...the possibility of directional gene transfer, analogous to traditional restriction enzyme cloning. Sticky-end...and requires only one RNA rather than the two (tracrRNA and crRNA) needed by Cas9 for cleavage. In certain... Cas9’s shortcomings - namely its blunt double stranded cleavage and G-rich PAM requirement. Zetsche et...target. Cpf1 also does not require an additional tracrRNA. Once they had determined the minimal elements...Sticky-end mediated gene transfer would be particularly helpful for targeting non-dividing cells, which are...favored by SpCas9. Since Cpf1 doesn’t require a tracrRNA, crRNA guides are only ~42 nt long. Direct synthesis...significantly cheaper than that of the ~100 nt crRNA/tracrRNA hybrid guides needed for Cas9 function. Since ...

...proteins needed for the virus to replicate,  and the transfer plasmid which contains the viral genome. In today...and cap are expressed from a separate plasmid in trans, freeing up space in the viral genome so that a ...Replication initiates from the AAV genome ITRs on the transfer plasmid, which contain regions of dsDNA. To generate...its endonuclease activity to generates a ssDNA substrate which is then nicked by its endonuclease domain...capsid. Rep proteins are also involved in the transcriptional regulation of the AAV p40 promoter which drives...into a capsid other than AAV2. This production strategy is called cross-packaging.  Capsid (cap) gene ...promoter. Alternative splicing and differing translational start sites generate the three VP proteins. ...

...Pubmed PMID: 24463574. Pubmed Central PMCID: PMC3988262. 4. Hart, Traver, et al. "Measuring error rates...flaws of RNAi screens are well characterized and strategies exist to control for these faults. However, it...to patterns seen in single-guide studies? Many strategies exist for reducing Cas9 off-target effects, but...DNase hypersensitivity, or are in an enhancer, transcription factor binding site, or a coding region. Targeting...on- and off-target effects, but also presents a strategy to control for these effects. By testing thousands...generalizable conclusions about off-target activity, demonstrate safe-targeting guides as a potential control,...8 (2017): 15178. Pubmed PMID: 28474669. Pubmed Central PMCID: PMC5424143. 2. Tsai, Shengdar Q., et al....

...difference. Traces The Traces tab shows the edited and control, non-edited Sanger traces in the region...as determined by comparing the edited trace to the control trace. In the ICE algorithm, potential editing...the targeted gene.   Analyze Complex Edits   Traditional Sanger sequencing-based analysis tools are unable...and Sanger sequenced. ICE compares these sequence traces to give a detailed analysis of CRISPR editing. ...result from single or multiplex CRISPR-Cas9 double-strand DNA breaks using SpCas9. More features will be ...three tabs - Contributions, Indel Distribution and Traces - provides particular details about the indel profile...the reference sequence derived from the control trace file. On the plot, the green (edited sample) and...

... to the administration of postdocs, often at a departmental level rather than being centrally administered...research in the U.S. and postdoctoral training has not been demonstrated to be required for the jobs that ...scientists are paid above this threshold or for tracking the hours that these scientists work and paying...across academia). Increased postdoc wages, overtime tracking? What are institutions doing? Institutions are...exactly how postdoctoral salaries are changing. Transparent salary information for postdoctoral positions...their salary raised 101% if hours are not to be tracked. University of Buffalo and Ohio State both have...exemption level. Not only is this in direct contradiction to explicit policies set up other institutions...

...abscessus is first transformed with Cas9 on an integrative plasmid containing tetracycline repressor TetR ...resistance selection marker KanR. This strain is then transformed with the sgRNA cassette on a plasmid ...luminescent reaction with the substrate furimazine. Right: Demonstration of PILATeS. Cell adhesion to ...multiple myeloma drug mezigdomide and ubiquitination substrate Ikaros (IKZF1ZF2) (B). Image adapted from Kroupova...9% of colonies (102 to 104 times higher than traditional homologous recombination methods). By using mCherry... mCherry for selection of sgRNA plasmid transformants, they avoid M. abscessus’ high rate of spontaneous...workflow in M. abscessus. A) Sth1Cas9 mediates double strand break at the target site, which results in either...

... protein of interest, such as a sample transiently transfected or stably infected with the protein of ...downregulated, examine a protein’s stability or post-translational modifications, or study how a target protein... two ways. Both methods utilize the same basic strategy: immobilize the antibody or antibody-target protein... two different approaches allow for optimized strategies for differing amounts of target proteins or antibody...strengths.    . Steps of an IP: 1) Protein extracts from cells or tissues contain a complex mix of... needed. Since many factors, including salt concentration, ionic strength and pH, affect binding the lysis...antibody and is immobilized. The ideal antibody concentration to use will vary between capture antibodies ...

...plasmids, investigating different viral production strategies or developing novel methods for data analysis...inhibitor sucrose. Previous work by Stoneham et al. demonstrated that the entry of AAV2 into target cells is ...formulation buffers, this sugar may be a more attractive choice for labs looking to improve their AAV ...wanted to be certain that this supplementation strategy would have no effect on the purity or infectivity...preparation. Through sugar detection assays, in vitro transduction, and silver stain approaches we show that there...effect on our overall yield, we were unable to demonstrate that these results were, in fact, due to impaired...test the endocytosis of a fluorescent dye and transduction of GFP-expressing AAV particles we did not observe...

...antibody stability, half-life, tumor penetration, inadequate concentration, mutation of cell surface targets...infections, or other strenuous biological changes. In contrast to the acute immune responses that eliminate early...associated with the onset of colorectal cancer. Strategic immune evasion mechanisms - The tumor microenvironment...relapse. Chemical and radiation therapies cause a transient shutdown of the immune system, hampering its ability...of even these conventional antitumor treatment strategies can be enhanced. Types of immunotherapeutics ...being injected back into the body in 3 doses intravenously. B cells and T cells activated ex vivo are also...susceptible to elimination by T cells. Immunotherapy strategies that synergize with these positive impacts on...

...from the OD and concentration of the known samples is used to calculate the concentration of protein in ...cells. They bind to proteins on the surface of extracellular pathogens such as parasites or microbes, or ... methods. Each type of antibody and production strategy has advantages and disadvantages compared to each...from the animal is drawn and the antibodies are extracted. These antibodies recognize a variety of epitopes...Hybridomas are formed by fusing B cells that have been extracted from animals injected with protein antigen to ...molecular weight by SDS-PAGE, and are subsequently transferred to a membrane. A primary antibody recognizing...appropriate controls for normalizing the protein concentration. To get even more details, check out our article...

...that green fluorescent protein through transcription and translation. Unfortunately, having live cells in...being able to actually hold all those seemingly abstract biological reactions that I learned about from... like gel electrophoresis to separate DNA or transforming cells with green fluorescent protein, these ... Making proteins outside of a cell - cell free extracts Typically, if you want to make a protein – say... into a host cell (through a process called transformation), essentially giving the cell all the instructions...perhaps consider using some elements of BioBits to transition your undergraduate students from the classroom...give your undergrads hands-on experience with the central dogma while also giving them practice with practical...

...Library amplification seems straightforward: E. coli bacteria are transformed with the library DNA, they... Each Addgene library has its own depositor transformation and amplification protocol, but the overall...Maintain representation of the library during transformation First, use enough DNA and competent bacteria...and we use electroporation to ensure a good transformation efficiency. For a typical library, Addgene ...growing the cells on solid plates, where each transformant forms its own colony, uninfluenced by the growth...concern, but Addgene still recommends plating the transformation mixture on solid media. The colonies are then...then scraped off the plates and pelleted for DNA extraction. Prepare library product for next-generation ...

...a repressible binary system for transgene expression, lineage tracing, and mosaic analysis., Cell 141,...more detailed level, the Gal4/UAS system is a transcription activation system co-opted from yeast (1). It...workhorse to turn genes on or off. Gal4 is a transcriptional activator that binds to UAS enhancer sequences...sequences found in DNA. It then recruits transcription machinery to the site to induce gene expression. Thus... my next post, I’ll explain the relatively straightforward process of generating your own fly lines. In...Kagan, Environmental Stress Causes Lethal Neuro-Trauma during Asymptomatic Viral Infections, Cell Host...9, 47–55 (2012). PubMed PMID: 22205518. PubMed Central PMCID: PMC3574576. 7. S.-L. Lai, T. Lee, Genetic...

...the hACE2 transgenic mouse strain, for example, is sustained by the availability of this strain from The..., they remain accessible during these times. Centralized reagent repositories, also known as biobanks,... Biological repositories that distribute mouse strains, plasmids, cell lines, and much more have been ...19 plasmids and resources! Case study 3: Mouse strains Reagents developed during the SARS-CoV pandemic...University of Iowa (McCray et al., 2006), the hACE2 transgenic mice were designed after it was discovered that...appearing with regards to SARS-CoV-2, this mouse strain is regaining popularity. The Jackson Laboratory...institution, has been able to rapidly propagate this strain, making it available to scientists around the world...

...antibodies from an Andean mammal called alpaca could neutralize the SARS-CoV-2 virus. But while the early results...facility needed to perform the experiments to demonstrate that this approach was useful in humans. Then...preprint repository BioRxiv. This example clearly illustrates the power of collaboration, which despite the...help researchers working on COVID-19. Its origins trace back to March 2020, when the first coronavirus lockdown.... None of us works directly with viruses or transmissible diseases, so we were left with no choice but...simpler and the volunteers helped with things like translations, bibliographic searches, etc.     Expanding...we call “asymmetric collaborations.” These are transient partnerships where a researcher makes a small ...

... beliefs. I asked participants to read short abstracts comparing the mortality rates between two controversial...peer-reviewed study. The second group read the same abstract but with the names reversed, meaning that the ...reversed. The third group read a version of the abstract where the real medical conditions had been swapped...asked to evaluate the quality of the study those abstracts represented. The point of this study was to see...see if their evaluations of the abstracts were driven by the quality of the study design or by their preconceived... significantly more favourable ratings to the abstracts that affirmed their personal beliefs even though...identical. Likewise, when participants read an abstract for a study with findings that challenged their...

...RNaseP. Although the Gao Lab demonstrated efficient MBE with such a strategy, the need for lengthy promoters..., lengthy genetic payloads and insufficient transcription on the array have limited the scalability and...multiplex gene editing. Moreover, existing multiplex strategies have been facing difficulties in pairing with...both efficiency and multiplexity, with these constraints? The Gao lab first thought to enable MBE by ...efficient base editing. They then worked to develop a strategy that would independently generate multiple gRNAs...kinds of CRISPR tools, without compromising their strategic advantages in gene editing, while keeping the ... Led by PhD student Qichen Yuan, the Gao Lab demonstrated that the tRNA-gRNA array can drive the expression...

...Washington Post: Trump administration tells EPA to freeze all grants, contracts The Guardian: Trump to...Formally, a government can be defined as a social contract citizens enter into in order to protect their ...science: “humility in the face of evidence” (4). Frustration among scientists has been rising as evidence ...general public agrees (5, 6). Many of the new administration’s actions, including taking down the climate...lab and march. Unintended consequences While frustration and anxiety have inspired people to rally for... marchers or supporters be scientists – on the contrary, anyone and everyone is welcome. Why? There are...following subjects: Climate change Vaccines GMOs Transgender issues Reproductive health Immigration Pollution...

...plasmids for use in CRISPR and Tol2 transposase induced transgenesis.  Xenopus Xenopus, or frogs, are one...organism in microbiology and biochemistry. Certain strains have been specifically developed to thrive in laboratory... laboratory settings. One of its most attractive features is its stability and easily obtainable growing... areas of microbial research like metabolism. Transport, biofilms, and much more. Because of it’s BSL-...ability to regenerate, the axolotl has several attractive characteristics that make them an ideal model...have created molecular biology tools to generate transgenic axolotls for an array of different studies. Elly...for human disease research. One of their most attractive features are their eggs. Frog eggs are large,...

...dDetermined as effective NIR fluorescence in transiently transfected live HeLa cells with no supply of exogenous...2015). The effective brightness reflects the concentration of BV-containing molecules in a cell and depends...and xenon lamps are the best for working in this spectral range. You can also check that no filters and/...multiscaling NIR-shifted spectrum is perfect for use in spectral multiplexing (Shcherbakova et al., 2018). NIR ...low autofluorescence, low scattering, and deep penetration of red-shifted light. The same NIR FPs and biosensors...in your applications.  There is a whole set of spectrally distinct (e)miRFPs available. The numbers in ...names correspond to the maxima of their emission spectra. These probes can be combined in two-color and ...