...B978-0-409-90042-2.50021-0  Toh, W. K., Teo, Y. L., Tor, X. Y., Loh, P. C., & Wong, H. L. (2023). Development...Cloning Vectors for Gram-Negative Bacteria. In R. L. Rodriguez & D. T. Denhardt (Eds.), Vectors (pp. 287... inform us on how to create better recombinant vectors. In this blog post, we’ll define plasmid host range...features to create broad host range recombinant vectors.  A plasmid’s host range is determined by the ...groups (Jain and Srivastava 2013).  Recombinant vectors that are made from broad host range plasmids are...different bacterial hosts (Doran et al. 1998). Regulatory elements within the ori also control copy number...The plasmid has its own replication initiation factors and proteins independent of the host. Host range...

...your experiment using the lentiviral vector system. Viral vectors have been increasingly popular in fundamental...primary cells. Amongst the different vectors used, lentiviral vector constructs have proven very useful ...fluorescent sensors. Retroviral vector background Until the development of viral vectors, gene transfer into mammalian...un-transfectable cells, labs use viral vectors and particularly retroviral vectors because they target a broader...attractive choice for a gene transfer vector. Lentiviral vectors Although retroviruses are still in use...generation of lentiviral vectors consisted of three plasmids encoding 1) the lentiviral vector genome which was...lentiviral vector system offers the best safety profile in terms of RCL generation because this vector requires...

...First, some history. In 2008, the Auricchio lab decided to solve the issue of “tiny vector, big transgene... overviews of three split vector strategies plus one additional split vector strategy for increasing the...homology directed repairs (HDR). Split AAV vector approaches Split vector approaches increase the size of the...copies of both vectors. Examples of use: Halbert et al., 2002 used an overlapping vector with 440 or 1000bp...splice site donor and acceptor sequences and the homologous region of the two vectors can be from one of ... hybrid vector, as measured by β-galactosidase activity, was 81% of that from a single vector, but significantly...ideal split vector system. Overall the efficiency of transgene expression from split vectors has been inconsistent...

... the perfect voltage indicator and optogenetic actuator pair. Ideally, indicators should possess fast ...the voltage indicator, QuasAr, and actuator, CheRiff, in the bicistronic Optopatch vector, the Cohen lab... our community of depositors. With over 40,000 plasmids avaliable in the repository, we can't give all...University of Wellington, Victoria, has developed an adaptable bacterial expression vector which gives close ...science.aaa3380 MuLE vectors for customized lentivirus production Finding a lentiviral vector backbone with ...custom-cloned vectors in a Multisite Gateway reaction with a desired destination vector expressing a separate...a final lentiviral vector containing up to 5 different elements. Lentiviral vectors constructed with the...

...genes, the laboratory of Jason Moffat at the University of Toronto has created the Toronto KnockOut (TKO...both laboratory and prototrophic strains. The vectors were based on the original EasyClone vectors (Jensen...coupled receptors (GPCRs) yet one third of the GPCRs in the human genome are orphan receptors meaning ...tools, compounds, and assays to monitor activation of these receptors. Bryan Roth's lab has a newly available...expression vectors with various promoters suitable for in vivo expression, and/or produce vectors to make... all TAL effector scaffolds previously constructed for the Golden Gate TALEN and TAL Effector Kit 2.0.... These vectors contain uracil-excision based (USER) sites, flanked by ADH1 and CYC terminators for easy...

...Updated mini-transposon vector for bacterial mutagenesis or gene targeting Victor de Lorenzo's lab has... existing transposon and vector systems and creating an all-synthetic vector that included only the elements...features to be monitored to study mitochondrial dysfunction. Easy-to-use tools to monitor these features... into a destination vector in a predefined order. Finally, the destination vectors are used with Multisite... eight entry vectors can be used for each Multisite Gateway compatible destination vector for a maximum...optogenetics tools – light-activated receptor tyrosine kinases Receptor tyrosine kinases (RTKs) are a large...step, compatible Level 0 vectors are directionally assembled into a Level 1 vector creating a single transcriptional...

...polycistronic vector or multiple single-gene vectors can be used to package reprogramming factors. An important... OSKM reprogramming factors: Mouse Expression Vectors Human Expression Vectors Carey et al Hockemeyer...reprogramming vectors available from Addgene. Another drawback is that lentiviral vectors insert in different...Cre-lox lentiviral vectors: This approach is the same as using a standard lentiviral vector, but transient...Non-integrating iPSC delivery methods Viral Vectors Adenovirus: Adenoviral vectors infect dividing and nondividing...different adenoviruses, each expressing one factor. These vectors don’t integrate into the genome and are ...mutagenesis, adenoviral vectors are considered a safer way to express reprogramming factors for therapeutic applications...

...building successful mentor relationships, asking for mentor support, and choosing your mentor. What isn’t discussed...receive technical mentorship from a ‘bench mentor’. In some instances, the bench mentor is also the scientific...Scientist Edition Mentoring for Scientists: I Have a Mentor, What Now? Will You Be My Mentor? Finding and ...often enough is the hands on mentoring you receive after you’ve found your mentor, though. Typically, you ...scientific mentor…but usually the bench mentor is a staff scientist, postdoc, lab manager, technician, or...more, depending on the mentoring style within the research group). The bench mentor-mentee relationship ...lot more face time with your bench mentor than your scientific mentor. Thus, this relationship will probably...

... 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...

...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),...