...which can reduce the potential image resolution of super-resolution fluorescence microscopy. Their size...about 2 nm, which can lead to higher resolution for super-resolution microscopy. Nanobodies can be labeled...animal sacrifice Poor tissue penetration Less resolution due to larger label displacement Require separate...penetration Can be labeled with multiple dyes Greater resolution due to lower label displacement 2° incubation... Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein...

...vector solution, and generally these methods measure different characteristics of the solution. These ...can also be altered by freeze-thaw of the viral solution (Lock et al., 2010). Infectious titer: the ...quantified by electron microscopy of a viral vector solution. Since this technique is intensive and requires...requires an electron microscope, it is not routinely performed on all new viral vector preparations. Addgene...facilities report the physical titer of a viral solution (Figure 2). Because physical titers are used for...starting template is the purified viral vector solution (which contains viral capsid proteins) as opposed...

...Blainey, P.C. (2024). CROPseq-multi: a versatile solution for multiplexed perturbation and decoding in pooled...valuable tool for microglia research and broader therapeutic applications.  Find microglia-targeting AAV plasmids...https://doi.org/10.1101/2023.12.12.571321.  Revolutionizing Gene Therapy: The REVeRT Dual AAV Vector System...has developed REVeRT, for gene delivery using reconstitution via mRNA trans-splicing. By splitting a large...Illustration of split fluorophore assay to test reconstitution via mRNA trans-splicing. A cis-splicing vector... The authors used REVeRT to functionally reconstituting coding sequences for a range of applications...mechanisms of delivery and the potential for therapeutic success. Find REVeRT plasmids here! Riedmayr...

...the complex interactions between cancer cells, therapeutic drugs, and the rest of the body than studies ...or cell culture. For example, Scott Lowe’s lab utilized mice to study how different mutations in leukemia...important human disease model with potential for therapeutic drug discovery as it is estimated that 75% of...they can also be used to easily screen multiple therapeutic compounds in one experiment. Zebrafish (Danio...to name a few. For example, Andrew Leifer’s lab utilized the worm to study calcium signaling in the entire...al. "Whole-brain calcium imaging with cellular resolution in freely behaving Caenorhabditis elegans." Proceedings...Drosophila melanogaster and the role of the fly in therapeutic drug discovery." Pharmacological reviews 63.2...

...Structural problems in academic research and potential solutions During the first couple of sessions of the symposium...his or her chosen career path,” but individual institutions can have many different names for the positions...From Panel Session 2: Defining the Postdoc: An Institutional Perspective. This lack of complete information...workforce, and we are beginning to identify potential solutions. Keynote speaker Paula Stephan did a fantastic... articulating many of these problems and some solutions. The structural problems seem to boil down to ...here). Importantly, the panel offered some great solutions to these problems and some that stuck out to me...those at the FOR symposium and will enact their solutions as soon as they become actionable. Additional...

...cytosine-to-thymine substitutions in target sequences (Banno et al., 2018). These substitutions were limited ...the utility of cytidine and adenine base editors that enable C-to-T and A-to-G base substitutions respectively... is done with recombineering, a technique that utilizes phage recombination machinery to promote homologous...especially for larger modifications. What’s the solution to this inefficiency? Use CRISPR to make it a ...introduce point mutations in P. aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, and Pseudomonas syringae...respectively. Substitutions seem most efficient when using a nickase version of Cas9, but were also successful...

...common solutions to avoid background staining when doing species on species IHC. (A) One solution is to...fluorophore) directly attached to it. (B) Another solution is to combine your primary and secondary antibodies...aren’t free to bind endogenous IgGs. (C) A third solution is to use Fab fragments against the sample species...still see non-specific staining! Hopefully, these solutions will help get your experiments back on track.  ... or immunocytochemistry (ICC). Not all of the solutions described here may be as effective for other techniques...

... super-resolution or single-molecule techniques.   Figure 2: Two-color super-resolution imaging...which reacts with O2-benzylcytosine(BC) ligands (Gautier et al., 2008). Although based on the same original...multiple proteins but label each separately before reconstituting, choosing your colors depending on the needs... want to use far-red rhodamine dyes for super-resolution imaging, HaloTag gives better performance than... (2019). Labeling Strategies Matter for Super-Resolution Microscopy: A Comparison between HaloTags and...7), 547–556. https://doi.org/10.1021/cb900062k Gautier, A., Juillerat, A., Heinis, C., Corrêa, I. R., ...

...The most commonly used yeast by biotech and pharmaceutical companies is K. phaffii (formerly known as ...the system. Since then, multiple biotech and pharmaceutical companies (along with some research laboratories...It’s also a methylotropic yeast, meaning it can utilize methanol as its sole source of carbon and energy...distantly related to S. pombe and S. cerevisiae; evolutionarily it evolved much slower and bears more similarity...the expression of recombinant proteins by the pharmaceutical and biotech industries as a protein expression...greatest hurdle for genome engineering. A unique evolutionary feature of K. phaffii, relative to S. cerevisiae...

...display-based directed evolution to create APEX2. They have now applied the same directed evolution technique to...compartments is via proteomics--the study of the distribution and interactions of proteins within these structures...small-molecule substrate to map the spatiotemporal distribution and interaction networks of specific proteins...paper, they describe TurboID and miniTurbo, which utilize non-toxic biotin, but label nearly as quickly as...escape detection. The Alexander Green lab found a solution: a low-cost, cell-free detection assay that can...

...chemiluminescence, precipitate the protein out of solution, or isolate cells expressing this protein. Read... The hybridomas are then subcloned by limiting dilution, which produces an immortalized, clonal population... curve. This curve is generated using a serial dilution of a known amount of protein that is also detected...the protein of interest and pull it out of the solution. Once the unbound protein is washed away, the ...2014.   In vivo uses For both research and therapeutic purposes, antibodies can be used to affect cellular...functional grade antibodies. Some of the research and therapeutic uses overlap: antibodies can bind to and inhibit...investigating signaling pathways or developing therapeutics. Experimentally, antibodies conjugated to fluorescent...

... that present as global developmental delays or autism spectrum disorder. It is estimated that CTD is ...could greatly enhance our ability towards CTD therapeutic development,” says Iyer. Since the RESOLUTE plasmid...understand so that we could possibly develop pharmaceutical interventions that would inhibit IL-36 release...says Mellett. This would help the lab identify therapeutic targets in the future. Congratulations Mark Mellett...of developmental delay, and is characterized by autism-like symptoms, seizures, and muscle weakness. So...To study cellular transduction events, we will utilize BRET-, FRET-, and intensity-based signaling biosensors...

... gene expression. On the other hand, using a constitutive promoter in the same background resulted in ...over-represented compared to those further away. While constitutive promoters drove maximal gene expression near...during shifts in growth rate as compared to a constitutive promoter. To simulate the increase in plasmid...When they increased concentrations of IPTG, a constitutive promoter upstream of GFP increased expression...stabilized promoter while gene expression from the constitutive promoter did not. Similar levels of expression... al. placed the genes under the control of a constitutive promoter and then moved them into the genome...

...human conditions, opening up new avenues for therapeutics that treat disease at the molecular level. For...patients have achieved persistent immunological reconstitution with a normally functioning T cell repertoire...design to clinical trials involve identifying a therapeutic gene, finding a means to deliver it and identifying...issue by developing new capsids through direct evolution, capsid shuffling, and peptide displays. These... and other ongoing clinical trials support the utility of AAV as a broadly applicable gene delivery tool...understanding of disease pathogenesis will enable robust therapeutic outcomes. In contrast to other gene delivery ...

...can be removed either by filtration of the virus solution (through a 0.45 µm or 0.22 µm filter) or by a ...sterile PBS.  Alternatively, to concentrate a virus solution without ultracentrifugation, you can reduce the...the virus is released, a more concentrated virus solution can be obtained. 4. Enhancing virus-cell contact... in single-use aliquots. Never use a polybrene solution that has been thawed more than once. Fibronectin... packaging cells. This check can be performed routinely and without disrupting the flow of your experiment... of the viral vector using the harvested viral solution as a PCR template. Regardless of the type of virus...

...co-founders of Revolution Bioengineering.  Last year we started a company. Revolution Bioengineering ... as food but support GMOs that result in a new utility, was there a business opportunity out there for...changed color throughout the day. It would be a beautiful GMO, one that people would actually want to have...out of our careers: to build something new and beautiful, to engage with the public in a meaningful way... much knowledge and experience that goes into executing a successful business that the learning curve ... in the United States Air Force. She left active duty to pursue a degree in a synthetic biology laboratory...

...Creating xCas9 through directed evolution Hu et al. used phage-assisted evolution to generate and select for...of phage-assisted evolution, generating xCas9 2.0-2.6. This second round of evolution included continuous...lab, have gone one step further, using directed evolution to create xCas9, an enzyme recognizing a broad...broadest-targeting variants. For the final round of evolution, they used a no-G PAM library to enrich for Cas9...

...Collectively, the pool of CRISPR software embodies solutions that facilitate most experimental applications... engineering. When compared to that collective utility, it’s easy to fault the lack of features in a single... sgRNA design tools into database and de novo solutions. Database tools allow us to view and get a sense... workbenches such as MacVector or Benchling. Utility functions: These functions help speed up the sgRNA...single-method design”. Trends: The most common utility functions are batch design or multiplex features...repertoire. Organism agnostic tools are the better solution but are often packaged as offline tools requiring...use. The R based package CRISPRseek offers great utility and coupled with Bioconductor should be the first...

... DNA and proteins in solution. The pH is then lowered using a renaturing solution, which causes the proteins...genomic DNA to precipitate. Plasmid DNA is free in solution. Proteins and genomic DNA are removed by centrifugation...placed in a chaotropic salt sodium iodine (NaI) solution. NaI has two purposes in this protocol: 1) it ...: nucleic acids are broken out of cells with a solution of salts, detergent, and ball bearings. Then a...nucleic acids, so placing the dipstick into a wash solution removes contaminants while nucleic acids stick...

...create. Wildlife biology and ecology can make for beautifully compelling photos and videos, while molecular...platforms can splinter audience. Primarily digital distribution (though radio is possible.) Written works...works Low Flexible. Low barrier to entry. Ample distribution channels both digitally and physically. Easy...overwhelming your audience. Digital or print distribution.  Static. Can be difficult to convey an entire...highlighting, or omitting details. Digital or print distribution.  Static. Often requires both artistic and scientific...overlooked as a means of scicomm. Digital or print distribution.  Typically limited to a single page of information...