...include Flp-dependent DREADDs viral vectors. Both inhibitory and excitatory DREADDS from the Gether lab are... project vectors available as ready-to-use viral preps with more on the way. S5E2 regulatory element containing...subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These articles...deployed around the world to combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus...encompassing all possible amino acid mutations of the receptor binding domain (RBD) of the viral spike protein...angiotensin converting enzyme 2 (ACE2) cell-surface receptor. The spike protein RBD responsible for binding...2021.   After validating this method through monitoring GFP degradation, this protein compartment was...

...Cas9. Adenoviral CRISPR vectors (left) or CRISPR vectors and donor DNA vectors (right) are transduced ...need to use a two viral vectors system: one vector for Cas9/sgRNA and one vector for your specific DNA ...gRNA and Cas9 using adenoviral vectors As compared to other viral vectors, AdVs can provide a large transgene...adenoviral vectors Since then, other groups have successfully developed and used AdV vectors to edit genes...updated Dec 10, 2020 by Benoit Giquel.   Adenoviral vectors (AdVs) have been used for many years for in vivo...of oncogenicity or genotoxicity seen with other vectors (Lukashev et al., 2016). The protein capsid encapsulating...have already a packaging capacity larger than AAV vectors. This capacity is large enough to carry the Cas9...

...Subcloning - To move a gene from one vector to another simply amplify the vector and insert with separate primer...promoter of the pHLsec vector. In this scenario, IVA can be employed by cutting the vector with restriction ...García-Nafría from the MRC Laboratory of Molecular Biology. Jake Watson is a postdoctoral researcher in neurophysiology...  Javier García-Nafría is an investigator scientist at the MRC Laboratory of Molecular Biology. He is ... Watson and Javier García-Nafría from the MRC Laboratory of Molecular Biology. Plasmid cloning is an essential...with successful recombination reported in all laboratory E. coli strains tested so far. The use of this...technique fail to gain traction? It is likely that both vector amplification with low fidelity polymerases and...

...process, which was a major barrier for depositors and requestors alike. We transformed this process by ...ready-to-use viral vectors from some of our most popular lentiviral and AAV vectors, as well as developing...privilege to find and share what stories we can, and to hear the stories others have to tell.  "Also thanks...amazing years in front of us. So in our last celebratory moment, we wanted to walk through some of our...service would allow people to add a gene to a repository others could request from — Addgene!  Addgene...meant we could grow accessibility alongside our repository.  “I’m sure you hear it all the time, but Addgene...took almost six years to reach our first major repository milestone — 10,000 plasmids deposited and available...

...cheaper to use Cas9 activators. This larger scale of activation allows for exploratory-based research as...of the gene, Cas9 activators efficiently solve these problems.   Which Cas9 activator should I use? There...plastic tool and has taken the scientific world by storm. While Cas9 has been most widely used to create ...significant work on constructing Cas9 transcriptional activators. These constructs allow for the upregulation ...no longer activated. However, there are several factors that may prevent your gene from being up-regulated... we would recommend using a cDNA overexpression vector rather than going through all the troubleshooting...genome-wide studies One of the best uses for Cas9 activators is in genetic screening. gRNAs targeting every...

...validation carried out by the commercial repository. Repositories generally ensure cell line identity and...which can be stored in liquid nitrogen long term, preferably some of these vials will be stored in a separate...in a cryostore log. Keeping good passage records allows you to track a cell line’s age and monitor any ...and money but cells lose provenance (this is the history of the cell line consisting of records of the origin...invariably much older than those available from biorepositories leading to genotypic and phenotypic artefacts...easier to conduct high quality cell culture. Biorepositories around the world ensure that the cell lines...The oldest, largest, and most diverse of these repositories is the American Type Culture Collection aka ...

...makes AAV vectors especially amenable to correcting disease-associated mutations. AAV vectors maintain...gene delivery by affinity-purified receptor-targeted viral vectors." Nature communications 6 (2015): ncomms7246... Check out our Viral Vectors Featured Topic Page Learn about CRISPR-AAV vectors Learn about Addgene AAV... is caused by mutations in the common cytokine receptor gamma chain (c) and is characterized by a lack...thereby correct the mutations using retroviral vectors. However, many diseases require techniques that...target cells has been challenging. A variety of vector delivery technologies have been investigated including...usability in clinical settings. On the other hand, vectors derived from AAV have recently gained popularity...

...are similar except that the vectors carrying the transdifferentiation factors are directly injected into... Kotton Four Factors that Differentiate the Stem Cell Field Learn how lentiviral vectors can be used for... involves overexpression of four reprogramming factors- OCT4, SOX2, KLF4, and C-MYC - that induce a differentiated...another cell type of choice. The four reprogramming factors can be delivered and expressed in multiple somatic...supplements, bioactive small molecules, and growth factors are used to control the cell fate of iPSCs and ...         Adipocytes [28] Keratinocytes [29] Photoreceptors [30] Otic hair cells [31] Neurons [32, 33... intermediate pluripotent state is not always obligatory in cell conversion processes [35]. Rather than...

...In-Fusion cloning molar ratio calculators Lab math calculators These calculators can help you with common...lab. Molarity calculator Promega's Biomath Calculators Primer resuspension calculator (sign-in required...data repository for validation data on Addgene/CLOVER viral vectors.  Caspedia: A data repository for ....)  DailyCalcs Science Calculator app for iPhone Reagent and Data Repositories These links can help you... for plasmid design and mapping. ApE (A plasmid Editor): A free, donation-based plasmid analysis tool ...enzyme cut sites Open reading frame finder  Addgene vector database: Find sequences for plasmids assembled...Golden Gate Reactions Cloning ligation reaction calculators These links can help you calculate how much insert...

...Stable   For cloning into and storage of lentiviral and retroviral vectors or cloning or repeated sequences...derived. For cloning into and storage of lentiviral and retroviral vectors or cloning or repeated sequences... HB101. For cloning into and storage of lentiviral and retroviral vectors or cloning or repeated sequences...cells for cloning and storage of unstable DNA and lentiviral/retroviral vectors. Used by Addgene for pooled... your plasmid or carrying out your experiment. History of E. coli strains E. coli are gram-negative, rod... DH5alpha and DH10b (also known as TOP10). The history of B strain is a bit more convoluted due to researchers...researchers sharing and renaming samples throughout history. It was likely isolated in 1918 but was first referred...

...reaction to generate high-throughput targeting vectors. These vectors can be used to tag endogenous genes and...the final targeting vector (Fig. 1, Step 4), and co-injection of the targeting vector and a Cas9 expression...site destination vectors for inserting a tag at multiple target sites. SapTrap vectors can be easily modified...cells. To use CRISPaint, users need 3 vectors:  1) a gRNA vector that targets the gene of interest, 2)...PCR or cloning, as digestion of the destination vector with SapI yields 2 sites- the first site accepts...screening of the insertion event, and a variety of connector modules (linker sequences between the tag and ...with SapI allows the tag, selectable marker, and connector to be released (Fig. 1, Step 2-3). Since donor...

...are transfected with both vectors. Flex vectors are the solution. Flex vectors expressing fluorescent cytoplasmic.... It became possible to design FLEx vectors (Flip-excision vectors) that only allow one inversion event...concentrations of vectors expressing the required recombinase. The high concentration of FLEx vectors guarantees...experiments. Mutated FRT and loxP sites in Flex Vectors – inversion only once All recombination events ...between target sites of the same sequence in FLEx vectors, the DNA sequence that should be inverted is flanked... target sites are cleaved out, leaving behind a vector with the inverted DNA flanked by non-compatible...non-compatible target sites (Schnütgen et al. 2003). Flex vectors revolutionize neuroscience Manipulation of specific...

...has been exploited to localize factors such as transcriptional activators and repressors to target genes...target genes using dCas9-based activators The simplest of the dCas9 activators consists of dCas9 fused to ... dCas9 and antibody-activator fusion proteins. The Synergistic Activation Mediator complex, generally ...promoter region. Cas9 activators include Cas9 fused to VP64, VP64 along with other SAM factors, and the Suntag...with CRISPR–Cas nucleases, base editors, transposases and prime editors. Nat. Biotechnol. 2020 387 38, ...array of Cas9s (many available through Addgene’s repository) and make it easy to find your perfect match....precise edit without even making a break.  Base editors are dead Cas9 (dCas9) or nCas9 enzymes fused to...

...imaging microscopy (FLIM) method. Acceptor photobleaching method The acceptor photobleaching method is simple...don’t measure acceptor fluorescence, this method is also less sensitive to direct acceptor excitation artifacts...interactions in living cells using a microscope. Historically, molecular interactions were detected by indirect...fluorophore (the donor) to another fluorophore (the acceptor) by means of intermolecular long-range dipole-...dipole-dipole coupling." The emission of the acceptor fluorophore can be measured using microscopy techniques...photons) and the extinction coefficient of the acceptor (linking the quantity of absorbed light, at a ...highest quantum yield donor, the highest absorbing acceptor and fluorophores with significant overlap in their...

...responsible for storage.  If datasets are too large to transfer to a journal repository, they are likely...publishers. Nonetheless, the initial release caused a storm as the organization left open the question of how...for advice about the scope of the policy. End of story? Not quite. The PLOS policy on data access is a...statistical methods, omission of some data, etc.  The firestorm that followed implementation of the policy comprised... with limited acknowledgement of the initial contributors. This is probable to some extent - but that ...tools and assumptions to reveal findings that the originator was oblivious to, no matter how much time they...they had. It could be argued that data generators may, in light of the policy, hold onto data longer before...

... years ago and developed cloning vectors to harness it. These vectors, called pKIL18 and pKIL19, contained.... If, however, an investigator were to successfully clone an insert into the vector, the ccdB reading ...cells that contained non-recombinant vectors (re-ligated empty vectors, for instance) would still express...ccdB makes cloning easier by selecting against vectors that did not take up your insert. But exactly how...ccdB accomplish this? Let’s start with a brief history of the gene and how molecular biologists have harnessed...potent toxin... The ccdB gene, located on the F sex factor plasmid of E. coli, is part of a toxin-antitoxin...from Bernard, P., et al. showing the pKIL 18/19 vectors and demonstrating the concept of restriction enzyme-mediated...

.... Viral vectors undergo DNA rearrangements. Depending on the composition of the viral vector and the ... researchers prefer to store freshly harvested virus at 4 °C instead of storing it briefly at -80 °C. ...of a recombinant adenoviral vector: optimization of conditions for storage, transport and delivery." Japanese...about Lentiviral Vectors Additional Resources on the Addgene.org Check out Our Viral Vector Guide Pages ...perform complex biological tasks, both of which are factors in the technical challenges they can present. Many... one point or another. After amplifying a viral vector, collecting virus, and transducing target cells...the repeat sequences present in the LTR, the vector may undergo genomic rearrangements during replication...

... space is the Victoria Makerspace in Canada, which I belong to. The team at the Victoria Makerspace is..., we intend to focus on motor neuron knockouts so that we can perturb factors related to worm motion. ...student and then instructor at Singularity University at NASA Ames, and an instructor at Biocurious.org...was contributed by Derek Jacoby from Makerspace Victoria, CA Over the decades science has become increasingly... of a bigger movement in a variety of research sectors to provide open source technologies and spaces ... are constantly moving so we need good ways to monitor and track them. We have a number of microscopes...instance, we use a mutant known as CX51 that exhibits motor defects above 25 degrees. Computer scientists and...

...counterselection vector is the pDS132 sacB-based allelic exchange vector. This vector contains sacB, an...This post was contributed by Jacob Lazarus, a postdoctoral researcher at Harvard. There’s an astounding...selecting for an antibiotic resistance marker from the vector (abR, Figure 1). The resolution step (“double crossover...leads to the desired mutation and excision of the vector backbone. This step requires “counter-selection...colonies isolated are those where the allelic exchange vector has undergone single crossover and integrated into...recombineering (Khetrapal et al., 2015). They created vectors for Lambda Red that contained one of eight bacterial...aeruginosa. These toxins were a mix of Type VI secreted effector toxins, toxin-antitoxin type toxins, and phage...

... to be nice, the mentor fails to effectively mentor the underrepresented - the mentor over praised and...received a doctoral degree (or equivalent) and is engaged in a temporary and defined period of mentored advanced...to your peers and other mentors who can help you. You Can Talk to Your Editors I wish I had been more...well-meaning mentors often fail to judge the work of underrepresented groups under their mentorship with the...research community to develop a plan to make postdoctoral pay/benefits commensurate with work training...image below). Slide of information gathered by Postdoctoral Associations around Boston - From Panel Session... staff scientists. This results in a glut of postdoctoral trainees who, even though they are aware that...