...signaling is likely to have). Anterograde tracing outlines neurons from their cell bodies to the terminals...terminals of their axons; while retrograde tracing outlines neurons in the opposite direction, from the terminals... dynein, while anterograde transport relies on kinesin. Different rates of speed for various forms of ...predominately anterograde tracing, biotinylated dextran amines (Veenman et al., 1992) or fluorescent molecules... achieved by using optogenetically controlled transgenes, in combination with conditional transgenic mice... M., and Schnell, M. (2008). Concepts in the pathogenesis of rabies. Future Virol 3(5), 481–490. PubMed...Ho, A., Nicholas, E., Hirokawa, K.E., Bohn, P., Joines, K.M., Peng, H., Hawrylycz, M.J., Phillips, J.W...

...producing landslide after landslide, only adding more stones to the field. Fortunately, our bodies have been... (Th) cells. These cells produce inflammatory cytokines - interleukin (IL)-12 and interferon gamma (IFNγ... promotes the production of VEGF to increase angiogenesis around the tumor. This in turn regulates the...cancer. Surgical removal causes increases in tumor cytokines like TGFβ and IL-6. However, when strengthened...response. Antibodies targeting the tumor-augmenting cytokines produced in the tumor microenvironment TGFβ, IL...necroptotic tumor cells have also been tested as vaccines and therapeutics. Their uptake by DCs is increased...marker blockade and suppression of tumor-aiding cytokines, for example, will more broadly activate the immune...

...are tumor microenvironments made of?  A tumor’s genesis is divided into 3 phases: initiation, progression...active regions, microenvironments shape a tumor’s evasiveness, evolution, and response to treatment. Some microenvironment...transport miRNA from drug-resistant cells to sensitive ones. Though this process is not fully understood, a ...formation by inhibiting cell growth, invasion, and angiogenesis (Allinen et al., 2004). In fact, Sanford H. ...influence tumor microenvironments: immunology and angiogenesis (Witz et al., 2009). Then, scientists began ...Fernando Schmitt. "Microenvironment in breast tumorigenesis: Friend or foe?." Histology and histopathology...

...experiments. Test dTAG molecule toxicity in new cell lines Treat parental cells (or cells expressing a control...FKBP12F36V-fusion chimera of interest. In the cell lines we have evaluated to date, we observed little to...FKBP12F36V fusion chimera degradation. In the cell lines we have evaluated to date, we have not observed ...was employed to evaluate degradation of ENL. Cell lines were engineered to express ENL-FKBP12F36V and endogenous... employed to evaluate degradation of MELK. Cell lines were engineered to express FKBP12F36V-MELK and endogenous...was employed to evaluate degradation of YY1. Cell lines were engineered to express YY1-FKBP12F36V using ...

...either through a chromogenic or chemiluminescent reaction. Chemiluminescence, which is light produced by ...the primary amine groups from lysines, sulfhydryl groups from cysteines, or carbohydrates from glycosylation... found throughout the antibody. 2) Endogenous cysteines, such as those that make up the disulfide bonds...antibody (Mueller, et al., 1988). If some of those lysines are in the antigen binding region or even just ...

... to, ones targeting muscular dystrophy, cardiovascular disease, inflammatory diseases, blindness, and ... cancer treatment by traditional gene therapy pipelines (viral), and researchers are currently working...many more in the earlier stages of the pipeline, ones that have experienced hiccups (either clinically...challenges with therapies involve patient safety, effectiveness, and delivery. Patient safety is important for...concerning when using a systemic approach. Regarding effectiveness, if the editing frequency isn’t high enough ...

...stripping to remove, so start with the weaker binding ones first to preserve your sample. Pro tip! Because ...reprobing works best with western blots that use chemiluminescent or fluorescent detection, like horseradish...protein retention. Following this step, most PVDF membranes recommend re-wetting with methanol before rinsing...the membrane you are using. Dry nitrocellulose membranes are brittle, so proceed with caution. Unlike PVDF...your own stripping solution using a recipe like the ones shared by Bio-Techne, ThermoFisher, or Millipore...remains, whether that involves adding fresh chemiluminescent substrate or imaging your fluorescent probe...

...into ‘split-vector systems’ by separating viral genes and transgene expression across several plasmids...that allow functional screening of thousands of genes in genome-wide experiments. Gamma-retroviral vs...direct the cellular RNAi machinery to silence target genes. This empty MuLV vector from the Weinberg lab enables...find more lentiviral expression vectors (empty backbones) here. Cloning and expressing new shRNA sequences...lentiviral backbone from the Zhang lab expressing S. pyogenes Cas9 and a gRNA cassette. It can be used to produce...studies. The ability to simultaneously alter multiple genes via infection with a single ecotropic or amphotropic...

...expression yielded editing of 80–90% in human cell lines and primary cells. Although cell entry of a Cas9...minimal toxicity, including in clinically-relevant lines such as CAR-T cells. Plus, this method is fast! ...break up the dimerization without losing that brightness and stability. Here’s a quick overview of these...variants:   Figure 2: Inner mitochondrial membranes imaged with structured illumination microscopy.... This variant has similar photostability and brightness compared to the original, though its maturation...versions. Thanks to Christophe Leterrier, whose FocalPlanes piece on mStayGold inspired and informed this...

...So how can you use CRISPR to introduce large FP genes into C. elegans? By using a split FP approach. Split-wrmScarlet... fluoresce. The team engineered C. elegans cell lines to express wrmScarlet1-10 unattached to any other... validated this tool by targeting six different genes whose protein products have distinct localizations... library that targets human glycosyltransferase genes and other proteins related to cellular glycosylation...deletes human alternative cassette exons and disrupts genes with alternative exons. Get the library from Addgene... format! The library inhibits over 18,000 human genes. Find the lentiviral preps at Addgene hSyn1-SIO-stGtACR1...

... constructs for tagging the kinesins KIF5A and KIF5B as well as backbones for generating other SNAPf-,...the conformational dynamics of the motor protein kinesin-1, the Twelvetrees Lab took advantage of the self-labeling... will be useful not only for future studies of kinesins but for many other proteins of interest too! Find....A.S., Craggs, T.D., & Twelvetrees A.E. (2024) Kinesin-1 is highly flexible and adopts an open conformation...mouse monoclonal antibodies targeting the human genes and are recommended for use in immunohistochemistry...

...Joining (NHEJ) DNA repair mechanism to knock out genes as a result of an insertion, a deletion or a frameshift...-transformed cells yielded rates of targeted mutagenesis similar to those achieved by isogenic AdVs encoding...Homology Directed Repair (HDR) to knock in corrective genes. For this you would need to provide a DNA template...successfully developed and used AdV vectors to edit genes in several biological systems. It has been used ...plasmids at Addgene: pAdSh.PGK.Cas9 (expresses S. pyogenes Cas9 from the PGK promoter) and U6 promoter-driven...CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse array of human cells. Sci Rep 4:...

... (24-30°C). Brightness: Brightness is a measure of how bright is the emission. Brightness is calculated...contributed by Gal Haimovich of greenfluorescentblog. Be honest. Do you really know how fluorescent proteins glow...the protein, divided by 1000. In many cases the brightness is compared to that of EGFP which is set as 1...proteins are very dim (e.g. TagRFP657, which has a brightness of 0.1) and this should be taken into account...

...namely that they can cause cancer via insertional mutagenesis (which HIV does not do). Furthermore, molecular... are, in most cases, interested in transferring genes to mammalian cells (i.e. transferring DNA), yet ... is (thankfully) gutted of many essential viral genes. How it is, exactly, that heaps of viral vector ...the only component that is not dependent on viral genes. It is derived from the host/producer cell membrane...full “life cycle” of expression of selfish, lazy genes of a non-living viral particle, and how we as dedicated... N. (1971). Noninfectious ARK mouse embryo cell lines in which each cell has the capacity to be activated...

...changing guidelines and regulations impacting grant management, we track grant opportunity deadlines, and ...organization to evaluate the organization’s grant readiness, identify grant opportunities, secure grant awards...schools, healthcare facilities and with private businesses. How do you get the expertise needed to enter...such as research, collaboration across multiple disciplines, writing, project management, and program evaluation...process or the whole process, including grant readiness, funding opportunity research, application writing...

...that recognize codons found in highly expressed genes (Emilsson and Kurland, 1990). Controlling gene expression...changing conditions - Organisms often need to express genes at different levels under different conditions. ...specific tRNA pools. For example, tRNAs used in genes encoding amino acid biosynthetic enzymes may be ... the problem of codon choice is to resynthesize genes in such a way that their codons are more appropriate...properly. You may be able to avoid getting your genes of interest codon optimized by ordering plasmids...use the same expression system for many different genes without having to create new constructs. However...

...love with biology because of an image that was honestly quite boring. My Bio 101 professor displayed a...mammalian vectors are even less similar to bacterial ones. The mammalian analogue of bacterial transformation...uptake, but this approach requires compatible cell lines that express viral machinery. Alternatively, some...selection is useful for producing stable mammalian cell lines, but it isn’t common for simple transfection experiments...by transformation and transfection and left the business of transduction to our Viral Vectors 101 series...Resources on Addgene.org Collection of empty plasmid backbones ...

... transition Base change between purines (A ↔ G) or between pyrimidines (C ↔ T).   Base transversion ... ABE would allow it to act on newly-deaminated inosines. Pro tip! The nucleoside inosine consists of the...this promising start, the team used rational mutagenesis to produce the adenine transversion editor AYBEv3...and ABEs, they could be useful for saturation mutagenesis, and they provide a strong proof of concept for...empty space. Over several rounds of rational mutagenesis, the team progressed to gGBEv6.3, which was capable...

...in mouse embryos to generate gene modified mouse lines. Hopefully this post will help get your in vivo ...that later). Find Validated gRNAs to Target Mouse Genes To begin the in vitro screening process, you should... at the same time, and choose the most effective ones to increase your chances of gene modification in...a rapid acceleration in the production of mouse lines that could be used to study a wide variety of phenomena... et al. "Feasibility for a large scale mouse mutagenesis by injecting CRISPR/Cas plasmid into zygotes....generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering." Cell...

... the speaker and offer your business card (yes, you should have a business card; it’s a bit more informal...smaller talks within the bigger conference, especially ones that are more career-focused. After these talks,... will probably be willing to accept your CV or business card. Be warned, though – someone accepting your... formally apply for the position. This post on Business Insider nicely summarizes good job fair practice... Reading and Outside Resources http://www.businessinsider.com/how-to-get-the-most-out-of-job-fairs-2014...

...activators and repressors to target genes. Repressing target genes using dCas9-based repressors Early ...repression or knockdown allow you to study essential genes whose knockout would cause cell lethality. The Krüppel...purpose. dCas9-KRAB plasmids for repressing target genes in a variety of species and cell types can be found...found on Addgene’s website. Activating target genes using dCas9-based activators The simplest of the dCas9...transcriptional activator. Plasmids for activating target genes in a variety of species and cell types can be found...

...engineering pipelines, we selected Nextflow, a workflow management system for scientific pipelines, including... existed who had created a shared resource of pipelines for genomic data. Thanks to the flexibility inherent...separate Nextflow process. In Nextflow, a process defines which command or script will run and can be thought... connect each subsequent process to the existing ones. Once our first process was working as expected,...check, we repeated our strategy and copied a few lines of our existing script into the section of the next...

...job it is to disagree with the group. They are the ones who ask for rationales behind ideas and decisions...fill out the form at any time and a member of our Business Development team would review it. This survey ...frequently observe in a scientific setting.  Powerlessness It’s hard to be creative when you don’t believe...ideas obviously worth pursuing, evaluate the “bad” ones. Bad ideas are easier to work with than nothing ... mistakes that kill your productivity. Harvard Business Review. Retrieved January 14, 2022, from https...exercise to spur innovation on your team. Harvard Business Review. Retrieved January 14, 2022, from https...

...Commercial Plasmid Prep Kit Amount 1000 Lanes 1000 Lanes 1000 Lanes Cost $200-$500  <$10 $20-30 Cost...commercial ladders cost between $200-$500 for 1000 lanes worth of ladder. The Penn State ladders are significantly...prep protocol, it cost less than $10 to make 1000 lanes worth of the 100 bp or the 1 kb ladder. Using a ...

...productive discussion first defines the material resources needed, then determines how much those resources...possible, this even includes clocks, timers, and phones.  As for reagents and consumables (such as pipet...time to decide what resources to keep, and which ones to abandon. It’s critical to make these decisions...1. First make decisions based on experimental timelines and specific aims. 2. Then make decisions based...productive and manageable laboratory environments. His business skills enhance his passion for science. Follow...

...I really brought to the table was empathy, a willingness to listen, and an ability to remember the right...amount of conflict is actually critical in the business world. Companies and organizations that avoid ...involved, and also for the labs, organizations, or businesses as a whole. When I’m attempting to resolve a ... actively runs away from it. That can mean a willingness to make yourself temporarily uncomfortable for...while giving people the tools to resolve necessary ones on their own is even better. The Thomas Kilmann ...conflict resolution based on various levels of assertiveness and cooperation: avoiding, competing, accommodating...

...526) that (1) enables efficient knockout (KO) of genes in immune cells; (2) is not immunogenic, a necessity... optimize conditions for a screen that assessed genes that regulate antigen-specific CD8+ T cell responses...screen, we used the pRDA_526 vector to knock out 30 genes putatively involved in CD8+ T cell responses to ... Ptpn2, Pdcd1, and Zc3h12a, demonstrating the robustness of the system and utility of the pRDA_526 vector...introduction of the model antigen, Ovalbumin, into cell lines to study antigen-specific responses. Find FITS plasmids...

...transcriptional effectors (LITEs) designed to bind specific genes and turn them on or off, literally at the flip of...are able to react and learn. All of this requires genes to be regulated dynamically.” To understand how ... tools to manipulate the expression of specific genes in the brain in reversible fashion. And that’s exactly... of living mice both quickly and significantly. Genes under LITE control showed a rise in transcription...researchers applied the LITE system to 28 different genes to establish it as “a novel mode of optogenetic ...

...focused on choosing a lab, but almost all of these guidelines can apply to any workplace or job. It amazes ...will need is about you. It’s worth taking time to honestly evaluate your own style of work and communication...below, the first question to ask yourself is “which ones matter to me?”  and hone in on the factors that ...Overwork – including undue pressure, impossible deadlines, unnecessary disruptions Destabilization – including...author order PI lets lab members fight it out PI intervenes if there are interpersonal conflicts PI has no...can be very helpful in uncovering strengths and weaknesses of the options. Use this blog as a guide. Start...

...and many plant scientists want to transfer large genes, smaller binary vectors with a larger cloning capacity...than that from the commonly used 35S promoter. Transgenes are also efficiently inherited in both transgenic...expression. Sahoo et al. have tested the system with genes up to 4 kb in length and confirmed successful protein... assays as well as stable expression of foreign genes in plants." PLoS one 9.6 (2014): e98988. Pubmed ...Plant Synthetic Biology Quick, Versatile Plant Transgenesis with GreenGate Plasmids Interview: Nicola Patron...

...dictator or laissez-faire; theory or practice; business-like or artistic; tech, anti-tech or pragmatic...employers love PhD students to have: working to deadlines, writing up clear and attractive reports, being...students and postdocs go on to work in patent law, business development, teaching, etc. Church also encourages...science and engineering (STEM) expertise in law, business, journalism, entertainment, politics, K-12, etc... passion, hard work, collaboration, and staying honest with personal goals. Stay tuned for the follow ...

...explosion SUperNova," SunTag can help you turn up the brightness in your fluorescent imaging experiments. Fluorescent...managed to track run lengths of the motor protein kinesin across microtubules. Tanenbaum et al. then tested...to traditional fluorescent proteins Improved brightness and signal-to-noise ratio Less chance of phototoxicity...used to improve CRISPR-based activation of target genes, but we’ll save that application for another day...on Addgene.org Find Fluorescent Protein Empy Backbones Visit Our Fluorescent Protein Guide Page Find ...

...naturally occurring genes for cloning purposes (antibiotic resistance genes, toxin genes, and the sacB gene...gene of interest, but others will uptake plasmid backbones that re-ligated back on themselves. Therefore,...Plasmid cloning: Creativity with naturally occurring genes  At this point in the blog post, you may have realized...

...both introduce genes into an organism’s genome (see figure) and to disrupt endogenous genes at the site ...by transposition assays of antibiotic-resistance genes conducted in HeLa cells). In their Nature Genetics... the rapid generation of stable transgenic cell lines which can robustly express your gene-of-interest...transposons rapidly generate stable transgenic cell lines. Biotechnol J. 2015 Feb 4. doi: 10.1002/biot.201400821...

...research project must have innovative yet attainable milestones that contribute to intellectual thought and discovery... and skills do you need to use to reach these milestones? Can you get assistance from people who have ...Think about what your personal goals are and set milestones along the way so that you don't get stuck doing...been developed. Can you think of a project that combines an exciting new method or approach with another... Passion fuels the drive to continue persuing milestones and working hard. Being patient, being selective...

...validated simultaneously in the brain and the intestines of mice. The result? A very cool tool to advance...polymers are heated up and pulled to a desired thickness. The fibers are drawn with microelectronics included...engineering road map of sorts, giving an idea of the stiffness the fibers would need to be, as well as other ...fiber implants. Due to the soft nature of the intestines and the requirement for digestive motility to...excited a class of enteroendocrine cells within the intestines known as neuropods. They observed increased vagus...

...Now that we know all about antibiotic resistance genes, let’s consider another basic element of any plasmid...focused only on those found in bacteria. Some common ones you might see include ColE1, pMB1 (which comes in...20-40 CloDF13 (CDF) D Relaxed This table defines common cloning vectors, their copy number, ori,...copies of plasmids if they contain large inserts or genes that create a toxic product. The E. coli strain...for single- and multiple-unnatural amino acid mutagenesis in Escherichia coli. Biochemistry. 2013 Mar 12...

.... Yet, we cannot prosper in a world where our medicines, technologies, defense, and governing policies...more scientists in our leadership, and raising awareness regarding the pitfalls of accepting alternative...chromosomes. Montreal, QC, Canada 45.5017°N 73.5673°W – Vanessa Sung Hundreds of scientists and supporters in ...well as allies outside the scientific community. Vanessa Sung is currently a PhD student studying breast...restorative justice.  Follow her on twitter @sung_vanessa. Albany, NY, USA 42.6526°N 73.7562°W – Nicholas...denying science, from celebrities falsely claiming vaccines cause autism to politicians saying that climate...

...mice Transgenic mouse lines have also been engineered to express various opsin genes [7-9]. The advantage...other tool in a scientist’s vast toolbox, its effectiveness depends on how and why it’s used. There are ...behavioral output is sufficient to assess the effectiveness of your optogenetic manipulations (though a ...has genetically manipulated a number of the opsin genes to alter the light spectrum for activation and the...advantage of using these lines is, if one is crossed with an appropriate mouse line expressing a particular ...

...Julio Ernesto Valdivia Silva, Universidad de Ingeniería y Tecnología (UTEC), Peru Julio Ernesto Valdivia...her current research, Sheen aims to identify new genes related to PZA-resistance by using different approaches...experience in the field of bacterial and fungal pathogenesis. Her research focuses on a collection of pathogenic...growth will then be measured to link the deleted genes to specific functions in biofilm formation. The...