...GBC) 85968 Barcode Human Weissman 3rd N/A N/A Retroviral Mouse Genome-wide CRISPR Knockout Library 104861...104861 Knockout Mouse Teichmann N/A (retroviral) 5 90,230 RNA-Binding Protein Pooled CRISPR Knockout Library...

...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 ...

...20 bp in length and paste them into contiguous stretches of DNA known as CRISPR arrays. Separate Cas proteins.... PMID: 28931002 Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini ... FJ, Terns RM, Terns MP, White MF, Yakunin AF, Garrett RA, van der Oost J, Backofen R, Koonin EV. 2015...

...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...

...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 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...

...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...

... 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...

... 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...

...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...

...below. ID Plasmid Gene/Insert Mutations Industry Return to Top MJFF Shared Research Tools Labs affiliated... ID Plasmid Description Gene/Insert Industry PI Return to Top...

...bio 32407 CD200RCd4d3+4-blac 32408 secretedBirA-8his 64395 secreted BirA-Flag...