... laboratory tool often employed to establish cell lines that stably express a gene of interest. While ...background of the lines you are using, it is best to start from scratch. Obtain a vial of the desired cell ...production and infection.     Great transduction start with great virus If having happy cells is the first...extreme changes in osmotic pressure and pH can destabilize the viral envelope. One drawback of the multiple...The effect of freeze-thaw cycles on lentiviral stability depends on a variety of factors such as the transgene...al. found that lentivirus produced at pH 6 are resistant to multiple freeze thaw cycles. Consequently, ...prep!) and infect. While this approach is a great start to a successful infection, one point to consider...

...new sample I always start by asking two questions: 1) Is the sample dynamic or static? 2) Is the sample ...acquired all fluorophores throughout the sample are constantly exposed to light, not just those in the focal...can a a high enough density of red-photons be establish for this to happen.Using red light has a second...within the depth of field of the objective (the distance in the axial dimension that will appear in focus... effects of high light doses to the cells. Thin static samples Ex: Fixed monolayers of cells or thin (... requirement for high temporal imaging speeds. Standard fluorescence widefield microscopy can be combined...require both optical sectioning, imaging over large distances and high temporal resolution. Traditionally, this...

...conclusion. Advice for Telling a Good Story Start at the start Any good story needs a solid point to kick...at. Whatever you’re working on, start wherever it makes sense to start and try to be as chronological ...almost all of us are programmed to appreciate and understand. We build narratives around the self, too — ...author. When I raised this, he was pretty angry and started arguing with me. First I calmed the situation down...for all of us.” This is a classic example of the STAR (Situation, Task, Action, Result) interview technique...and engage with and what parts you can leave out. Start viewing your work in the form of a story, and you...telling it. End at the end A story needs a good start point and it needs a good end point. One thematically...

...contributed by guest blogger Joachim Goedart, an assistant professor at the Section of Molecular Cytology...interference is known as steric hindrance. For instance, GFP may occlude catalytic sites or obstruct binding...Organized Smooth Endoplasmic Reticulum (OSER) assay (Costantini et al., 2012) measures homodimerization of FPs...This assay is quantitative and is currently the standard for assessing oligomerization in cells. In addition...protein characteristics such as the brightness, photostability, maturation time, and acid sensitivity of various...parts of the fusion from N- to C-terminus. For instance, Lifeact-mTurquoise2 (Plasmid #36201) or mScarlet-tubulin...blogger, Joachim Goedhart! Joachim Goedhart is an assistant professor at the Section of Molecular Cytology...

...conformational states: bent-closed, extended-closed, and extended-open. In the two closed states, integrins...its open state, activating the receptor. Ligands can bind to integrins in their closed states (A) and ... ligands with high affinity. This extended-open state is key to strengthening ligand binding, mediating...in the extracellular environment. This further stabilizes the extended open integrin conformation. During...B) but with much lower affinity than their open states (C). Figure courtesy of IPI. IPI anti-integrin...rat or mouse variable regions fused with rabbit constant domains. All bind specific integrin alpha subunits...recommended for use in flow cytometry. Based on the established applications of the original hybridoma monoclonals...

...feelings, low morale, poor decision making, and stalled projects. You want to identify and address these...also be categorized as either “personal” or “substantive”. A personality-based conflict occurs when two...a lab or work environment if left unchecked. Substantive conflict occurs when people or teams who normally...”) when in fact many sources of conflict are substantive (“Person C told Person A one thing and inadvertently...in my current role revolve around missed tasks, stalled projects, and disagreements over prioritization...through multiple channels is not reasonable or sustainable. That said, when you know that a person or team...can indicate confusion around... Ownership: Misunderstandings about who is responsible for what tasks or...

...post-translational modifications, identifies a staggering ~250,000 phosphorylation events in the proteomes...proteomes of higher mammals (2). How can we begin to understand the importance of any of these phosphorylation...not reversible, and thus cannot reflect the true state of protein modification within the context of its...difficulties with kinase specificity in vitro. The establishment of a system in which purified, recombinant protein...provides researchers with a valuable tool for understanding the contribution of phosphorylation to enzyme...and second, through a gel shift assay using the PhosTag system (see below). This plasmid backbone can also..., one should optimize protein production using standard protein purification strategies; expression should...

...postdoc salaries as opposed to Staff Scientists Increase the Starting Salary for Postdocs to $50,000 ...symposium will prove incredibly important to the stability of the biological research enterprise going forward...need to know in order to even assess the current state of research became more clear throughout the various...information about the postdoc work force, problems start with the question of “What is a Post-doc?” The National...cost-effective for PI’s to pay postdocs as opposed to staff scientists. This results in a glut of postdoctoral... De-incentivize going into PhD programs as the standard option for scientists by requiring graduate students...$50,000 (the current median postdoc starting salary as reported by Paula Stephan is $40,000) Increase Knowledge...

...Macaques N/A Stanton et al., 2023 AAV-MDV1A CNS Mice No (LY6C1) Stanton et al., 2023 ...The blood-brain-barrier (BBB) exists to prevent substances in the blood, like proteins and viruses, from...into the brain. While helpful from a biological standpoint, it makes delivering AAVs to the brain difficult...interest, and health. While it’s always helpful to start with a literature search to see what others have...considerations you’ll need to be aware of before starting an experiment. Controlling expression As systemic...sensors, CRISPR elements, etc... *These are only starting recommendations; you will still need to experimentally...are a good fit for your experiment, it’s time to start selecting and testing systemic capsids! Figure 2...

...Kostman.   Important considerations before you start an experiment with CRISPR The hammer, the jigsaw... an alternative ATG downstream of the annotated start codon. Likewise, we avoid target sites that code...undertaken lightly! Indeed, truly achieving the gold standard requires not one but two rounds of single cell...in a 5 - 10 nt window relative to the PAM, and bystander edits are possible if there is another target ...target a ~100nt window upstream of the transcription start site (TSS), while for CRISPRi, a ~100nt window downstream...the target (e.g. accessibility due to chromatin status). No computational prediction is ever perfect, ...nevertheless still lead to activity has been established, and can be used to identify sites that are likely...

...$47,476 per year, under updates to the Fair Labor Standards Act (FLSA). This was due to the threshold at which...to now - across a wide consensus of academic stakeholders. However, while most institutions are indeed...the lapsed mandate (or are being compelled at the state level, in the case of some public institutions) ...do so. Immediately after the injunction, as we started to hear of institutions cancelling plans to raise...their raise were: postdocs at institutions like Stanford and the Whitehead Institute, where salaries had...continuing to reach out to institutions with a statement indicating that we encourage them to keep raising...across geographical regions When we originally started gathering this information, many assumed that those...

...academic scientists are starting to see the advantages of using social networking to start, build, and track...tell when some start-ups were about to be bought by seeing certain groups of people start to update their...my seven best tips beyond the basics to get you started. 1) Be visible and open to connections This is...add a personal note, you waste an opportunity to start things out right. Also, many people on LinkedIn ...include news, inclusion, culture and connections. Start your interview research there! 6) Your LinkedIn ...good or better than your resume Don’t make the mistake of having a shoddy profile and not updating it....changes. If you are working, and all of a sudden you start updating your profile, everyone will think you are...

...recognize the constant region. Nonspecific binding, caused cell receptors for the constant region, is also...about an affinity reagent, they’re talking about a standard antibody: An ~150kD protein with both light and... consistency in the final product. Because the constant (non-epitope binding) region is the first thing...connected by a short linker peptide. The lack of constant region makes them less immunogenic, but they have... protein nanostructures. Diabodies readily co-crystalize, a major advantage over antibodies (Kwon, 2019...signals due to their monovalent nature. They are stable up to 80°C and can refold correctly after denaturing...domain as their scaffolding instead of the antibody constant region. They are generated using combinatorial...

...antibodies). By the end, you’ll have a better understanding of what makes polyclonal antibodies unique and...protein. Whereas production of monoclonal antibodies starts with selecting just one of these B cell clones ...immune response. Once the initial antibody response starts to wane about a month later, the animal is given...target protein. While this can increase background staining (see next section), it may be advantageous if ...protein in low abundance or whose conformational state may change. If your protein might become slightly...greatly increasing signal amplification.  When to stay away from polyclonal antibodies The most prevalent...might experience variability in the strength of staining and background noise. If you do use different ...

...increases when target DNA strand separation is stabilized (2,3). Stable strand separation is maintained by 2 sets...HF1 each stall in an inactive form after binding to a target site and propose that this stalling leads to...biological disciplines, the base technology itself is constantly improving. Cas9 variants have been modified for...and the negatively charged DNA and therefore destabilize basal strand separation. This decreased strand...separation and target DNA binding to the Cas9 nuclease. Stable strand separation is maintained through interactions...mutations in the mobile Rec3 domain facilitate this stall. With this new found structural knowledge, in collaboration...publication at Addgene. 2. Nishimasu, Hiroshi, et al. "Crystal structure of Cas9 in complex with guide RNA and...

...MMLV-derived Retrovirus Integrating High Low Efficient and Stable Genome integration Insertional mutagenesis, transgene... Lentivirus Integrating High Low Efficient and Stable Genome integration Insertional mutagenesis, residual... vectors: This approach is the same as using a standard lentiviral vector, but transient expression of...for the oriP/EBNA1 plasmid to be maintained as a stable extrachromosomal piece of DNA. Reprogramming efficiency...large nucleoli, and little cytoplasm. Positive staining for alkaline phosphatase, expression of embryonic... the ability to generate teratomas is the gold standard instead. Teratoma formation requires injecting...two categories: 1) studies focused on better understanding the mechanisms of reprogramming, and 2) studies...

...connections to the starter cell, RABV was modified to only infect user-defined starter cell types, as well...’s architecture and plumbing are important. We started with learning the head is connected to the neck...the whole brain, it first infects one cell (the “starter cell”). It then replicates within that cell, and...retrogradely infect neurons that project to the starter cell. Once this first order infection occurs, subsequent...). Based on this, cell type specificity of the starter cell can be achieved by using EnvA-pseudotyped ...selectively supplying the G protein in trans only to starter cells (infected with G-deleted RABV). In this case... case, G-deleted RABV will infect the starter cell. RABV replication will then occur and the G protein...

...Hurt. Understanding how individual parts can be assembled into TUs is key to understanding MoClo. By...another optimized platform. Gene Stacking As the name implies, gene stacking is the use of a combination of...transcriptional units, and multigene constructs. Let’s start by examining individual parts and transcriptional...plant biology to achieve traits such as disease resistance. MoClo is a great tool to deliver many combinations...MoClo Interested in using MoClo? You don’t have to start from scratch! As you can imagine, there is an array... many different MoClo kits have already been established. Keep an eye out for our next blog on choosing... Marillonnet S. A modular cloning system for standardized assembly of multigene constructs. PLoS One. ...

...a primary research article, it’s important to understand that presenting your work in poster format differs...figures. Next, the poster should be legible from a distance. This means big font and fewer words. Further,...those sections. This strategy helps your sections stand out, and makes the entire poster more navigable....templates with different column numbers for a good starting point. Adobe also offers free poster templates...display your data most prominently, making it the star of your presentation. Portrait. This style displays...catchy because it is generally less common and will stand out; however, be careful not to hang your poster...Morrison, a psychology doctoral student at Michigan State University. The idea is that the main research finding...

...to minimize rearrangements such as NEB Stable or use a standard strain, like DH5α, grown at 30 °C instead...save you time, and allow you to standardize your experiments later. Stay tuned to the Addgene blog and ...that have helped me troubleshoot some technical obstacles when working with virus: 1. Viral vectors undergo... to a target cell by reducing the repulsive electrostatic forces between these two negatively-charged ... will be selectable with the antibiotic whose resistance gene is encoded in the viral vector. This antibiotic... may be some low-level fluorescent protein or resistance marker expression following transfection, higher-level...Central PMCID: PMC84922. 5. Ugai, Hideyo, et al. "Stability of a recombinant adenoviral vector: optimization...