...AAV plasmids contain several important elements, but among the most important are the inverted terminal ...Adeno-associated viruses (AAV) are an important biological tool that allow scientists to express a gene...modifications and types  Since ITRs are one of the most important elements of AAV and rAAV vectors, modifications...site reduce Rep nicking. Since Rep proteins are important for viral genome replication, these mutations ...is still limited, but this area of research is important for downstream AAV applications.  Tips & FAQs ...

...to scientists and open your eyes to the many opportunities available to those who are scientifically minded...actively require these skills, including: Science Reporter Scientific Sales Epidemiologist Scientific Recruitment... actively use these skills include: Science Reporter Scientific Illustrator Research and Development...desirable in a range of roles, including: Science Reporter Technical Writer Statistician Bioinformatician...job title actually involves, other than what is portrayed in the media and in films, which can often be ...skills? Other things to consider are wage, opportunity to advance, and how many jobs are normally available...

...go about starting a lab? Obviously, funding and support are crucial parameters, and familiarity with equipment...a PhD student in his first year. “It was very important to me to be with them in the lab when they started...spending more time in his office, he gradually incorporated regular group meetings, journal clubs, and focused...the lab and to take things slowly! It is more important to carefully select first hires who are well-suited...seek out advice from seasoned PIs who can offer support. “It is an exciting and emotional time!” George...a challenging process. Each PI emphasizes the importance of slowly building up your lab and selectively...

...phosphatase-positive assay) as well as stabilization (Nanog-GFP reporter activation and silencing of factor-linked mCherry...are available at Addgene. Kim, et al. Stem Cell Reports. 2015. PubMed PMID: 25772473 Targeting and manipulating...Genetically modified organisms like transgenic mice are important tools for understanding the function of cell types...enhancer elements and combined it with a variety of reporters (i.e. eGFP) and effectors (i.e. DREADDs). The ...CRISPR edits and improves editing efficiency. Importantly, the point mutations created by Agudelo et al...et al. also mutagenized the cytidine deaminase portion of the base editor to create SpCas9 base editors...reagents shared via Addgene. Narayan S, et al. Cell Reports. 2017. PubMed PMID: 28813671 Listen to the podcast...

...individual cells. Electroporation on the other hand uses electrical pulses to create transient pores in the cell...plasmid of interest. Scientists can also use electroporation, the application of an electrical charge to...lysogenic cycle, the phage’s genetic material is incorporated into the host’s genome at a particular integration... and stable cell lines, where foreign DNA is incorporated into the cell’s genome and is thus passed down...charged cell membrane. Physical methods such as electroporation or microinjection actually pokes holes in the...

...strategies for finding what they need, and we wanted to support all of them — searching, browsing, drilling down...experts, our software team tailored Elasticsearch to support the different usage patterns the larger team identified... selecting a suggestion — how should these be supported and combined?  Luckily, we had the advantage of... information in our search indices in order to support different kinds of matches: exact words, partial... terms.  Weighting We determined the relative importance of different bits of information for a given ...that make our new search engine unique. Most importantly, with this new implementation, we laid the foundations...

...increased complexity comes the need (and/or opportunities) to modify the device for individual experiments...openUC2, which I am a co-founder of), that aim to support the community in developing better, more sustainable...difficult to overcome. Universities may not have the opportunity to buy parts through low-cost sources such as...accessibility. Through this approach, the Planktoscope, a portable device for studying plankton using flow cells,...quickly with the printer, act here as a kind of "teleporter," since a part developed in Germany, for example...-based models and the use of taxpayer money to support this research. But there is a move towards more...

...off for the day. 1:45 PM--Write final report. Work on a final report for a user research project I led. ...field-based software product manager at Oxford Nanopore Technologies. If the first thing you hear when...streamlined manner. Now, in my current role at Oxford Nanopore Technologies, I work with our customers in the...believe that more and more people will have the opportunity to explore a career in product management.  A...currently a Field-based Product Manager at Oxford Nanopore Technologies for the North America region. She...

...Harvard University. No matter whether you are a sports photographer at the Super Bowl, a medical technologist...the first fluorescence microscopy technique to incorporate optical sectioning. Optical sectioning refers...their fast movements. Depending on the sample, a temporal resolution on the millisecond scale may be needed...plane of excitation light that is thinner than the portion of the sample that is in focus. This technique ...resolution due to the lack of a requirement for high temporal imaging speeds. Standard fluorescence widefield...sectioning, imaging over large distances and high temporal resolution. Traditionally, this type of sample...technique for many of these samples due to its fast temporal resolution and decreased phototoxicity. Lightsheet...

...still many important questions in this exciting field of research. One of the most important: How will ...commute to and invade multiple organs, and most importantly escape the immune mechanisms of the host. The...against cancer. Sipuleucel-T by the Dendreon Corporation is currently the only FDA-approved DC therapy...therapies themselves can be made more effective. Some important factors researchers are beginning to focus on ... of cell types. This cell heterogeneity is an important element of successful tumor progression. A tumor...), and ability to metastasize. There have been reports of clinical trials going amiss and patient deaths...

...nonspecifically soak up your fluorophore package. One important concept in FRET experimental design is that the...donor first where labeling specificity is most important. Donor labeling and the need for specificity Donor...protein molecules contain the donor and, more importantly there is no fluorescence background resulting...in the ribosome, resulting in site-specific incorporation of the attached fluorophore. However, since ...chemical warfare [Vilensky & Redman, 2003]). Most importantly, these compounds cross cell membranes, thus enabling...Ebright, and Richard H. Ebright. "Site-specific incorporation of fluorescent probes into protein: hexahistidine-tag-mediated...protein kinase dynamics in living cells with FRET reporters." Methods 40.3 (2006): 279-286. PubMed PMID: 16908183...

...designated work areas and come with health risks. Importantly, with this method it is not possible to follow...azidohomoalanine (AHA). In the technique, AHA is incorporated into newly synthesized proteins instead of methionine...endogenous protein of interest that also had incorporated the AHA tag after pulse labeling of a determined...FUNCAT-PLA provides both quantitative and spatio-temporal information about the turnover of specific endogenous..."Puromycin inhibition of protein synthesis: incorporation of puromycin into peptide chains." Proceedings...Gabriel. "Inhibition by puromycin of amino acid incorporation into protein." Proceedings of the National Academy...

...changes. In addition to supporting in-person conference attendance, the award now supports virtual conference...research.   Abhi Aggarwal: Glutamate sensing reporters Abhi Aggarwal is a postbaccalaureate research ...third-generation intensiometric glutamate sensing fluorescent reporter (iGluSnFR3) to study glutamate, a signaling molecule...in pMinDisplay (PDGFR anchoring domain), in electroporated in rat hippocampal cultured neurons, imaged...hydropersulfides modulate protein activity and have important roles during oxidative stress. To monitor this...

...want to turn things on or off, permanently or temporarily, we’ve got you covered. Defining your control...requirements To pick the best tool for the job, it’s important to know what the job requirements are. Here are...inducible? How long do you need control? Will temporary control (a few days) be enough, or do you want...than making a knockout and is a great way to temporarily reduce expression of essential proteins (when...expression (more on this below) will both achieve temporary repression of protein expression. Addgene has ...via a custom gRNA, to your gene of interest to temporarily increase expression. There are several flavors...

... an ERK kinase translocation reporter with a mRuby2 fluorescent reporter (ERK KTR-mRuby2). The ERK KTR...control of kinase activity can provide greater spatiotemporal resolution than pharmacological or genetic ...everything downstream of MEK1), bypassing the upstream portion of the pathway and focusing on the downstream steps...protein of interest. If you plan to try this, it’s important to check that the function of the protein and ...Activity with GFP Resources at Addgene.org Find KTR reporter plasmids Find photoactivatable fluorescent proteins...

...workforce working group report. http://acd.od.nih.gov/biomedical_research_wgreport.pdf Resources at Addgene... Training (BEST) awards, which are designed to support new approaches that address the need to train our...Labor Market. On this topic it is clear that many important questions, areas for informed discussion, debate...University of Maryland College Park, is a strong supporter of increasing the number of postdoctoral scientists... collection and analysis. He is also a strong supporter of science Master’s programs. This will be the...

...Faculty of Sciences of NOVA University in Lisbon, Portugal. She leads the Glycoimmunology group based at ...glycans. Accordingly, she joined efforts with the Portuguese Association for Congenital Disorders of Glycosylation...PMM2 gene. Videira’s group recently reviewed and reported that PMM2-CDG patients suffer from immunological...wherein it's possible to delete the functional portion of the Nfix gene using Cre recombinase and to generate... construct NFIX expression vectors as well as reporters of NFIX function. They will then mutagenize the... the Glycoimmunology group at UCIBIO, FCT-UNL, Portugal. Her main work focuses on unraveling the pathophysiological...

...delivery include plasmid transfection, RNP electroporation, and viral transduction; but these methods ...Gag protein fused to a Flag-tagged SpCas9 is incorporated into Nanoblades, just like the unmodified MLV...MLV Gag protein. After Gag-Cas9 is incorporated, the MLV protease, supplied as part of the MLV Gag-Pol...into Nanoblades by association with the the Cas9 portion of the Gag-Cas9 fusion protein. A single Nanoblade...gene, while editing was not detected in cells electroporated with RNPs using the same guide. Nanoblades ...influence the level of off-target effects, with electroporation of RNPs generally having lower off-target edits...

...of the yeast TREX-2 mRNA export complex: components and anchoring nucleoporin. Lu et al. Plant J. 2010... (abundant in eukaryotes) A Near-Infrared BiFC Reporter for In Vivo Imaging of Protein-Protein Interactions...green-yellow or orange-red fluorogen A split fluorescent reporter with rapid and reversible complementation. Tebo....  Controls  When designing experiments, it’s important to include proper controls to assess the behavior...target proteins, especially if the termini are important for their interaction. You can read our previous...

... the known protein concentration. Importantly, you’ll want a portion of your standard curve to plot to...measured using a microplate reader, is directly proportional to the amount of enzyme present in the well ... If your standard curve does not have a linear portion or the OD of your experimental samples has reached...HRP-TMB reaction more quickly. Just make sure to incorporate the dilution factor in your calculation from ... you choose. Once you’ve identified the linear portion of your standard curve, you can calculate the concentration...

... your skills from the start. Just like with any sport, hours of practice are required and no one lab or... grant early! First off, and perhaps the most important bit of advice I can suggest, is to give yourself...Originality is essential for grant success The two most important things reviewers will be looking for in your research...from accounts or HR on the judging panel. It’s important for this person to get a clear and concise explanation...going to be an element of luck involved so it’s important not to throw in the towel if 1 or 2 funding bodies...submission panel. Some funding bodies provide opportunities to attend an overview of what they are looking...

...science advocates is an important way to push the balance from challenge to opportunity. We hope to see you...philosophy of academic science to date. Advocacy opportunities for the future No matter your opinion on these...scientific enterprise will provide challenges and opportunities for us all.  For example, implementation and...challenging. At the same time, there is much opportunity to make headway on long-term efforts to better...we are able to successfully advocate for the opportunities they present. Similarly, ongoing issues that...

...under-transferring.  Membrane pore size Membranes come in different pore sizes for different proteins:...to take. If you’re not sure if you need a smaller pore on your membrane, run a double-membrane transfer...membrane, that’s a good indication that the p membrane pores are too large.  Transfer method   Wet transfers ...increases (Figure 3A).  This step is especially important for highly expressed proteins, including “housekeeping...don’t want to skimp on your washing steps! It’s important to let them wash for the full amount of time, ...blot, you may want to consider using a different reporter or, if you don’t need to compare the protein amounts...Electroblot/dry transfer system Different membrane pore size Adjust SDS:alcohol ratio in transfer buffer...

...you’ll hopefully have the opportunity to do some great science in a supportive environment. Both paths ...on which company you choose. If publishing is important to you, make sure that your project will not involve...AstraZeneca, scientists who get postdocs and do not support or manage them well are simply no longer allowed...communicate your science to others and advocate for the importance of your work. In my experience in industry, you...paths will give you myriad opportunities to help you with the next step in your career.   Many thanks to ...

...but enhancers and chromatin looping play important supporting roles. Enhancers are cis regulatory DNA ...networks’ of 157 super enhancers.  3. Measuring temporal changes in chromatin interactions A third application...application for CAPTURE 2.0 is detection of temporal changes in chromatin conformation, like those that ...interactions. The chromatin story told by CAPTURE 2.0 is important, but it’s not the only plot line in gene regulation...Multiplexed capture of spatial configuration and temporal dynamics of locus-specific 3D chromatin by biotinylated...

...experiments didn’t provide any information on other important factors such as what is the best promoter to use... was far from an isolated case. Our scientific support team answers hundreds of AAV questions every year...successfully replicate published works. Of particular importance to these efforts is the open sharing of negative...Figure 2.) At the same time, when scientists who support open science, but do not share their own data -...experimental settings. It is a curated set of technical reports on AAV vectors from Addgene and the CLOVER Center...Center, each one submitted by researchers like you. Reports are reviewed by Addgene scientists for clarity ...

...vector with a reporter, such as a fluorescent protein, to visualize vector uptake. These reporters might be...tumefaciens, a plant-infectious bacteria that can incorporate foreign DNA into plant genomes. Eukaryotes are...stable transfection, DNA from the plasmid is incorporated into the host cell’s genome, so no special sequence...select for your vector. Some antibiotics block important cellular pathways shared by bacteria and eukaryotes...t common for simple transfection experiments. Reporters Finally, in some cases a researcher may choose...

...FLP-FRT. In addition, a variety of means to spatiotemporally control FLP and Cre expression have been developed...These differences in recombinase efficiency are important to keep in mind when deciding whether to use the... only occur at a specific time point? To allow temporal control, ligand regulated Cre and FLPe recombinase...control of Cre recombinase. After in-utero electroporation, this plasmid mix results in sparse labeling... brain area can be transfected by in utero electroporation. To achieve sparse labeling of individual neurons...instance) can be controlled with FLP-FRT. For further temporal control, Tamoxifen inducible Cre versions can ...

...genes important for mesoderm differentiation Now, let's turn tinman and bagpipe, two genes important for...protein Groucho, a transcriptional co-repressor important in development. Groucho family proteins are found...Dana Farber Cancer Institute! Sonic Hedgehog, an important regulator of embryonic development The largest...video game character but rather to an extremely important gene in animal development. To understand where... Tinman, 2016). Bagpipe on the other hand, is important in the formation of the visceral mesoderm which...

...tag. The MBP signal peptide allows the NB to be exported to the periplasm of bacteria where it can be secreted...the hPSC genome using AAV1 and contain an EGFP reporter. Expression of the dCas9-KRAB activator or dCas9...promoter. The gRNA piggyBac plasmids include a mRFP reporter. With this system, they could quantify and track...dCas9 targeting plasmids! Hazelbaker et al., Sci Reports. 2020. https://doi.org/10.1038/s41598-020-57500...Find the plasmids at Addgene! CRISPR RNP electroporation and AAV donor infection (CRISPR-READI) enables...GRABNE) sensors allow for sensitive and high spatiotemporal measurements of norepinephrine. These norepinephrine...

... are more likely to consider cover letters an important part of the application. Even within an organization...core skill, considers cover letters to be very important. On the other hand, our app/dev team rarely, if...that organization or group. This is particularly important for labs and nonprofits, who tend to prioritize... research], and have excellent [list the most important skills for the position]. I am enthusiastic about...about [something that you love and is important for the job]. Notice that the intros above allow you ...that they considered cover letters to be more important for higher-level positions. Many did not require...

...valuable lessons along the way. We think it’s so important that we have written plenty of articles on building...successful mentor relationships, asking for mentor support, and choosing your mentor. What isn’t discussed..., or a graduate student. This is the person you report or talk to on a daily basis who will help you get...bench mentor-mentee relationship is extremely important – without a good set up it could be very difficult...mentor learns where you are at and the level of support you need from them, and you can understand the ...Be My Mentor? Finding and Asking for Mentoring Support   ...

...Joanne as she answered questions to provide moral support and to monitor user replies, but I can’t thank ... basic questions and to provide a small bit of support (you are not alone!) for those going through the...see graduate and postdoc programs beginning to support diverse career path development--but the science...Finally, as I said in the AMA, it’s incredibly important to find good mentors and incentivize good mentorship...and your resources for science communication opportunities like an AMA. One of the best things an early...before, but I also highly recommend taking every opportunity you get to speak publically about your work (...

...of Different Methods for Generating iPSCs. One important note: in this table, reprogramming efficiencies...high is based on the reprogramming efficiencies reported in González et al. A percentage is not listed ... be used to package reprogramming factors. An important milestone of reprogramming is the silencing of...cells for the presence of transgenes is still an important control. Additionally, Sendai virus doesn’t rely...comprised of OCT4, SOX2, LIN28, NANOG, and a GFP reporter. Minicircles are removed from cells by dilution...Characterization Once generating iPSC clones, it’s important to determine how stem cell-like they behave. iPSCs... iPSCs Find gene-specific plasmids (including reporters and RNAi) for:  NANOG  OCT4 SOX2 MYC KLF4 LIN28...

... optogenetics is renowned for enabling precise temporal and spatial control. Optogenetics uses genetically... on and off gene expression while imparting spatiotemporal control, reversibility, and repeatability. ...system, Ye’s lab could edit a tdTomato fluorescent reporter gene in mice using Minicircle DNA vectors encoding...codon, TAG. (To learn more about site-specific incorporation of amino acids using pyrrolysl tRNA synthetase...seen in the figure below. Next, by using a dual reporter fluorescence assay, Hemphill et al. demonstrated...) A Singular System with Precise Dosing and Spatiotemporal Control of CRISPR‐Cas9. Angew Chem Int Ed 58...Rauen RR, Kahn SI, Tsang M, Deiters A (2020) Spatiotemporal Control of CRISPR/Cas9 Function in Cells and...

...Chapter 4: Crystal's approach to management and the importance of realizing that different people have different...learning from peers, and being receptive to those who report to you. Chapter 5: The types of science that Crystal...projects, and how she sets metrics for success. The importance of thinking about the big picture and not just... who are planning on working in industry. The importance of recognizing and leveraging your unique skills...skills and how they've helped her at Novartis. The importance of mentorship in developing career skills. Outro...

...the mitochondrial calcium uniporter (Martell et al., 2012). It is important to note that the biotin-phenol... of exogenous biotin allowing for more precise temporal control.  Find plasmids for TurboID and miniturbo... where two proteins are fused to complementary reporter proteins that can only assemble when in close ... ascorbate peroxidase as a genetically encoded reporter for electron microscopy." Nature biotechnology...proteomics approach to monitor the composition of spatiotemporally defined protein complexes." Nature communications...

...of interest and co-expressing a cytosolic ZtSH2 reporter, they observe fluorescence localizes to the plasma...And orthogonal sensors with different ITAM/SH2 reporter pairs allow multiplexed measurement of different...plasmids here!   Farahani PE, et al. pYtags enable spatiotemporal measurements of receptor tyrosine kinase signaling...10.1101/2022.08.13.503850.  iGECInano NIR calcium reporter Near-infrared (NIR) fluorescent proteins promise...photochemical characteristics than previous NIR calcium reporters iGECI and NIR-GECO2G, and its small size (~1500...sensors Whatever your analyte of interest, it’s important to choose a biosensor that has a binding affinity...

...infection, including both common infections and opportunistic infections not usually seen in healthy individuals... genome had been excised, leaving only a small portion of the LTRs joined together. Clonal Cas9/gRNA-expressing...populations were also immune to HIV-1 reinfection. Importantly, no off-target effects were detected. Kaminski...patients. Although this is an exciting finding, it’s important to note that these HIV-1 patients were ART-naive...Cas9. While these results are discouraging, it’s important to note that these experiments were done in cell...propagate and infect adjacent cells. It will be important to determine if CRISPR/Cas9 escape occurs in an...

...optimized, it’s important to ascertain how long the rescue phenotype will last, and more importantly, if it does...genetic disease in a postnatal mouse model, an important proof of concept for future preclinical and clinical...does not cause obvious germline modification, an important finding given the controversy associated with ...great step forward for CRISPR gene therapy, it’s important to realize that DMD is a simpler case than other...PMID: 26721684. 4. Reardon S. Gene-editing summit supports some research in human embryos. Nature. 2015 Dec...