...immediately prior to their transduction has been shown to increase AAV transduction efficiency by approximately... 3. Transduction efficiency depends on viral titer Both of these can be increased by concentrating a viral...unavoidable for some vectors) you can concentrate your virus by ultracentrifugation of the viral stock, followed...before proceeding to ultracentrifugation to avoid contaminating your transduction with the packaging cells... by ultracentrifugation (75,000 - 225,000 g for 1.5 – 4 hours at 4 °C). After ultracentrifugation, virus...PBS.  Alternatively, to concentrate a virus solution without ultracentrifugation, you can reduce the volume...immediately following transfection. In other words, when exchanging the medium after transfecting cells, replace...

..., exo-AAV2 in mouse retina after intravitreal injection to transduce bipolar cells and photoreceptors ...22314290. PubMed Central PMCID: PMC3345986. Meliani, Amine, et al. "Enhanced liver gene transfer and evasion...PubMed PMID: 29296848. PubMed Central PMCID: PMC5728288. Schiller, Lara Timantra, et al. "Enhanced Production... vectors. They found that they have a higher transduction efficiency, i.e. the same number of AAV particles...particles is able to infect more cells (as demonstrated in different cellular environments such as retina...also found that exo-AAVs were more resistant to neutralizing anti-AAV antibodies. This is particularly useful...vivo where endogenous anti-AAV antibodies often neutralize the therapeutic efficacy of AAV vectors. How ...

...friendships. I put “neutral” in quotations here because I would not have considered myself neutral in any of their... blog Management for ScientistsTransferable Skills Guide: LeadershipTransferable Skills Guide: Teamwork...stopped talking to each other if I was not there to arbitrate for them. They were different people with some... them would acknowledge later that without my “neutral” presence to alleviate those differences, they ...the right time. Like many of the posts in our Transferable Skills series, this post is not meant to be ... that you could strengthen. The right set of transferable skills, coupled with your technical skills, ...The source of those conflicts can frequently be traced back to one of a few trouble spots:  Communication...

...researchers feel they lack formal training in a variety of transferable skills. At Addgene, we've set out...evaluate potential strategies to resolve the problem.  Step Three: Decide on a strategy and implement it... highlighting that researchers do learn MANY transferable skills while working in the lab and by offering...where you might need some help. Today in our transferable skills guide: Problem-Solving. I don’t know ...potential sources of error and then decide on a strategy to fix the issue. I was problem solving!  You ... careers, I always felt that descriptions of transferable skills seemed fuzzy or ill-defined. In this ...it.  Step Four: Assess how effective your strategy was. If you didn’t quite fix everything or revealed...

...express a transposase or reverse transcriptase so they can transpose. Both class I and II transposons can be...eukaryotic genomes.  Components of a transposon system Transposon: The transposon is the DNA sequence that moves... encodes the proteins the transposon needs for transposition. Transposition protein: This is the protein...themselves as RNA transcripts and then rely on a reverse transcriptase enzyme to reverse transcribe back into ...reverse transcriptase and integrase enzymes which are required for LTR retrotransposon transposition. Non-LTR...reverse transcriptase activities.    Figure 1: Overview of retrotransposon transposition. Retrotransposons...2021. To mobilize a transposon, first the transposase enzyme binds the transposon’s long terminal repeats...

...Posts in the Transferable Skills Guide Developing Transferable Skills During Science Training Resources...researchers feel that they lack formal training in a variety of transferable skills. At Addgene we've set out... highlighting that researchers do learn MANY transferable skills while working in the lab and by offering...where you might need some help. Today in our transferable skills guide: Collaborating with others outside...of my position at Addgene I am now involved in Strategic Partnership Management, a very interesting and...being clear about what was expected, and keeping track of speakers. During your graduate or postdoctoral...perfect platform for practicing initiative and for training in teamwork and collaboration management. For ...

... The higher the titer, the better. AAV transport and transgene expression It’s clear that we need high... the issue of low titer by concentrating your virus. See our Concentrating AAV Protocol (Step 10) for ...PubMed Central PMCID: PMC2574934. 3. Zingg, Brian, et al. "AAV-mediated anterograde transsynaptic tagging...contact with the cell is to increase the AAV concentration. In this flowchart, you can see the dwindling...be required. For example, retrograde AAV gets transported up the cell’s axon. While the mechanism for this...yet fully understood, we can imagine that this transport is probably not 100% efficient. So the few AAV...particles that have entered the cell now have to travel up the axon, through the cell, and potentially ...

...researchers feel that they lack formal training in a variety of transferable skills. At Addgene we've set out... highlighting that researchers do learn MANY transferable skills while working in the lab and by offering...where you might need some help. Today in our transferable skills guide: managing a team. You’ve just been...about management skills during your scientific training. I never thought about management skills before...grad school. Both were hard-working scientists-in-training who got the job done, but I struggled with what... he graduated. Years later I attended a formal training for new managers and I had a “Eureka” moment while...around the fact that new team members need to be trained, which may seem like a waste of your time at first...

...researchers feel that they lack formal training in a variety of transferable skills. At Addgene we've set out... highlighting that researchers do learn MANY transferable skills while working in the lab and by offering...where you might need some help. Today in our transferable skills guide: Time Mangement for Scientists....to-do lists at the end of the day so they can jump straight into tasks the next day. If you work in a job ...useful to avoid such procrastination: Remove distractions: Probably the easiest way to do this is to leave...internet and all it’s enticements are far too distracting. (As a side note, I’d recommend the book, “Bored...more fantastic reasons to remove yourself from distractions every once in awhile.) Reframe the task in a...

...Target strand The DNA strand that base pairs with the guide RNA.   Non-target strand The DNA strand that... non-target strand is excluded as ssDNA. DSB Double-Stranded Break A cut to both strands of DNA. Indel...editing generates double-strand breaks causing deletion, transversion and translocation. Nature Cell Biology...a job for a single enzyme. Base transitions Base transversions C → TG → A A → GT → C C → G                   ...to base pair with the DNA target strand, leaving the non-target strand unpaired and accessible on the outside...binding. Pro tip! In CRISPR, the non-target strand is the DNA strand that does not base pair with the gRNA.... it the “edited strand.” Uracil excision repair leads to the first base transversion editor The story...

...apply to programs, and apply for travel grants. Good luck and happy travels! Resources Resources on the ...remote collaborations Transferable Skills Guide: External collaborations Transferable Skills Guide: Teamwork...current institute. A common reason is hands-on training in a technique that only a few labs have expertise...this expertise will provide you with guidance and training so that you yourself can become an expert and ...for another reason. Visiting would allow you to train and acquire the data you need on the new instrumentation...new collaborations and fresh perspectives. For trainee level researchers, it can expose them to a new ... Identify reagents you will need, any required training, and a priority list of experiments you hope to...

...resources on the Addgene blog Transferable Skills Guide: Time Management Transferable Skills Guide: Conflict ...of being a scientist and an incredibly useful transferable skill. Being able to stand up in front of an... audience and convey your ideas clearly and strategically is powerful in almost any career. It allows ... applies: what can go wrong, will go wrong. The trash removal service slams dumpsters outside the open... turn your slide deck into a chalk talk. Your translator doesn’t know the term “diving board” so you end...messing up?–and use the three tips above to make a strategy that can help. Even something as simple as telling...remember to breathe!)   Public speaking as a transferable skill The worst thing about doing public speaking...

...effort from acknowledging its existence. Studying ancestral group dependencies  Take, for instance, a recent... essentialist connections between biology and ancestral groups or, by extension, race. Many researchers...diligent scientist, they sought to understand these strange results by digging deeply into their data.   ...report the likelihood of any given sgRNA having an ancestral bias. Next, they worked with John Doench, David...looks like, which led to the incorporation of ancestral bias into genome-wide screens, introducing bias...doi.org/10.1101/2022.11.18.517155   Resources on ancestral bias and the human genome Wang, T., Antonacci-...

...easier to track translation in two different ways: by monitoring the first round of translation or by tracking...of relative reporter translation. Single molecule continuous translation tracking TRICK allows you to ...various stimuli on global translation, but a visual technique to study translation remained elusive. Two ...destination co- or post-translationally. TRICK: Visualizing the first round of translation While ribosome profiling...in the fluorescent signal: untranslated RNAs appear yellow, and translated RNAs appear red. To enable ...Using translation inhibitors cycloheximide and puromycin, Halstead et al. verified that translation is necessary...to see the first round of translation, but it doesn’t allow continued tracking of a given mRNA because ...

...have been used to track protein localization, cell structure, intracellular trafficking, and protein turnover...with SapTrap The Jorgensen Lab recently developed a modular plasmid assembly toolkit called SapTrap to ...selection marker for screening modified strains. With SapTrap, the user first designs either oligos or...fission/fusion, nuclear import, and neuronal trafficking. Although FPs have enabled discovery of many ...of the endonuclease Cas9 to create a DNA double stranded break (DSB) at a genomic site specified by a guide...challenging to screen for the genetically modified strains that contain your newly tagged gene. Most insertions... improve CRISPR genomic tagging in C. elegans. SapTrap offers scientists a convenient one tube assembly...

...an article describing a sucrose supplementation strategy to improve yield. In addition, as described in..., the team developed a viral genome sequencing strategy to confirm the identity and purity of all AAV ...! AAV purification by iodixanol gradient ultracentrifugation This video goes over AAV vector purification...protocol page for more detailed instructions. AAV titration by qPCR using SYBR green technology Here’s some...used to generate a standard curve, visit the AAV Titration by qPCR written protocol page. Getting started...

...competitive transplant assay in the mouse intestine, demonstrating its utility in long-term tracking in vivo...provides a combinatorial indexing strategy that simultaneously tracks clonal history and cell identity ...linking ancestral gene expression patterns with reprogramming outcomes (Biddy et al., 2018). Tracking lineage... tool that combines scRNA-seq and lineage tracking to track the dynamics of gene expression and clonal... of CellTag The Morris Lab developed CellTag to track the direct reprogramming of mouse embryonic fibroblasts...reprogrammed into iEPs by retroviral expression of transcription factors Foxa1 and Hnf4α, a process that like...reprogramming (CellTagMEF), at the end of the three-day transcription factor reprogramming treatment (CellTagD3), ...

...fluorescent protein (XFP) genes in a transgene. When multiple copies of the transgene cassette are introduced, stochasticity...delivery is more common than conventional transgenic strategies. In such vector-based gene delivery systems... XFPs in the original method, we used the Tetracycline trans-activator system to boost the expression ...expression levels, and we named the system Tetbow (Tetracycline trans-activator Brainbow) (Sakaguchi et al., 2018...Bio-Protocol 8 (2018): e3046. Livet, Jean, et al. "Transgenic strategies for combinatorial expression of fluorescent...troubleshooting your Tetbow experiment Plasmid concentrations and AAV titer - The XFP genes have to be introduced...highest color variations. Adjust the plasmid concentrations or AAV titer for the best results. The timing...

...NEB’s new teaching space at Lab Central to learn all about it. LabCentral is a lab and office space shared...because both LabCentral and Addgene aim to make scientific advancement easier. NEBNext Ultra FS DNA Library... prior NEBNext Ultra II DNA Library Prep Kit for Illumina, one was only able to extract libraries from...New England Biolabs (NEB) hosted workshop at Lab Central and discuss why these types of workshops could ...in a lab.   Participating in a workshop at lab central We’re working on a project where we aim to do large...throughput DNA prepping by trying out NEB’s new NEBNext Ultra II FS DNA Library Prep Kit for Illumina sequencing...people to take a break and relax. Companies at LabCentral get access to a variety of experimental services...

...your AAV packaging transfection. In my lab we regularly use the ratios below: 1 Transfer Plasmid : 1 pHelper...prevent future headaches. To help you plan PEI transfections for AAV packaging, considering using this PEI...Calculator. AAV packaging typically requires transfecting three plasmids at specific molar ratios. To ...PEI calculator for AAV packaging Use the PEI transfection calculator for AAV packaging Select the plasmid...pHelper: 1 RepCap OR 3 Transfer Plasmid : 5 pHelper: 2 RepCap Select what type of tissue culture dish ...dish you will be performing the transfection in: 15 cm dishes OR CF5 stacks Select how many dishes or ...or stacks you plan to transfect: 1 to 10 Fill in experimental details on the spreadsheet: Name your...