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...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...

...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...

... 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...

...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...

...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 ...

...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 ...

...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...

...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...

...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...

...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   ...

...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 ...

...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 (...

... 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...

... 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...

...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...

...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...

...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...

...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...