reporters for measuring kinase activity, KTRs have...KTRs also have some disadvantages when compared to FRET reporters. First, KTRs monitor global kinase activity...

...function improperly. 3. Incorporation into a FRET biosensor A FRET biosensor is a reporter that undergoes a...include the quality control of secretory proteins in the Endoplasmic Reticulum and live cell imaging approaches...molecule are part of a single fusion protein, as in a FRET biosensor, then the local concentration of FPs around...structures including organized smooth endoplasmic reticulum (OSER) whorls. 2)A dimerizing protein of interest...potential to form large stable polymers of dimers. 3) FRET biosensors that contain two dimerizing FPs can exist...For a membrane POI localized to the endoplasmic reticulum, the normally spiderweb-like pattern of tubules...membranes termed Organized Smooth Endoplasmic Reticulum or OSER if a dimerizing FP is fused to the POI...

... Energy Transfer (BRET). BRET is similar to Förster Resonance Energy Transfer (FRET), in which a donor...acceptor. TRUPATH uses an updated version of BRET called BRET2. Figure 1: TRUPATH measures heterotrimeric...resulting in decreased BRET2 signal. With increased concentration of drug, the BRET2 signal decreases. ...excite an acceptor fluorophore. The difference for BRET is that the energy transfer is between a luminescent...bioluminescence resonance energy transfer 2, or BRET2. With binding of the drug, the alpha and beta subunits... The TRUPATH BRET2 biosensor system was developed by constructing 14 luciferase donor Rluc8-Gα chimeras...emission. The activation state can be measured by a BRET2 ratio, which is the ratio of GFP2 emission to RLuc8...