...regulation thats as sophisticated as a natural network. We could build a material as sophisticated as wood. To... perspective and you can layer them for more complicated functions. Those things are easy with CRISPR....submitting parts from another paper for resource allocation. Those will allow you to set the amount of transcriptional... carbons and oxygens. Nothing too much more sophisticated. There are also proteins, like antibodies. But...just using what biology has given us. Wood is a complicated material and we can access it, but we are stuck...able to design new materials and chemicals as sophisticated as those that living cells and animals make....

...flexibility in the location of the cut and subsequent edit. In these applications, location takes priority...make a choice about what is your top priority: location or efficiency. If you are just trying to shred...still leave you choosing between efficiency and location.   Nearly PAM-less editing   The Kleinstiver lab...in double stranded cleavage and base editing applications, with high editing capacity in both contexts...randomly selected genomic loci will have a PAM located next to it. The SpRY Cas9 can also work with NYN...

...containing genes with many modifications. We look for point mutations, truncations, and insertions that could...significant amount of QC. First, we perform Picogreen quantification on each and every isolated plasmid sample and... preparation process and includes Picogreen quantification of various intermediate steps, a sizing electrophoretic...electrophoretic gel, and qPCR quantification of the final libraries. A broader view of the whole plasmid...often find a few mismatches in the origin of replication or other common backbone elements. Since we’ve...annotated Genbank files that are useful for more complicated plasmids, like those that contain synthesized...

... from E.coli, named TadA7.10 in ABE7.10)  a catalytically impaired Cas protein (dCas or Cas nickase)  ...accidental insertions and deletions (indel) by the catalytically impaired D10A nickase from ABE7.10 can be a ...the authors created ABE8 constructs using a catalytically “dead” S. pyogenes Cas9 (dC9-ABE8.x-m/d). The...these variants, ABE8 could target many additional locations in the genome. Compared to ABE7.10, NG-ABE8.xm... reduced off-target editing frequencies. For applications that require high DNA editing specificity, for...deamination, an important attribute for therapeutic application. ABE8s demonstrate an overall improved base editing...editors with increased activity and therapeutic application. Nature Biotechnology. https://doi.org/10.1038...

...channelrhodopsin, fused to a fluorescent GFP marker (CatCH-GFP). The lab deposited AAV plasmids containing ...expression. Surprisingly, <1% of synthetic promoters replicated the expression specificity of the wild-type promoters...reproducibly labeled retinal cells can be found here.  Applications for synthetic promoter AAVs Besides restricting...synthetic promoters have a few other potential applications: Using promoter combinations for targeted expression... promoters expressed in. The lab tested this application using one promoter to drive expression of Cre-mCherry...drive expression of a Cre-dependent fluorescent CatCH-GFP reporter. Expression of GFP will only result... rates of labeling are ideal of gene therapy applications, promoters that produce sparse labeling would...

...visit the Cell Catalog on the Allen Cell Explorer, where you can also find the certificate of analysis provided...signal that makes it hard to visualize the precise location of the protein or structure of interest. Endogenous...technology, inserting large tags into a precise location in the genome is still inefficient, particularly...from these cell lines can be found in the Cell Catalog on our data portal. The plasmids should be functional...

...in the brain region you wish to stimulate 2) Fabrication and implantation of the optical fiber into the...Channelrhodopsin2 (Chr2), a blue-light sensitive non-specific cation channel, activation of which results in depolarization...optogenetics. Successful stereotaxic surgery relies on identification of the bregma and lambda sutures and subsequent...bregma which allows you to quickly and reliably locate your injection site. We recently purchased this... fluorescent protein, which allows for easy identification of opsin expression, extent of viral spread...express for too long (synapsin was privately communicated to me as one of these extremely high expressing... and memory in opioid addiction and science communication/science writing. Derek is also a pianist and...

...single-molecule biophysics Fast and responsive voltage indicator ONE-GO biosensors kit Antibodies for neurodegeneration... an enhanced pU6cm promoter, viral replicon amplification of cargo, and heat treatment.  Figure...2024.12.20.629623.  A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic...and more responsive genetically-encoded voltage indicator (GEVI) than previous versions. ASAP5 can be imaged...The initiation site identified in panel (C) is indicated; red line is exponential fit. Adapted with permission..., M. Z. (2024). A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic...activation triggered by endogenous GPCR. Example applications include large-scale pharmacological profiling...

...development programs for bioscience educators. Using these hands-on tools, educators introduce their students to...biologists have always struggled to effectively communicate the nuances of protein structure in a flat 2-...dangerous. Subscribe to CRISPR Blog Posts How do educators and students use molecular models?   We created...something tangible. Under the guidance of a trained educator, these instances of focused attention become magical... their labs. Soon after we began working with educators in our Center for BioMolecular Modeling, we spun...through flexibility It is common knowledge in the education community that All models are wrong….Some models...

...setting—especially if job applications require teaching experience or high numbers of publications, which may take...their funding. Communication: In both industry and academia, you will have to communicate your science ...people involved is staggering. The process is complicated. If you’re interested in the drug discovery process... proprietary compounds or information. Where publication is expected, the process is similar to academia...science to others and advocate for the importance of your work. In my experience in industry, you’re more likely...

...stem cells are: Their ability to self-renew or replicate while maintaining the undifferentiated “stem” ...Unipotency: As the name suggests, cells under this category can differentiate into only one cell type. Muscle...differentiate only into myogenic precursor cells [5]. Classification of stem cells Stem cells are classified into...somatic stem cells, these multipotent stem cells are located in small quantities in specific “stem cell niches...body. These generate specialized cells for their location, mostly to maintain cell numbers or replenish ...joined Cell Press to pursue a career in science communication and publishing after completing her postdoctoral...Extraction of Pluripotent Embryonic Stem Cells: Implications for Regenerative Medicine and Reproductive Medicine...

...experts in science communication in real time! I follow great online science communicators like Phil Plait...the public, so I reached out to people who were educating the public and organizing events. I contacted ...about any topic I like and gain experience in communication, editing, marketing, and website organization...when I was trying to find local informal science educators, and ever since, the State Museum has been the.... She is particularly interested in science communication and fundamental molecular biology.      Additional...  science communication...

... unique compared to the rest of the genome and located immediately 5’ to a protospacer adjacent motif ...coli, and T. gondii genes.  2) What genetic modification are you trying to make? Addgene carries CRISPR...next-generation sequencing to check the quality of your amplification. If you've obtained a ready-to-use lentiviral... As such, sequencing CRISPR libraries after amplification and after a screen requires the use of next-...experiment, the use of appropriate controls, multiple replicates and several cell types can strengthen your results...independently validated to ensure that the desired modification produces the phenotype you screened for in the...information on CRISPR screens check out Addgene’s 2016 publication “Practical Considerations for Using Pooled Lentiviral...

...excited to announce we now offer an export license application for rabies materials! Some of the materials in...export-controlled materials in the USA, where Addgene is located.  The export license, if granted, allows us to ...international requestors. We also offer export license applications for VSV and SARS-COV-1 plasmids. Export control... applied to cover processing costs. Once the application is approved, Addgene then ships the material ...offered VSV and SARS-COV-1 plasmids under this application process since May 2020. We find this process ...whenever possible, as we cannot expedite the application process.  More information about export control...

...subfields.  Plasmid requests correlate with paper publication The dataset includes 11,426 plasmid requests ...14%). Spikes in plasmid requests correspond to publications of popular papers. Figure 1: Plasmid requests...effect on plasmid requests Before we began any sophisticated analyses, we first visualized our data in various...orders and new COVID-19 cases for each week by location. We observed that as expected, plasmid orders ...independence assumption of the Poisson process. This modification also eases the interpretation of the parameters...effects fairly well. However, we observed one complication in the China data – there is a period of 1 month...fundamental scientific discovery and high-impact applications.       Alvin Hsu is a graduate student at Harvard...

...containing a phage origin of replication that can be packaged into replication-incompetent phage particles...essential processes: cell wall synthesis, DNA replication, or protein synthesis. Because they target such... and medical organizations are working to help educate the public about proper antibiotic usage. Although...mammalian viral vectors in that the vectors are replication-deficient. Thus, a very large number of phagemids...CRISPR antimicrobials combines them with both replication-competent phages and antibiotics. Phage therapy...bacterial host range. To further potential clinical applications, Ando et al. recently engineered synthetic phages...

...transgenic mice. Purification: GFP can be used as a general epitope tag for protein purification and a number...their experimental needs, and some of the common applications in the lab. Why Green Fluorescent Protein?...to maintain fluorescent activity; very little truncation is tolerated, however, point mutations are acceptable...Re-engineering GFP to increase its range of color and application Almost as soon as its sequence was elucidated...others) Neutral mutations    A multitude of applications Due to its size and ease of use, GFP and other... just scratched the surface of the potential applications for GFP. It has also been used to identify particular...

...Addgene has a growing catalogue of viral preps available to request. In this post, we’ll walk you through...minimized by using an endotoxin-free plasmid purification protocol. To minimize the immunogenic properties...posted on the material page for the corresponding catalog item (see maps section for images).     ...Lentivirus Production Lentiviral preparations ("-LV” catalog items) are produced in 293T cells using the 2nd.... Concentrated lentiviral preparations (“-LVC” catalog items) are generated from lentiviral preparations...typically prepared using endotoxin-free plasmid purification kits.     Figure 4: Schematic of the... Addgene's Data Hub Digital Droplet for AAV Quantification Viral Vectors 101: An Introduction to AAV References...

... are not ideal for every application. Disadvantages include their duplicated (i.e., redundant) genome,...mutant, which regulates a developmental pathway implicated in congenital heart defects and cancer. These...DNA into the genome is useful for a variety of applications. For example, specific tissues such as blood...pathways control this process. Additional zebrafish applications and conclusions Zebrafish are also popular outside...—have proven useful in a variety of research applications. Lineage tracing studies highlight how these...Contemporary zebrafish transgenesis with Tol2 and application for Cre/lox recombination experiments." Methods...

...control, 1; very dark). Brighter fluorescence indicates a more successful gRNA design. Adapted from Mashiko...plasmid using standard cloning techniques (The XX indicates where your target is inserted, disrupting the ...effective ones to increase your chances of gene modification in vivo. Find Plasmids for Testing Your gRNAs...et al., 2014). The plasmid is sturdier than the delicate RNA and therefore there has less chance of contamination... the validation assay should give an accurate indication of how many pups will contain a mutation and ... months from the day you decide to do a gene modification experiment in mice to the day you have your ...reproductive biology and genetics. She loves to communicate the latest and greatest in scientific content...

...compartments undergo significant post-translational modifications including glycosylation, disulfide bond formation...are equally susceptible to these non-native modifications, which can all potentially affect function [...N-glycans [9]. Both of these posttranslational modifications can disrupt FP folding rendering the protein... non-optimized parental proteins. Our recent publication illustrated the quantitative increase in fluorescent...and Jack Griffith, her research focuses on the replication machinery of Kaposi’s sarcoma-associated herpesvirus... for diverse cellular environments." Nature communications 6 (2015). Pubmed PMID: 26158227. Pubmed Central...pathway."Biochemical and biophysical research communications 430.3 (2013): 1114-1119. Pubmed PMID: 23257162...

...very little data on which might work in your application. It can be quite difficult to decide on which...be searched and sorted by gene/target, name, application, pass/fail rating, sample species, antibody species... how the antibody performed in the submitted application, along with a brief description of the experiment...experiment. If that data was included in a publication, you'll find that information in the report as well... section will vary slightly depending on the application — flow cytometry, western blot, immunohistochemistry...data showing how the antibody worked in your application. The submission process is easy, with simple,...

... citations for recent articles. Of course, a publication in Science or Nature is always nice, but in the... lesser-known journal as it is in a top-tier publication. Accessibility is surely also behind the increase...increase in citations for older publications. (As an interesting aside, Nature recently reported that the...in a solution.) But are citations and journal publications really the best way to measure impact? As that...and what it’s made of. Increasingly, journal publications alone aren’t enough. Scientists are sharing ...consider and quantify the impact of scientific publications. Ventures like Impact Story seek to find ways...

...procedure enables quantification of antibody performance in major research applications. eLife, 12, RP91645...that the antibody being published doesn’t always indicate the presence of supportive data. To be supportive...that the antigen being detected in the intended application — here, flow cytometry — is the desired target...fixation-permeabilization protocol. Although the expected location of the target is an important factor in deciding... saponin. 400,000 cells were stained with the indicated Synaptotagmin-1 antibodies and corresponding Multi-rAb...this approach should also allow for the clear identification of the cell types of interest. By correlating...of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72. eLife...

... science communication and outreach - if you'd like to learn more about Science communication check out...microscopes and give a similar optical quality (magnification of 140X and 2 micron resolution). I have worked...creative approaches to visualize bacteria in an educational setting. The Symposium on ‘Creative Approaches...from hand-drawn comics (Dr. Sanja Saftic) to educational gifs created by the Amoeba Sisters. Here’s one...Bacterial Genome Engineering Learn about Science Communication using Twitter  Resources on Addgene.org Check...

...decades. Widely used across various research applications, they have become indispensable tools for manipulating... easily, without the need for time-consuming purification processes, and are powerful, due to their ability...-lasting changes in cells. However, for some applications, integration might be considered a double-edged...dividing cells because they lack an origin of replication (Banasik and McCray, 2010). However, IDLV genomes... certain types of clinical or basic research applications. Considerations for IDLV use If you’re reading...Integrase-defective lentiviral vectors: progress and applications. Gene Therapy, 17(2), 150–157. https://doi.org...lentiviral vector: prospects for safe clinical applications. PeerJ, 10, e13704. https://doi.org/10.7717/...

...efficiency constructs are great for in vitro biocatalytic applications where toxicity doesn’t matter. Plus, for... binding of the plasma membrane. In previous publications, they described tools to control formation of.... Restricting the effect to a specific time or location can be critical when, say, the gene is essential...that trap the fusion protein away from its usual location, preventing it from carrying out its proper function...) and to introduce unnatural head groups for applications like fluorescent labeling via click chemistry...substantial PA accumulates in the plasma membranes, indicated by the PA probe colocalizing with SuperPLDx30....

... do need multiple first/corresponding author publications in quality journals. It helps to come from the...a job. The currency of scientific success is publications and grants. 2. Write succinct and easy to understand...humbling. My conviction that I had a flair for communication was rapidly dispelled. She would ask me, "Is...succinct. Slide decks leads to the opposite and can suffocate an exciting idea in a swamp of unnecessary details...essential for human health. Failure to do so is implicated in several human diseases. Proper patterning ...disease treatment, etc., again in clear terms. "Implications for human disease" is vague. Instead, consider...interviews. For point of reference, I submitted my applications all by snail mail and only had phone interviews...

...aptamer soup by exploring the development and applications of fluorescent RNA aptamers in Addgene’s collection...target cellular RNA to specific intracellular locations using Cas9 and track the localization of the complex...biotin (the addition of biotin allows for the purification of specific RNAs from cells using this tag, ...TO1-biotin and TO3-biotin contain an acetate modification that reduces nonspecific binding with RNA by...non-coding RNAs in mammalian cells." Nature communications 9.1 (2018): 656. PubMed PMID: 29440634. PubMed...fluorescent rna aptamer spinach2 for imaging of replication and immune-mediated changes in intracellular ... Shanker Shyam S., et al. "Ribonucleoprotein purification and characterization using RNA Mango." RNA23.10...

...the FOR website, the FOR Boston 2015 organizers indicate that the main question they wanted to focus on...oriented, scientifically minded, and able to communicate complex information - all traits you are likely...Publishing Panel brought in editors from a variety of publications as well as young PIs and all gave advice on ...blogging, write reviews, or write for a university publication, improving your general writing skills will help...academic publishing goals and make you a better communicator overall. Don’t Write in a Vacuum Your research...cognizant of this during my PhD. Submitting a publication doesn’t have to be like throwing it into a black...Science The Future of Research Website Additional Publications of Interest Can Be Found Here Resources at ...

...sequences To design experiments for further plasmid verification Identifying plasmid features While our quality...promoters, selectable markers, tags, origins of replication, and fluorescent proteins that are already included...clue about where the gene of interest might be located. By easily using BLAST to check the translated ...reference sequences. Using Snapgene for additional verification experiments: Primer and restriction digest design...repetitive regions. These regions require additional verification experiments that can be partially designed using... plasmid verification...

...talking to people about and communicating science. The path to science communication This may come as somewhat... I would ultimately want to go into science communication but thought that I would be best poised to share...PhD myself. To continue to foster my science communication skills, I took on TA positions, tutored fifth...largely my work developing myself as a science communicator that got me here! Thus far being an Outreach... one of my supervisors warned me, the swift gratification of publishing posts can be addicting. Even more...

... Foundation Round Table. In any field, good communication skills help when it comes to finding a job. ...an argument for taking the time to hone your communication skills. So, where can a graduate student or ...opportunities than ever before for scientists to communicate about their work and their interest in science...your area or at your institution; the news or communications office is a good place to start. It’s not unusual...about science writing as a career, the Science Communication Program at the University of California, Santa... your research stories. Consider practicing communicating your research by guest blogging here, just leave...

... perspective – a plasmid-containing cell must replicate the plasmid in addition to its own chromosomal...fully synthetic compounds. Antibiotics can be categorized based on whether they directly kill bacteria ...bacteriostatic); however, the distinction between the two categories may be a bit of a gray area as some bacteriostatic...Blocks 50S ribosomal subunit; inhibits aminoacyl translocation Bacteriostatic 50-100 ug/mL in EtOH Polymyxin...Binds 50S ribosomal subunit; inhibits peptidyl translocation Bacteriostatic 5-25 ug/mL in EtOH *In prokaryotes...and can be used in place of Ampicillin in most applications. Antibiotics vary in their sensitivitly to heat...

...“Off-target effects” a vague catch-all term for unintended consequences in an experiment. For CRISPR ... example, if there are 10 putative off-target locations you can 1) determine if editing at those sites...sequencing. While this may be unnecessary for many applications, it is the only comprehensive method available...binding by Cas does not always correlate with catalytic activity at those sites, especially when DNA mismatches...For a candidate site-specific approach, mRNA quantification or western blot of putative off-targets would...you’ve tried everything listed here. For some applications, this is ok, but it still must be controlled... typically confound an experiment due to their location (e.g. in a gene in a similar pathway, a growth...

... also been developed which don’t fit in either category, but those are a subject for a future post! ... GAL80 under different promoters and create sophisticated patterns of UAS-driven gene expression. To further... PMCID: PMC2942869. Find plasmids from this publication at Addgene. del Valle Rodriguez, Alberto, Dominic... PMCID: PMC2883883. Find plasmids from this publication at Addgene. Wei, Xing, Christopher J. Potter... PMCID: PMC3846601. Find plasmids from this publication at Addgene. Riabinina, et al. “Improved and ... PMCID: PMC4344399. Find plasmids from this publication at Addgene. Riabinina O, Potter CJ. The Q-System...expression of genes for metabolic engineering applications in Saccharomyces cerevisiae.” FEMS Yeast Res...

...scientific and technical training is that these educational paths prepare you for a vast selection of career...tech transfer, management consulting, science communication, policy and the diverse options in nonprofit...science is much like industry but the “product” is publications—the pressure to produce is the same You get ...and teach students Collaborations may be less “complicated” - for example, easy to initiate a collaboration...is different about non-academia? Scientific publications are a nice to have, not a need to have Must ...every 3-6 years—a short and exciting ride Complicated matrix structures are common Small integrated...

...contains the proteins needed for the virus to replicate,  and the transfer plasmid which contains the ... by the AAV. On a plasmid map, its sequence is located between the ITRs. It can be anything you like as...expression of your transgene to a specific species or location. Promoters can also drive different levels of ...sequences that can flank the transgene and are located between the ITRs. Transfer plasmids often have ... the expression of your transgene. The WPRE is located at the end of the AAV genome, just upstream of ...promotes RNA transcript longevity. The polyA is located at the end of the AAV genome, just upstream of ...

...infection, because this setup allows for experimental replicates for each of the gRNAs. The idea was to assign...cassette is removed, the effector is expressed. By truncating one of the recombination sites in the construct...intermediate (d), and sparse (e) labeling of the calcium indicator, GCaMP6f, in the Drosophila optic lobe. Image ... reporter (blue) and stained with PI (pink) to indicate cell death. Image from Diessl et al., 2020. ...https://dx.doi.org/10.15698%2Fmic2020.04.713 Truncated TALE-fluorescent protein fusions as a DNA staining...protein to the DNA. They created plasmids for purification of tTALE-eGFP and tTALE-mCherry. In comparison...

...these tools, which aren’t always easy to find in publications, so this is a much-needed community resource...resource of over 100 entries is driven by the dedicated community of data-contributing researchers who...is helpful to report the genetic background and catalog number or RRID if it exists. This is helpful because...exactly which strain you used so if they wish to replicate your experiment, they know exactly where to get...detection methods. For example, you can provide location, titer dose (Example: 1E13 GC/mL) and subject ... we’d like to give a shoutout to all of those dedicated scientists who have contributed to the AAV Data...