... 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...

...Transposable elements (TEs): Retrotransposons  Class I TEs are also known as retrotransposons. They transpose by...those without (non-LTR TEs). LTR retrotransposons are like retroviruses both in their structure and mechanism...which are required for LTR retrotransposon transposition. Non-LTR retrotransposons contain two open reading... Figure 1: Overview of retrotransposon transposition. Retrotransposons are mobilized by a ‘copy-paste...of transposition: Class I TEs, also called retrotransposons, and Class II TEs, also called DNA transposons...into new target sites. This is similar to how retroviruses, like HIV, replicate. Class I TEs do not encode...integrates in human cells at rates similar to retroviral vectors. The hyperactive version of the SB transposase...

...Q1: Are retroviruses and lentiviruses the same thing? Can I package lentivirus with retroviral packaging...Lentiviruses are part of the retroviral family. What scientists often refer to as “retroviruses” are technically ...technically gamma-retroviruses - another, separate member of the retroviral family. While both lentiviruses and ...your gene of interest into cells. Unlike gamma-retroviruses that can only infect dividing cells, lentiviruses...and gamma-retroviruses use the same genes for packaging (that's gag, pol, and env), the isoforms of these...LTRs) are different. As a result, lentiviral and retroviral packaging vectors are not interchangeable. While... physiological difference between lenti- and retroviruses. Lentiviruses are able to enter an intact nuclear...

...Moloney murine leukemia virus (MMLV)-derived retroviruses. Retroviral vectors, such as pMXs or pMSCV, infect...Mechanism Efficiency Safety Pros Cons MMLV-derived Retrovirus Integrating High Low Efficient and Stable Genome...required for translational use. Viral Vectors Retroviral: Some of the first iPSCs were generated with ...generating iPSCs compared to other delivery methods. Retrovirus has a cloning capacity of ~8 kb, so either a ... corresponding endogenous pluripotency genes. Retroviral vectors are prone to incomplete silencing of ...with their differentiation potential. Finally, retroviruses are prone to insertional mutagenesis and random...integration creates variation between iPSC clones. Retroviruses are not considered safe for clinical applications...

...the best one for your experiment. Retro and lenti viruses Retroviruses are a family of viruses that stably...The most well-known retrovirus is HIV. Lentiviruses are a genus of the retroviral family that can infect...types of viruses used in mammalian research: 𝛄-retrovirus, lentivirus, adenovirus, and adeno-associated...the need for envelope disassembly. Both lenti and retro viruses can accommodate a cargo up to approximately... the host genome (non-integrating) as lenti and retro viruses do. They are generally used to achieve transient...muscular atrophy.   Type of Virus Pros Cons Retrovirus Stable Gene Expression Broad Tropism ...: PMC49545. 3. Coffin J, Hughes S, Varmus H. Retroviruses. Cold Spring Harbor Laboratory Press; Cold Spring...

...as in-stock preparations. AAV retrograde (AAVrg, also known as AAV2-retro) We’ve covered AAVrg in a previous...found to transduce skeletal muscle, cardiac, and retinal cells (Issa et al., 2023). AAV1 capsids are available...including liver, muscle, lung, CNS, kidney, and retinal cells (Issa et al., 2023; Wu, Asokan, et al., 2006...cardiac, pancreas, smooth muscle, CNS, kidney, and retinal cells (Issa et al., 2023; Wu, Asokan, et al., 2006...transduce liver, skeletal muscle, pancreatic, and retinal cells (Issa et al., 2023). AAV9 packaging plasmids... and engineered to infect neurons and undergo retrograde transport, traveling up the axon to the cell ... 2022 Pancreas AAV6, AAV8 Wang et al., 2006 Retina 7M8 Dalkara et al., 2013 Skeletal muscle MyoAAV...

... As an example, most secreted proteins are produced in the endoplasmid reticulum, modified in the golgi...golgi and then transported in vesicles via the secretory pathway to their subcellular or extracellular destination...proteins within complex structures. In order to interpret colocalization experiments in a scientific and...colocalization detected in microscopy cannot be interpreted as direct molecular interaction without further...more closely is Förster Resonance Energy Transfer, FRET. Additional opportunities: Tracking intracellular...Edition. 3.  CureFFI.org. Cell Biology 04: The Secretory Pathway. 4. Dunn, Kenneth W., Malgorzata M. Kamocka...Central PMCID: PMC3984138. Microscopy U. Basics of FRET Microscopy. Additional Resources on the Addgene...

...tail vein or retro-orbitally Gauge: 27–30 Volume: < 0.2 ml Skill required: beginner (retro-orbital) to ...restrainer or anesthesia Tail vein or retro-orbital 27–30 < 0.2 mL Beginner (retro-orbital) to intermediate (tail...2020). A retrospective analysis for different routes of administration in Mice-Percutaneous Retro-Orbital...vein injections.” However, they can also be given retro-orbitally, behind the eye. IV injections are used...needle in the correct vein without collapsing it. Retro-orbital injections are also thought to be less stressful...between the layers of the skin. While this could theoretically be performed anywhere on the body, somewhere...researcher preferences. It sounds complicated but has a pretty low learning curve! Restraint: scruffing Location...

...et al, 2020). Because retrovirus is closely related to lentivirus, many retroviral envelope proteins have...done on enveloped viruses such as lentivirus, retrovirus, and rabies virus. How to pseudotype To pseudotype...particles.   Why pseudotype? Lentiviruses and retroviruses typically target immune cells with their wild-type...glycoprotein targets neuronal cells and can travel in a retrograde direction from the synapse to the cell body, ...allows the lentiviral vector to be transported retrograde and also has higher neuronal transduction efficiency...lentivirus with rabies glycoprotein now serves as a retrograde tracer so that scientists can identify the neurons...the replicating rabies virus envelope, travel retrogradely, and jump the synapse to the previous neuron...

...criterion than theoretical brightness for selecting the brightest fluorescent protein. Theoretical brightness...higher the number, the higher the theoretical brightness. The theoretical brightness often appears in tables...EC) and quantum yield (QY) respectively. The theoretical brightness is calculated by multiplying EC by...Cranfill et al (2016) or Thorn (2017). However, the theoretical brightness ignores several important experimental...be expected in a 'real' application than the theoretical brightness. Consequently, it makes sense to identify...Amsterdam) where she develops genetically encoded FRET based biosensors.     Additional Resources on the... theoretical brightness...

... the FLIM-FRET acceptors here. See Addgene's FRET reference page for many other common FRET vectors. ...fluorescent proteins for use as FLIM-FRET acceptors  Listen to the FLIM-FRET acceptor podcast segment Article...allowing for minimized basal FRET variability, and is a good choice for FRET assays where a dark acceptor...Mork The Murakoshi lab has developed two new FLIM-FRET acceptors, ShadowG, a novel dark green fluorescent...photophysical properties and can be used in quenching, FRET (Förster resonance energy transfer), and FLIM (fluorescence...

... incredibly useful in FRET experiments. Professor Fessenden's example of a FRET experiment with all relevent...into a concrete context. I recommend this post to anyone wondering if and how they can apply FRET in their...forward.   Special Delivery Fluorophore Targeting for FRET Studies While I love all of our blog posts, my pick...Hospital. This post describes how to target and use FRET flurophores for a variety of applications. While...While editing this post, I learned a lot about how FRET can be used to study biological phenomena and about...NgAgo Special Delivery: Fluorophore Targeting for FRET Studies Gendered Innovations: Why Does Sex of the...

...produced in high-titer retroviral stocks generated by the transfer of a retrovirus into the packaging cell...CRISPR/Cas9 components, and fluorescent sensors. Retroviral vector background Until the development of viral...cells, labs use viral vectors and particularly retroviral vectors because they target a broader range of...into the genome is more efficient. The first retroviral vectors used were derived from the Moloney Murine...MoMLV, lentiviruses, a separate genus of the Retroviridae family including human immunodeficiency virus...transfer vector. Lentiviral vectors Although retroviruses are still in use, considerable efforts have ...possibility that mutation or recombination with human retroviruses could lead to RCL. Regarding transduction efficacy...

..., but at their core they can all be boiled down pretty simply – they are systems that measure biological...fluorescence in the presence of calcium. This includes FRET-based sensors such as Twitch. While calcium is king...calcium dynamics in the cytosol and sarcoplasmic reticulum are of interest to muscle biologists, while neuroscientists...Neurotransmitters Many canonical fluorescent as well as FRET-based biosensors have been engineered to detect ... vs. low in the cell the donor and acceptor of a FRET pair on actinin will produce different signals based...For one, many viral platforms, such as AAV or retrovirus, can either permanently integrate and/or express... sensor, whether you choose lentivirus, gamma retrovirus, or AAV as your vehicle, we’ve got you covered...

...specific response cytoplasmic retention insensitivity to other ions In BRET, A mutant Renilla luciferase...Synchronization of secretory protein traffic in mammalian cells The journey of secretory proteins, from their...that use Bioluminescence Resonance Energy Transfer (BRET) were developed by the Johnson Lab . Characteristics...luminescent siganls and a ratio is calculated (the BRET ratio), which allows for compensation of varying...the Institut Curie in Paris, has developed the retention using selecting hooks (RUSH) system. How does ...proteins fused to a streptavidin core that are able to retain reporter proteins fused to the streptavidin-binding...reporters which can then be tracked along the secretory pathway (Figure B). The RUSH system has already...

...Callaway EM. Retrograde neuronal tracing with a deletion-mutant rabies virus. Retrograde neuronal tracing...that cell, and nascent RABV cross synapses to retrogradely infect neurons that project to the starter cell...occurs, subsequent cycles of viral replication and retrograde transmission occur, spreading the virus through...RABV G protein is necessary and sufficient for retrograde transmission (Etessami et al., 2000, Wickersham...released from the starter cell and transmits retrogradely to first order neurons, via entry mediated by...Conzelmann KK, Lepier A, Götz M, Berninger B. Retrograde monosynaptic tracing reveals the temporal evolution... for Rabies Glycoprotein (G) expression  Find Retrograde AAV ...

...the interpretation of the parameters we infer, since scientific productivity is better interpreted as ...request activity appears to return to normal. The model fit these two discrete phases with an estimate of... impacted. The inferred weekend effect is, in retrospect, generally more dramatic than the COVID-19 effect...remind the reader that this is not a causal interpretation -- the effect would generally not be the same...just one lab, which limits how broadly one can interpret our analysis of COVID-19’s effect on scientific...instabilities in the China data, so we recommend interpreting the model results for China with more uncertainty...

...qualitative. The Organized Smooth Endoplasmic Reticulum (OSER) assay (Costantini et al., 2012) measures...homodimerization of FPs targeted to the endoplasmid reticulum based on the appearance of OSER whorls. This assay...considered a linker (figure 1). In fact, for several FRET biosensors (yellow cameleon 3.60, EPAC, and Galphai...fluorescent protein can be truncated to increase the FRET efficiency. Creating fusions at the N- and C- termini...technology and should always be considered when interpreting results.  The recent revolution in genome editing...Klarenbeek, Jeffrey, et al. "Fourth-generation epac-based FRET sensors for cAMP feature exceptional brightness,...PubMed PMID: 30886412. Mastop, Marieke, et al. "A FRET-based biosensor for measuring Gα13 activation in...

...interaction that increases FRET transfer between the fluorescent proteins. FRET is highly amenable to biosensor...biosensor creation, and custom FRET biosensors can be created using the cpFRET kit created by Olivier Pertz... increases in fluorescence of up to 11,000-fold. FRET biosensors are another common class of biosensors... Tsien’s lab contain two fluorescent proteins (a FRET pair) separated by CaM and M13. When calcium binds...such as the nucleus, mitochondria, endoplasmic reticulum (ER), vacuole or cellular membranes. Examples ...

...Cameleon-based FRET sensor D1ER, and GCaMPER. Bonus: There are also Cameleon-based FRET sensors available...CEPIA) or through FRET sensors, can reduce these unwanted variables. Twitch is a FRET-based calcium reporter...fundamental to muscle and nerve function, hormone secretion, blood clotting, and as we all know, a structural...available biosensors use a single GFP or CFP/YFP-derived FRET pair. If you need to free up crowded optical space...buffering and to increase sensitivity), and is a great FRET sensor option if you have the setup necessary for...