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... of Gateway cloning vectors Vector type  Vector features Purpose Donor vector attP sites for recombination...sequences into multiple vector systems. With the appropriate entry and destination vectors, one can use Gateway...ccdB gene is present in the donor vectors and the destination vectors prior to recombination, and it is...donor vector.   Method B: TOPO-cloning of the desired insert into an attL-entry-TOPO vector. TOPO...destination vector that best fits your experiment. This choice will depend on a number of factors, like your...Gateway vector to produce the desired expression clone. This is possible thanks to the Gateway vectors’ design...sites creates attL and attR sites.   Gateway vectors contain modified versions of the att sites so that...

... A cis-splicing vector served as positive control (cis-ctrl). v1, vector 1; v2, vector 2; BD, binding ...Improved prime editor PE7 Antibodies to facilitate SARS-CoV-2 research New viral vector preps of Voltron2...Voltron2 voltage sensors AAV vectors for targeting microglia The REVeRT Dual AAV Vector System Neurodegeneration...protection factor) promotes editing (Yan, J et al., 2024). They developed a new prime editor that incorporates...Brian O’Neill New viral vector preps expressing the Voltron2 voltage indicator, created by the Schreiter...voltage), the investigators found that administering these AAVs with a dilute Cre-expressing vector resulted...incorporated in an AAV vector (Figure 5). Efficiency and specificity of these AAV vectors for microglia was...

...the reprogramming factors Sendai virus- a non-integrating RNA virus Episomal vectors- extrachromosomally...nonintegrating hiPSCs where the vectors encoding reprogramming factors behave as separate extrachromosomal...Reprogramming efficiency of PBMCs with episomal vectors Episomal vectors are based on the Epstein-Barr Nuclear ...of the reprogramming factors. However, reprogramming of PBMCs by episomal vectors has yielded hiPSCs at... et al. generated a mixture of two vectors that encoded six factors - OCT3/4, SOX2, KLF4, C-MYC, LIN28...Table 1) encoding the reprogramming factors. The episomal vectors are introduced into cells by electroporation... the addition of reprogramming (or “Yamanaka”) factors, they changed the landscape of regenerative medicine...

...thawing a fresh vial. In many labs, the history of the cells in storage may be a bit dubious due to lack of... and can alter expression of receptors, ion channels, and growth factors resulting in changes to the cell...depends on a variety of factors such as the transgene expressed, the viral storage buffers used, and other...titrated. If storing for less than a day, lentivirus can be kept at 4°C. For long-term storage, viral preps...Rubinstein, M. “LDL receptor and its family members serve as the cellular receptors for vesicular stomatitis...About Viral Vector Elements Learn How to Do Genome-Wide Screens with CRISPR Lentiviral Vectors Additional...Lentiviruses are a powerful laboratory tool often employed to establish cell lines that stably express...

...-associated viral vectors: packaging capacity and the role of rep proteins in vector purity." Human gene...Cas9 and AAV Viral Vector Elements Additional Resources on Addgene.org Find AAV Vectors for Your Research...Location, Location, Location! Failure to Plan (for Storage) Is Planning to Fail Patience Is Bitter but Its...Future? I knew little about adeno-associated Viral Vectors (AAVs) before starting my graduate program at the...lab has been utilizing chemogenetics (Designer Receptors Exclusively Activated By Designer Drugs, DREADDs...in locomotion. We have relied heavily upon AAV vectors to deliver chemogenetic/optogenetic constructs ...Although they have a small packaging capacity, AAV vectors were suitable for our needs for the following reasons...

...some of the most frequently requested vectors in our repository and will discuss some of the features ...fusion protein. Mammalian vectors One of our most popular mammalian expression vectors, c-Flag pcDNA3, comes...will need accessory packaging vectors to generate the virus. Bacterial vectors This system is great for overexpressing...cases bacterial expression vectors are inducible (e.g. pBAD LIC cloning vector (8A)) and have an epitope...folding assays. Yeast vectors There are several types of yeast expression vectors. Consider your applications...shuttle vectors. You may prefer the more traditional approach of an autonomously replicating vector that ...conduct your custom experiment. Fly vectors The vast majority of fly vectors are based on p-element, random...

... Depositor Collections Rinehart Lab Phosphoprotein Reagents...Library #111704) is anticipated to be useful for laboratories performing phosphoproteomic experiments in need...can also be introduced into a second expression vector encoding a split mCherry fluorescent reporter, ...domain and the phosphosite, mCherry fluorescence is restored, so interactions can be discovered by fluorescence-activated... via partial UAG codon reassignment and release factor 1 deletion . FEBS Letters , 586 (20), 3716–3722...

...only occurs when donor and acceptor are close together. The binding of acceptors >100 Å away from the donor..., 2009). Donor and acceptors: Putting it all together The FRET donors and acceptors described above can...for donor and acceptor pairs that you can use in your FRET experiments. Donor Acceptor Tag R0 (Å)a Range...when you feed it substrate or add an essential cofactor? How many binding partners does it have and how...environment? If these are pressing issues in your laboratory, then FRET experiments are a viable biophysical...directly via resonance to the second fluorophore (the acceptor) if the two probes are close together (typically... a precise physical distance between the donor/acceptor fluorophores (Stryer, 1978), a measurement that...

...toolbox for adenoviral vectors in Addgene’s repository is smaller than for other vector types, we hope that...Viral Vectors 101: Viral Applications Adenoviral Vector Production and Troubleshooting Viral Vectors 101...fact, of how adenoviral vectors work. What are adenoviruses?   Adenoviral vectors are commonly derived from...adenoviral vectors all have a genomic capacity that is much higher than other viral vectors (tens of kilobases...has been reported when using vectors, and immune inactivation of the vector can still occur even in the... of existing vectors, as well as research into the development of new adenoviral vectors. This post was... viral DNA polymerase – it makes sense that vectorization strategies include creating an AdV transfer ...

...modular vector assembly process designed for a quick, plug-and-play approach to cloning CRISPR vectors (McGee... new vector to four days for an experienced bench scientist. New to the bench? Fragmid depositor John ...for each module, generate the vector file, and quickly clone their vector of interest. And it truly is ...destination vectors for various delivery methods, along with compatible inserts. All destination vectors contain...heavy lifting out of CRISPR vector design, making it easier to get the vectors that are just right for your...Fragmid destination vectors currently available. Table 1: Fragmid destination vectors Organism Delivery...designing a CRISPR vector the Fragmid way!    Figure 1: Selecting your vector type on the Fragmid...

...fusion to a receptor-transcription factor fusion. TEV protease cleave the transcription factor from the ... of two different receptors- angiotensin II type 1 receptor and the β2 adrenoceptor. GPCRs in chemogenetics...down the name "G-protein coupled receptor" into two parts: the receptor itself and the G-proteins it associates...associates with.  Receptor: GPCRs are also known as seven-(pass)-transmembrane domain receptors, due to the ...pharmacologically-inert, small molecules. These new receptors were termed Designer Receptor Exclusively Activated by Designer...of the five senses? GPCRs or G-protein-coupled receptors (GPCRs)! Aside from these three senses, GPCRs ...in inflammation, and neurotransmission. These receptors can be activated by an array of different ligands...

...twenty years at Addgene, we are sharing stories of our repository’s positive impact on science and scientists...three functional groups: nucleases, modulators, and chimeric effectors. Nucleases generate a classic double-stranded... distributed CRISPR plasmids. But modulators and chimeric effectors, made available in 2013 and 2016, ...community better. We therefore looked at depositors and requestors by country, and graphed the top ten countries...no surprise that the USA is the largest depositor and requestor of CRISPR plasmids. But many other countries...from Japan to Australia, are significant depositors and requestors. CRISPR tools are developed and used all...think so — and so do our requestors! CRISPR plasmids in the Addgene repository have been cited a total ...

...cytidine deaminase portion of the base editor to create SpCas9 base editors with editing windows as small as...some phages fight back using CRISPR inhibitors. Several such inhibitors have been characterized for type ...bacterial and mammalian expression vectors for these CRISPR inhibitors are listed in the table below. ...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These brief...posts for every great plasmid that comes into the repository, but be sure to let us know if you'd like to ...TagRFPt, EGFP, mVenus, mCerulean3, mKOFP2) and empty vectors that have standard cloning sites or gateway compatible...cell types directly linked to disease, including motor neurons (-3mnx1/Hb9), pan neuronal (elavl3/HuC),...

... Viral Vector Packaging Service AAV Biosensors Viral Vector Packaging Service: Biosensor...CaMKII Dlx EF1a GFAP/GfaABC1D minBG Synapsin E2 regulatory element Activity Cre-dependent Flp-dependent ...Karpova and Schaffer labs. See the Retrograde AAV Inventory Page . Don’t See What You’re Looking For? Our ... eligible AAV plasmids and we’ll make the viral vector prep you need. Or fill out our form to let us know...format. Please note this does not guarantee viral vector packaging service, but lets us know what viruses...

...into distinct expression vectors using the enzyme BbsI. These expression vectors each contain Type IIS restriction...Cas9 or dCas9-containing destination vectors. Destination vector options include humanized wt Cas9, dCas9...-VP64 (transcriptional activator)-containing plasmids. Each destination vector contains GFP, enabling ...create lentiviral vectors expressing wt humanized Cas9 and up to three gRNAs. Entry vectors containing the...wt hCas9 entry vectors are supplied with the kit, you can clone your own entry vectors containing other...containing vector using Golden Gate assembly, and up to 8 gRNAs may be expressed simultaneously. Vectors for ...Joachim, et al. “A versatile modular vector system for rapid combinatorial mammalian genetics.” Journal of ...

... with viral vector manufacturing facilities, such as the University of Pennsylvania Vector Core. Transfections...Pennsylvania Vector Core. In general, titering is performed by the facility that produced the viral vector lot....Empty vector particles can be identified after negative staining and appear darker than full vector particles...Electron micrograph of AAV vector preparation shows that the vast majority of the vectors consist of full particles...vivo studies. Preparations are then aliquoted and stored at -80 °C. Titer Titering is either performed by...to determine which facility produced your viral vector lot. At Addgene, AAV particles are titered by droplet...method.  Titering by the University of Pennsylvania Vector Core is (as of April 2016) also performed by ddPCR...

...students, and those from historically underrepresented groups. Through such mentoring programs, scientists...especially grateful to its mentors and supporters during this first year. Mentors Amanda Gunn, Bryan Gibb.... The teaching/mentoring bug never went away. While he has frequent bouts with Impostor Syndrome, Joe ... done at my first six ASM meetings. I told this story over a great dinner at Pittypat’s Porch with old...James et al., 2020. Help me stranger - Finding mentors for the meeting Just like analysis pipelines for... meetings. A key was to provide each bin with a mentor who had attended the meeting multiple times before...plan for and network at the meeting. We found our mentors by asking for help with the program on social media...

...community concerns AAV vectors are complex biological reagents; many factors, like storage buffers and conditions...when I started working with AAV vectors. I wanted to use an AAV vector to express ChR2(H134R) in A2 norepinephrine...to AAV vectors, many from people trying to answer the exact same question I was: What AAV vector is best...Working with AAV vectors in the brain and peripheral nervous system presents a special challenge to scientists...question and used it to publish!  Working with AAV vectors in neuroscience Let’s consider my first challenge...didn’t provide any information on other important factors such as what is the best promoter to use, or if...deal of time and effort to test and optimize our vector - overall, I spent two years testing and reformulating...

...Choose a moderator with a backbone. It is nice to have a subject area expert, but the moderator is not ...LinkedIn profiles of all panelists to the moderator. The moderator and organizers should develop a list of...interviews, the moderator should be taking good notes and discovering interesting stories, riffs or opinions...contribute. The moderator should identify the funny, provocative or interesting stories that will make ...bring out these interesting stories and remind the panelists of the stories or topics you know they can...(lapel mics are best). The moderator takes control It is the moderator's job to prevent anyone from ...published on LinkedIn. Follow Addgene on LinkedIn for repository news and updates. Panel discussions are becoming...

...designed their AAV tagging vectors (pAAV-SEPT-Acceptor and pAAV-TK-Acceptor) so that they can be easily...gene. pAAV-SEPT-Acceptor, the preferred vector, contains a promoterless splice acceptor-IRES-neoR gene,...cloning them sequentially into the chosen acceptor vector. The vectors are then packaged into AAV virions via...identical targeting vectors delivered through transfection of naked DNA. Other investigators who had been working...Check out our Viral Service Find AAV Vectors Browse all Viral Vectors ...well understood, researchers have been using AAV vectors for gene editing in mammalian cells since 1998....when delivered via AAV infection, gene targeting vectors with relatively short (1 kb) homology arms underwent...