We narrowed to 1,042 results for: URE

...the ability to easily cure cells of the plasmid after integration. Figure 2: (A) Streamlined VchINT...species not previously cultured or isolated (Rubin et al., 2020).  Figure 3: Multiplexed insertions...transposon end sequences (R and L ends).  Figure 1: (A) Components involved in each type of CRISPR-Transposon...produce off-target DNA insertions and intermediate structures known as cointegrates (Vo et al., 2020; Strecker...achieved with VchINT, at two different incubation temperatures. Data adapted from Vo et al., 2020. In E...targeting by a transposon-encoded CRISPR–Cas system. Nature 577:271–274 . https://doi.org/10.1038/s41586-019...–Cas systems direct RNA-guided DNA integration. Nature 571:219–225 . https://doi.org/10.1038/s41586-019...

...science communications: podcasts, blogging, videos, lectures, exhibits, social media… how do you choose? Which...Them All, consider the following four questions to figure out which method(s) work best for you, your audience...-speed internet. Before you begin any work, make sure you know if your audience will be able to access...never know what's going to work. After all, a stick-figure internet comic about math probably didn't sound...needed. And since we live in the digital age, make sure to research any software you may need: cost or freeware...accessible or most appealing to you. And if you’re not sure what all the possibilities are, check out our table... useful for data-focused stories. In-person lectures, presentations, etc… Low (except for space) Real-time...

...proportion of virulent S. aureus from 50% to 11% in just 24 hours.   Figure 1: Single-phage strategies...targeting sequences found only in certain bacteria (Figure 1). Citorik et al. targeted beta-lactam and quinolone...al. used a phagemid system to target virulent S. aureus, a common hospital-borne infection. Two rounds ...system to resensitize antibiotic-resistant bacteria (Figure 2). The first phage introduced is lysogenic, integrating...resistant bacteria have previously flourished.   Figure 2: Two-phage strategies to resensitize bacteria...CRISPR, we’re turning the tables on the bacteria. Future research will show if this new strategy can help...using efficiently-delivered RNA-guided nucleases.” Nature Biotechnol. 32(11): 1141-5. (2014). PubMed: PMID...

...infection with S. aureus (red). You can then see neutrophils swarming around S. aureus and eating the bacteria...largely absent in adult organisms, where tissues structures are more stable and local movements predominate...than the static microscopy images of chemically coloured lymphoid organs did in the past. How do researchers...electronic transition in a fluorescent molecule (see figure to the right). The excitation generated by these... a chosen focal volume and thus the microscope captures only fluorescence coming from this volume. The...environments. These studies provide quantitative measurements of cellular motility, interactions, and response...peripheral interfollicular region of lymph nodes." Nature immunology 9.2 (2008): 155-165. PubMed PMID: 18193049...

...minutes to point me in the right direction for my future work. She graciously took me for coffee and spent...conference for any reason was a Singleton. Figure 1: The Binning Singletons logo. Image: James et...that binning Singletons together would provide a structure and support network for people attending large...improvement. We will make a greater effort in the future to match people within similar fields (ASM Microbe...that all participants would benefit from more structure with regard to the Mentor-Singleton relationship...addressing. Clear expectations have been established for future participants regarding the activities and time ...scientific community can improve the diversity and culture of science. We want Binning Singletons to be another...

...requires stringent biosafety measures, thereby increasing the costs of the procedure. Episomal reprogramming...fibroblasts, which require a more invasive skin biopsy procedure. This becomes a major benefit when recruiting ...reprogramming success [1]. Fibroblasts need to be cultured and expanded in vitro for several passages to ...collection [2]. PBMCs display unique epigenetic signatures that are closer to hiPSCs than age-matched fibroblasts....03%) [3, 9]. In 2011, Chou et al. generated a mixture of two vectors that encoded six factors - OCT3/... system generated by the group is an optimized mixture of three episomal plasmids (Table 1) encoding the...cells with unique epigenetic and gene expression signatures. Cell Res, 2011. 21(3): p. 518-29. PubMed PMID...

... at the nucleotide level but share an overall structure of 13 base pair palindromic repeats separated ...efficient The first version of FLP discovered has a temperature optimum of 30 °C and is therefore inefficient...led to the identification of FLPe which has a temperature optimum of 37 °C (Buchholz, Angrand, and Stewart...Visualizing single neurons in the living brain   Figure 1: Plasmid mix to label neuronal morphology (eYFP...transfection efficiency. However, if the cellular structure of a neuron and its synapse should be visualized...Combing FLP and Cre for additional control   Figure 2: Expression of a morphological marker (eYFP) ...FLP Recombinase Evolved by Cycling Mutagenesis.” Nature biotechnology 16(7): 657–62. PubMed PMID: 9661200...

... Staphylococcus aureus Cas9. Nature 520:186–191 . https://doi.org/10.1038/nature14299  Senís E, Fatouros...bacteriophage and plasmid DNA. Nature 468:67–71 . https://doi.org/10.1038/nature09523  Ibraheim R, Song C-Q, ...SI, Dohmae N, Ishitani R, Zhang F, Nureki O (2014) Crystal Structure of Cas9 in Complex with Guide RNA ... pool of shorter orthologs, only Staphylococcus aureus Cas9 (SaCas9, 1053 amino acids) displayed cleavage...a distinct family of RNA-guided genome editors. Nature 566:218–223 . https://doi.org/10.1038/s41586-019...

...about citing: I wasn’t sure if I should cite reviews or the primary literature or both. I ended up citing...lists and pictures are the most useful parts of reviews. These could be in the form of figures/schematics... to write, to read efficiently, to search the literature, and to synthesize a large volume of information...well-researched field, you may need to get specific to make sure your article doesn’t turn into a textbook. Get the...active/well-known researchers in the field. I made sure to find information from genome-wide studies, as...appreciate the discussion sections of the primary literature as a resource that both summarized results and...Addgene Blog Writing Scientific Manuscripts: Literature Searching, Reading, & Organizing Tips for Improving...

...recombination sites (attL-left- and attR-right-, Figure 1). Under certain conditions, the attL and attR... regeneration of attP and attB sites.   Figure 1: Lambda phage integration and excision reactions...gene is excised from the donor vector.   Figure 2: The Gateway system adopts phage integration ... program to design Gateway plasmids.    Figure 3: Method A to create an entry clone: recombination...into an attL-containing entry vector.   Figure 4: Method B to create an entry clone: TOPO cloning...) of an attL-containing entry vector.   Figure 5: Method C to create an entry clone: Restriction...clone to create the expression clone.    Figure 6: Generating an expression clone. The reaction...

...This post was contributed by Future of Research Executive Director, Gary McDowell. On Dec 1st, the threshold... guidance issued by the Department of Labor. At Future of Research, our volunteers have been helping to...their postdoctoral salary scales to the NRSA scale (Figure 21, National Postdoctoral Association Institutional...legal justification of this have not been received. Future actions for junior scientists and for academia ...many postdocs will immediately lose jobs; that tenured professors/mid-career scientists/junior faculty...biomedical enterprise in the U.S. will decrease. At Future of Research, we think this is a good thing. Reliance...McDowell is currently the Executive Director of Future of Research. He is interested in advocating for...

...automatically using motion capture software such as EthoVision. Consult the literature to find all the applications... is an approximation of reward or the sense of pleasure that the animal perceives through taking a drug...may affect the animal; stimulation could induce seizures or some other unwanted phenomenon or it could ... elegans [10]. If used properly, optogenetics is sure to teach us much in all of these species. Happy ... nucleus accumbens facilitates reward seeking." Nature 475.7356 (2011): 377-380. Pubmed PMID: 21716290...direct comparative analysis of microbial opsins." Nature methods 9.2 (2012): 159-172.5. Goshen I, et al...."An optogenetic toolbox designed for primates." Nature neuroscience 14.3 (2011): 387-397. PubMed PMID:...

...scientists was Nature. Overall scientists tweet URLs with scientific domains <10% of the time. Figure 3: Top ...by biologists were: Nature, Sciencemag, Wiley, Cell and Scientific American Figure 4: Top Scientific Domains...have the most Twitter users. Biologists rank #8. Figure 1: Highly Represented Scientific Disciplines on...scientists on Twitter are male and 40% are female Figure 2. Gender Ratio of Scientists on Twitter. 60% of...scientific domains more often than other scientists   Figure 5: Portion of Tweets with Scientific Domains, Categorized...their fields than scientists outside their fields Figure 6: Follower Network of Scientists on Twitter. Each...

...Jaenisch, Nuclear reprogramming and pluripotency. Nature, 2006. 441(7097): p. 1061-7. PubMed PMID: 16810240...cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006. 126(4): p. 663...human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors. Proc Natl Acad...differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells. Cell Res, 2009...pluripotent stem cells into intestinal tissue in vitro. Nature, 2011. 470(7332): p. 105-9. PubMed PMID: 21151107...Differentiation of human induced pluripotent stem cells to mature functional Purkinje neurons. Sci Rep, 2015. 5: ...Dedifferentiation, transdifferentiation, and reprogramming: future directions in regenerative medicine. Semin Reprod...

...updated version of BRET called BRET2. Figure 1: TRUPATH measures heterotrimeric G protein dissociation...can be measured by a BRET2 ratio, which is the ratio of GFP2 emission to RLuc8 emission (see figure). What...? The TRUPATH collection allows researchers to measure the physical association of combinations of different... signaling pathways.  This dissociation can be measured using a technique known as Bioluminescence Resonance...Rluc8 was more challenging. The Roth lab used structure-guided protein engineering and experimental refinement...both antagonists and inverse-agonists could be measured using the TRUPATH system. Conclusions Although...

...post about workplace bullying featured on the always interesting Tenure, She Wrote blog. Unfortunately...little pre-work most PhD students and postdocs do to ensure the advisors they choose will be ones that help...choose a field that you may want to work on in your future career, so the research topic will be more important...scientists to change labs – do as much as you can to ensure a good fit in advance. When a lab is interviewing...interviewing the lab to find a good “fit” and to ensure the PI will be a good role model and teacher. Do...creates a level of familiarity and confidentiality to ensure more honest answers. Ask people who work in the... to speak to them more than once to get a full picture and listen for hints at the same concerns from ...

...methods termed proximity-based labeling to better capture these interactions in a vast array of biological... of biotin for avidin, proximity labelling can capture weak/transient protein interactions as well as ...exposed lysines on neighboring proteins. Thus to capture protein complexes, scientists can simply express...After a few hours biotinylated proteins can be captured on streptavidin affinity beads or matrices. The...labeling in living cells and organisms with TurboID." Nature biotechnology 36.9 (2018): 880. PubMed PMID: 30125270...for electron microscopy and proximity labeling." Nature methods 12.1 (2015): 51. PubMed PMID: 25419960....genetically encoded reporter for electron microscopy." Nature biotechnology 30.11 (2012): 1143. PubMed PMID: ...

...necessitating the development of a new technique. In a structure as tightly packed and interwoven as the brain,...previously visible with Brainbow-1 and -2. The general structure of Brainbow-1.0 is retained in Brainbow-3.0, but..., but can be detected via immunostaining. This feature facilitates screening of Cre-negative Brainbow ... H, Draft RW, Bennis RA, Sanes JR, Lichtman JW. Nature. 2007 Nov 1;450(7166):56-62. PubMed. A technicolour...the connectome. Lichtman JW, Livet J, Sanes JR. Nature Reviews Neuroscience. 2008 Jun;9(6):417-22. doi... P, McKellar CE, Hall D, Looger LL, Simpson JH. Nature Methods. 2011 Mar;8(3):253-9. doi: 10.1038/nmeth..., Alexandre C, Bell DM, Dickson BJ, Salecker I. Nature Methods. 2011 Mar;8(3):260-6. doi: 10.1038/nmeth...

...calcineurin activity measurements in stressed or aging cell populations. Figure 3: Micrographs of...library Article contributed by Susanna Bachle Figure 1: Initial steps in the CRISPR-pooled screening...infection (MOI) during cell library construction to ensure that the majority of cells receive one gRNA. The...Read our SPARC blog post for more details. Figure 2: Dense (c), intermediate (d), and sparse (e) ...target. Calcineurin reporters allow scientists to measure calcineurin dynamics and can serve as a drug-screening...high throughput screening, calcineurin dynamics measurements, or monitoring in vivo. To overcome these technical...dye Article contributed by Jennifer Tsang Figure 4: Double stranded DNA bound by tTALE-FP with the...

...10.1089/hgtb.2019.110 Pacouret S, Bouzelha M, Shelke R, Andres-Mateos E, Xiao R, Maurer A, Mevel M, Turunen...quality control (QC) mantra is to do our utmost to ensure that scientists are receiving the exact materials...substrates for tagmentation-based library preparation procedures. This finding eliminates the double-stranding...time and cost for AAV genome sequencing.  Figure 1: The viral genome sequencing workflow. Using... that looks for pre-defined serotype specific signature sequences in the data. Unlike previously described...not require any additional reagents or lab work (Pacouret, 2017). Moreover, the sequencing data from multiple...t new to Addgene, we are excited to share the procedures and Python scripts with laboratories and viral...

...many iconic papers, including the ones on the structure of the DNA, were not peer-reviewed (Baldwin, 2015...the study to be replicated or omission from the literature. I think that delivering constructive critique.... The general representation of data is... The figures are ... In general, I think that this is an (e....checklist for individual sections of the paper (see figures). After finishing my section-specific comments,...WormBase, Arabidopsis.info). These repositories will ensure that the results of your colleague’s hard work ...comments will learn from them and propagate this culture further.  Many thanks to our guest blogger Magdalena...salt-stress induced responses in Root System Architecture of Arabidopsis HapMap population in Amsterdam...

..., resulting in significant background staining (Figure 1A). Not all tissues will have the same levels ...species on species staining with no problem! But to be sure that your secondary is not binding endogenous IgGs...following three strategies might help!     Figure 1: Species on species staining can be a problem... secondary binding Direct immunohistochemistry (Figure 2A)  How do you avoid having your secondary antibody...Pre-formed primary and secondary antibody complexes (Figure 2B) Some labs have found that you can significantly...as your primary/sample, and then applying this mixture to your tissue (Tuson, et al., 1990; Goodpaster...within a problem.  Pre-block with Fab fragments (Figure 2C) Speaking of Fab fragments… Another strategy...

...from? Or why they are such a commonly used cell culture model? In this blog we will spill the beans on ...postdoc Frank Graham’s 293rd experiment, hence the nomenclature we use today: HEK-293. The exact origin of the...making it genetically female. HEKs are generally cultured as an adherent monolayer, though derivatives have...However, Frank Graham showed HEKs display many immature neuron-like qualities, which is inconsistent with...cell line. What the HEK is wrong with it?   All cultured human cell lines are susceptible to contamination...immortalized, but cell health can degrade over time in culture. This can affect factors that could skew research... etc.). Thus, it’s important to maintain short culture periods (less than 20 passages) to prevent these...

...affected by environmental conditions (such as temperature, pH, fusion protein, etc...).  There are many... making such a decision remains spread across literature in publications that introduce these tools. ...references, etc...) and a more robust database structure was needed.  It is also difficult to scale such...submissions also require a DOI referencing primary literature, and every effort is made to moderate and cross-check...cross-check all data with published literature as soon as possible after submission. Using FPbase The most...’ll find matching FP suggestions. If you’re not sure exactly what you’re looking for, the Table of Fluorescent...emission spectra when available, and will soon feature filter spectra and collection efficiency calculations...

...rough structure of the plasmid based on the predicted sizes and organization of different features within...and temperature compatibility when digesting with more than one enzyme. Consult the manufacturer’s manual...their defined recognition sequence. Most enzyme manufacturers make High Fidelity versions of the endonucleases...loading them. The glycerol in the buffer will make sure your sample settles in the gel well and the dyes...expected band sizes. Always run control uncut DNA to ensure your enzymes are working. When uncut plasmid DNA...approximately 75-80% of the way down the gel will ensure you keep smaller bands from running off; however...

...plasmid-based research using synthetic vectors and cultured mammalian cells. Of course, these mammalian vectors...Therefore, it is not always mission critical to make sure the plasmid replicates in the cell, as many of these...foreign material and will subsequently pass it on to future generations. Stably transfected cells may be considered...stable transfection, there should be a selective pressure to force cells to incorporate the plasmid DNA ... used in conjunction with positive selection to ensure your gene gets targeted to a specific location ...293HEK, Jurkat T cells Inhibits protein synthesis; premature chain termination 1-10 ug/mL Zeocin Sh bla HeLa...is often used as a supplement in mammalian cell culture to suppress bacterial growth, and is not appropriate...

...passaged from an infected host-cell culture to an uninfected culture which results in less overall dilution... without DNA cleavage. Nature 551:464–471 . https://doi.org/10.1038/nature24644 Richter MF, Zhao KT, Eton...for speed: base editing before Cas lets go Figure 1: David Liu gives a talk at the Cell Symposia:...from the infected to a fresh uninfected host cell culture). This dilution step represents the major difference...dilution and less stringent phage selection. Figure 2: Phage-assisted evolution of base-editing activity...improved Cas domain compatibility and activity. Nature Biotechnology. https://doi.org/10.1038/s41587-020...

... this class measures either factors as proxies for a cellular process or directly measure a cellular process...to the structure of actinin in low vs. high tension. These systems are powerful tools to measure real-time...boiled down pretty simply – they are systems that measure biological phenomena by generating an output which...microscope, plate reader, or any other instrument which measured GFP levels and converted that signal into a readable...will focus on those which can be applied in cell culture and animal models primarily. Fun fact: Almost all...tissue preps, in multiple species, and in cell culture models. Some of them have even been expanded for...

...the amino acid change based on maps of protein structure. Sometimes this provides a great deal of clarity...bioassays could accurately test for molecular signatures of variant function and cancer phenotypes. Broad...systematically as possible. In developing these assays, we figured that they would need to be generalizable and capable...capable of measuring a variety of molecular signatures or cancer phenotypes. These assays were designed...look-up-tables” of mutant function that could enable future clinicians to improve real time decisions when ...proof-of-concept. If such approaches are proven to improve future clinical decisions, we can work together to realize...of precision functional genomics and propel the future of cancer precision medicine for the next-generation...

...-peptide (represented as an orange wedge in the figure on the left) and inserted it into a plasmid, creating...note that IPTG is also included in the medium to ensure transcription of the lac operon.   Tips for blue-white...care in making your plates: X-gal is light and temperature sensitive and needs to be added to media after...autoclaving. If spread on top of pre-made plates, make sure it is evenly distributed and allow sufficient drying...analysis, like PCR or restriction digest. Make sure you use a proper E. coli strain (i.e. contains the...STBL4, JM110, and Top10 are a few examples. Make sure you use a proper plasmid (i.e. contains the α-complementation...selection methods. Combining selection and screening ensures that the white colonies you see are white due to...

...journal. Be sure to check the Author Guidelines of the journal you are submitting to and make sure your manuscript...lab resources, such as catalogue numbers and manufacturers’ details for antibodies, plasmids (like your...F1000Research, also ask for the raw data behind the figures and results. This is a relatively new development... of science publications. Peer reviewing To make sure your manuscript is perceived as a reliable source...when publishing a scientific manuscript Dos: Make sure your paper fits the scope of the journal you are...chances of getting your manuscripts published in the future. Disregard reviewer’s comments without explaining...Always check the journals Author Guidelines to make sure that your paper fits with the journal’s house style...

...Conclusions & implications One striking feature of this paper is the failure of the embryos to undergo editing...mutation in human embryos." Nature. Epub 02 August 2017. doi:10.1038/nature23305. PubMed PMID: 28783728. ...coverage. But the paper published August 2nd in Nature gives us even more to talk about, as it represents...but instead from the mother’s wild type allele (Figure 1). 72.4% of embryos carried two wild-type copies... proceeded through NHEJ rather than HDR. Figure 1: Homology-directed repair in targeted zygotes...broader applicability of CRISPR germline editing. Nature News mentions that the MYBPC3 gRNA used in the ...

...48 hours exposure to blue light also induced tail swelling in ~75% of worms (see below figure). Zhou et... current state (Green when off, dark when on). Figure 1: Photoswitchable MEK1 (psMEK1). Is it possible...is a serine/threonine kinase with a similar 3D structure to MEK1, but Raf1 and CDK5 have less similarity... pathways Negative feedback loops are a common feature of signal transduction pathways, but are often ...used a photoswitchable CDK5 in C. elegans. See figures 4B and C in Zhou et al for details on these experiments...no. 6327 (2017): 836-42. PubMed PMID: 28232577. Figures 2-4 were modified from this reference. 2. Regot...Carrasco, and Markus W. Covert. "High-Sensitivity Measurements of Multiple Kinase Activities in Live Single...

...antigens.      Figure 1: IgG antibody structure with important features labeled. V = Variable ...Fab Fragment? Consider the classic IgG antibody (Figure 1): it is composed of four peptide chains (two ...different fragments that retain antigen binding (Figure 2):  F(ab')2 - These fragments are produced by...Fragment Variable (scFv). Size: ~25 kDa.   Figure 2: Comparison between a full IgG antibody and common...tool box. As you consider which tools to use, be sure to think about what pieces of the antibody you do...Bates A, Power CA (2019) David vs. Goliath: The Structure, Function, and Clinical Prospects of Antibody ...

...guest blogger, Gary McDowell, executive director of Future of Research. On December 1st 2016, many postdocs...some cases reversing these decisions entirely. At Future of Research, we had been collecting data and documenting...published in F1000Research on November 2016. Due to the nature of the postdoc position, we viewed salary raising...FLSA updates was granted nationwide. Again, we at Future of Research believe that, while the mandate has... out about what their institution is doing from Future of Research over social media, not from their institution...actually getting paid, which we will be analyzing for future updates. But suffice it to say, salaries are showing...McDowell is currently the Executive Director of Future of Research. He is interested in advocating for...

...available, including those found at Addgene! Figure 1: Strategy for assembling the barcoded gRNA library...pool-clone them into the pAWp28 storage vector (Figure 1): Forward oligo: 5’- CACCGNNNNNNNNNNNNNNNNNNNGTTTGGGTCTTCGAGAAGACCTATTCXXXXXXXXC...the CombiGEM lentiviral vector backbone pAWp12 (Figure 2). Cloning from the pAWp28 storage vector to the...by NGS before beginning your experiments. Figure 2: Strategy for assembling barcoded combinatorial...and minimize off-target effects of CRISPR-Cas9." Nature biotechnology (2016). PubMed PMID: 26780180. PubMed...cells with a lentiviral CRISPR-guide RNA library." Nature biotechnology 32.3 (2014): 267-273. PubMed PMID...activation by an engineered CRISPR-Cas9 complex." Nature (2014). PubMed PMID: 25494202. PubMed Central PMCID...

...publishing, but still made it so she could “get exposure to the drug discovery process.” The ClonTracer...xenografts after cell implantation. Their goal was to figure out how to optimize protocols for in vivo screening...panned out quickly,” and was summarized in a recent Nature Medicine paper. In the publication, Bhang and coworkers...specifc, easily read sequences that can be used to measure their clonal abundance before and after a treatment... populations with resistance to both drugs. The future of ClonTracer Since starting her new role as an...populations are treated as they arise. In their Nature Medicine paper, Bhang and coworkers also looked...cancer therapy using high-complexity barcoding." Nature medicine 21.5 (2015): 440-448. Pubmed PMID: 25849130...

...workshops (see the Careers essays in the journals Nature and Science, for example). This blog post is about...write a manuscript. Consider questions like: What figures are missing? For example, are additional controls...been validated? How will the results fit into a future manuscript? You could easily spend a day or a ...experiment. However, by taking this time, you can help ensure that you understand the methodology and can have...the data and any recent relevant studies in the literature. Plus, presentations benefit from practice. If...and how your research fits in with the current literature. Finally, if you give your talk or poster to ...application and manuscript reviews, etc. You'll also meet future collaborators and get access to unpublished information...

...Transposons: The jumping genes. Nature Education 1(1):204. https://www.nature.com/scitable/topicpage/transposons-the-jumping-genes...retrotransposons are like retroviruses both in their structure and mechanism of replication. They contain two...and reverse transcriptase activities.    Figure 1: Overview of retrotransposon transposition. Retrotransposons... TEs have LTRs on both of their ends.    Figure 2: Overview of DNA transposon transposition. Modified...Transposon mutagenesis screens Transposons, by their nature, are mutagenic elements, which makes them a great...of DNA up to 10 kB in size in bacteria.   Figure 3: INTEGRATE system for RNA guided transposon insertion...Rev Genet 41:331–368. https://doi.org/10.1146/annurev.genet.40.110405.090448 Kumar A (2020) Jump around:...

...particles produced could not replicate. However, Mother Nature found a way to re-create a functional virus. Indeed...non-dividing cells is not restricted to in vitro cell culture as lentivirus-derived vectors are capable of transducing...your gene of interest into and is an important feature for your experiment; it must be chosen carefully...expression of your gene. The addition of Dox in the culture will then repress your gene of interest. This system...state and is expressed when Dox is added to the culture. This system is now used as it shows rapid gene...biosafety guidelines for more information and always be sure to consult your institution’s Biosafety Committee...cells start releasing viral particles into the culture supernatant and you can generally harvest the supernatant...

...  Figure 1: (Top) Polyclonal antibodies are produced in animals and consist of a mixture of antibodies...made from animals or hybridomas.   Figure 2: (Left) Figure 2: (Left) The light chain and heavy chain...Reproducibility crisis: Blame it on the antibodies. Nature 521:274–276 . https://doi.org/10.1038/521274a Begley...Raise standards for preclinical cancer research. Nature 483:531–533 . https://doi.org/10.1038/483531a Bradbury...Reproducibility: Standardize antibodies used in research. Nature 518:27–29 . https://doi.org/10.1038/518027a Bradbury... (2015) Antibodies: validate recombinants once. Nature 520:295–295 . https://doi.org/10.1038/520295b Bradbury...

...mCherry coding sequence in the antisense orientation (Figure 1, top). The entire DNA cassette would be flanked...include both Cre and FLP recombinases.   Figure 1 (modified from Schnutgen et al. Nat Biotech 2003...identical sites on one end of the DNA fragment (Figure 1, middle). A second recombination event then excises...DNA between the identical loxP or lox511 sites (Figure 1, bottom). Since only one loxP and lox511 site...switch conditional knock-in cassette. As shown in Figure 2, the cassette should contain a reporter DNA sequence...control expression of an optogenetic reporter would ensure that the reporter remains silent until a cell or...tissue-specific Cre is provided. Since the FLEx switch ensures Cre-dependent reporter expression, the potential...

...poly-uracil tract as shown in the figure to the right. The tertiary structure of the hairpin-DNA complex is...the transcript.   Figure 1: A predicted conserved secondary structure and sequence conserved Rho-independent...enzymes that cut RNA not protected by secondary structure. It is thought that poly(A)s are used to control...relies on a specific sequence and RNA secondary structure to induce transcript cleavage, similar to the ...motif and a downstream GU-rich region (shown in the figure below) releases the mRNA from the polymerase and...export and translation processes as well.   Figure 2: The conserved eukaryotic polyadenylation signal...

...field. Nobel Laureates and the ever expanding importance of RNA The second keynote lecture was presented...project on its 25th anniversary, how the NIH sees the future for genomics, and President Obama’s Precision Medicine...presented by Nobel Laureate Jack Szostak. Dr. Szostak’s lab continues its work on the origin of cellular life... two of the conference was kicked off by Nobel Laureate (and Addgene depositor) Craig Mello. Taking his...problem of antibiotic resistant bacteria. See the picture to the left for his list of the bacterial species...engineering. The final keynote presentation was by Nobel Laureate Thomas Steitz of Yale. He gave an update on new...conformation “not like DNA which has a boring structure.” That certainly summed up this conference—nucleic...

...little better. As a Master’s student, she had less pressure to publish and more freedom to attend conferences...legitimate brain exercise in developing a plan for the future. From good ideas to a SynBio startup At some point...them to see how feasible they were, conducting literature reviews and having weekly journal clubs outside...this process, we had an idea that survived the literature review, survived technical feasibility analysis... fearful of this technology and angry at the agriculture companies that produce GMO food plants. When ...idea, we knew it was a winner – although we didn’t figure out how we would accomplish the color change until...science, and to have a job that we enjoy and see a future in. And we decided to make education and outreach...

...not based on any existing protein structures, called Top7. Figure 1: Top7 receives the Nobel Prize...characterized it, producing crystal structures demonstrating “the structure is strikingly similar to the design...works and creating biological tools not found in nature. Dr. Baker’s team was the first to develop and ...wanted to create a new fold that is not present in nature (at least that we’ve seen so far!). They started...the biophysics of protein folding and selective pressures during protein evolution. The Baker Lab has deposited...

...antibodies that bind to the spike proteins in cell culture, and inhibit these viruses.  But llama immunization...proteins that may unfold due to camelid high body temperature (37.2 - 39.2°C). The Seeger lab found a way around...protein targets, similar to how antibodies do. The structure of the common antibody is heftier (~150 kDa) and...molecular crevices proteins on the cell surface. Figure 1: Comparison of common antibody, HCab, and nanobody...protocol, which cover 3 naturally-occurring nanobody structures (concave, loop, and convex). Each of these 3 ... to select for optimal sybody candidates. Figure 2: Outline of the process for selection of sybodies...anti-Myc antibodies bound to an ELISA plate to capture Myc-tagged sybodies. Then, the biotinylated target...

... like to make sure they are present at a very low concentration. So how do we make sure to catch all of...experiment because of poor reagent quality. To make sure our AAV vectors are of the highest quality, we undertake...mission of accelerating scientific research. To make sure that adeno-associated viral (AAV) vectors available...all the DNA packaged inside the viral particles (Figure 1). Briefly, packaged DNA is extracted from purified...plasmid #44362. Method validation, limitations, and future plans How did AAV-68544 become contaminated? Well...processed the mixed sample in our pipeline (see Figure 2, sample 2). When we blindly analyzed AAV-68544... the Addgene blog Check out our Viral Vectors Featured Topic Page Subscribe to Viral Vectors Blog Posts...

...to Our Podcast Interview with Alex Chavez Figure 1: Schematic of native (nuclease-competent) Cas9...of genome editing (Fig. 2 and 3) (5, 3). Figure 2: Gene activation with nuclease-competent Cas9...alterations observed at the target site.   Figure 3: Gene repression with nuclease-competent Cas9... systems. She is also a NASA Fellow studying biosignature preservation in Mars analogue environments. ...proteins for RNA-guided gene regulation and editing." Nature methods 10.11 (2013): 1116-1121. PubMed PMID: 24076762... parameters via a library-on-library approach." Nature methods 12.9 (2015): 823-826. PubMed PMID: 26167643...for genome editing, activation and repression." Nature methods 12.11 (2015): 1051-1054. PubMed PMID: 26344044...

...situations in cells are described below (See Figure 1).   Figure 1: Each problem with dimerizing fluorescent...follow. It's a matter of concentration Most FPs in nature are prone to dimerization (i.e. EGFP) [1, 2] or...together and can wrap the membranes into stacked structures including organized smooth endoplasmic reticulum...state, and an artificially active state due to closure by FP dimerization.   1. Transmembrane fluorescent...if a dimerizing FP is fused to the POI. These structures are large (microns), bright, non-physiological...whorl formation assay, are practical real world measures of dimerization potential that will determine ...GFP-based FRET, with application to intrinsically unstructured proteins. Protein Sci, 2007. 16(7): p. 1429-...