...capsids were packed with a GFP transgene and GFP intensity and transduction efficiency of neurons were measured...AAV.PHP.eB and AAV.PHP.S had 1.5- to 2-fold higher GFP intensity compared to the strains they were derived from...Additional Resources on the Addgene Blog Important considerations when using AAVs Learn more about using AAV ...

...inhibit cell cycle regulatory proteins responsible for the transition out of S phase, so cells stay in an...exogenous DNA can be used as well. Below are some considerations for designing donor DNA for successful HDR ...

...Fluorescent Protein? GFP is a ~27 kDa protein consisting of 238 amino acids derived from the crystal jellyfish...point mutation that increased the fluorescence intensity and photostability of GFP. This also shifted its... blog, you should! He's got great articles and insight on the newest tools and techniques in fluorescent...

... Wilson Lab also looked for the gene variant responsible for the PHP.B permissive phenotype in mice. They... Mychal S. Grames, and Ronald L. Klein. "More expansive gene transfer to the rat CNS: AAV PHP. EB vector...user tips from an AAV rookie  Learn about some considerations when using AAVs Learn more about using AAV ...

...exception) necessitates such molecular gymnastics for sensitive applications such as CRISPR screening. The plasmids...are often many more genetic strategies) and then consider whether traditional cDNA overexpression is suited...eBook Resources on Addgene.org Check out our comprehensive CRISPR guide Find CRISPR plasmids and resources...

...?" is an open-ended list of proteins. Instead, consider "how do proteins X and Y regulate the Z pathway...that is easily visible from the back of the room. Consider going beyond the whiteboard. You may wish to create...blood sugar. Find an energy bar that you like and consider eating one before your chalk talk. Bring your ...

...of the desired T-A base pair. Figure 1: Transition mutation (C>T, G>A, A>G, and T>C) installation...also constructed smaller vectors suitable for transient assays using protoplast transformation. Cytosine...72 hours post-transformation/transfection in a transient assay. For stable transformation, regenerated ...

...important to note that in Morgens et al., guides were considered toxic if they were depleted from the screen. ...sites that lack open chromatin marks, DNase hypersensitivity, or are in an enhancer, transcription factor... significant difference in depletion of ricin sensitizer genes identified with short and long guides. ...

... bins together at a mixer. We call this Quorum Sensing, after the phenomenon in bacteria that occurs when...gene expression in response to changes in cell density. At the mixer, they meet other Mentors and Singletons...briannadoesbio) June 22, 2019 Favorable conditions - Expansion of networks The last stage is Exponential Growth...

...When choosing beads, there are several factors to consider. Agarose beads are sponge-like and vary in shape...express the protein of interest, such as a sample transiently transfected or stably infected with the protein...appealing, but the disadvantages must be carefully considered before using. For example, the tag may affect...

... the virus and is less affected.  Altering sensitivity to serum. Different pseudotyped viruses are differentially... protein that binds phosphatidylserine and low-density lipoprotein receptors (LDLRs) on a cell surface...CD, Friedman T, McClure MO, Weiss RA (1997) Sensitization of rhabdo-, lenti-, and spumaviruses to human...

...pTargetF system consists of one plasmid that expresses the S. pyogenes Cas9, a temperature-sensitive replicon...changed the replicon of pCas into a non-temperature-sensitive replicon, and 3) added the sacB gene for counterselection...

...staining and the molecular weight and relative intensity of the viral capsid proteins are analyzed. The...preparation shows that the vast majority of the vectors consist of full particles (white arrowhead) relative to...targeting integrated copies of the lentiviral Rev responsive element (RRE) gene and the cellular ribonuclease...

... can be to get a “bad batch”); Customizable sensitivity and detection - since they frequently undergo...fluorescence for readout may render this approach less sensitive in most applications. Learn About Fluorescent ... sensor could save precious minutes in a time sensitive detection assay. In 1999, Grate and Wilson paired...

...clone. You will also need to use a E. coli strain sensitive to CcdB (e.g. DH5α, TOP10, Mach1). The ccdB gene... the CcdB protein inhibits the growth of CcdB sensitive E. coli strains, most colonies should contain ...sequences, start codon, stop codons, reading frame considerations, etc). This video demonstrates how to use the...

...place and give you some reasons why you should consider using Reddit to share your science. While Reddit...permanent reference by the Winnower. The only small downside to the AMA from my end as an organizer/promoter... general advice on how to meet people, how to transition out of academia, and other science career topics...

...in the door When I was first considering preparing for and transitioning to a career in healthcare consulting...fast turnover of projects, pressure to maintain consistently high levels of performance, and occasionally...

... to solve the problematically low fluorescent intensity of previous versions of Brainbow. Developed from...this system useful for visualization of cellular extensions, such as neurons in the fly visual system, which...cutaneous melanoma induced by mutant BRaf arises from expansion and dedifferentiation of mature pigmented melanocytes...

...donor (e.g., sister chromatid, plasmid, etc.), and consists of three pathways: classical double-strand break...you need to generate a knock-in.   General considerations for designing a repair template to create mutations...cells than their plasmid counterparts. Another consideration for dsDNA templates is their ability to randomly...

...material, which ensures the editor will only be transiently present in transduced cells, limiting off-target.... (2024). Engineered virus-like particles for transient delivery of prime editor ribonucleoprotein complexes...CRISPRoff system — a programmable epigenetic editor consisting of dCas9 fused to a DNA methyltransferase — that...

...to be examined on a case-by-case basis. GFP is sensitive to acid The chromophore of GFP can exist in a ...determined in vitro by measuring the fluorescence intensity of purified protein at different acid concentrations... its fluorescence and therefore GFP cannot be downsized by deleting residues. Only about 10 amino acids...

...vectors are then packaged into AAV virions via transient transfection of 293T cells with helpers, and used...disadvantages to the AAV-based approach that must be considered when deciding whether to use AAV or CRISPR for... is a Professor of Oncology at the Lombardi Comprehensive Cancer Center, Georgetown School of Medicine...

... social science to an audience that I thought consisted of all biologists. The social scientists in the...a solution or overcame obstacles. Take time to consider the order of your narrative. Scientists do this...are worried that this might sound condescending, consider explaining the term on a slide but not reading...

...repetitive sequences, but if present, a minor extension can usually ensure that the 3’-base(s) do not ...manufacturers’ PCR protocol. Be sure to adjust the extension time to match the size of your template. Amplification...PubMed PMID: 2448875. 4. Xia, Yongzhen, et al. "New insights into the QuikChangeTM process guide the use of...

...States government? Politics and science are intrinsically linked: governments exist in reality and science...include science in decision making, it will be considered a success. Below are resources for the march ... “politicized science” ABC News: White House considering gutting 38 EPA programs: draft budget  Science...

...really there are no positive results to be found. Consider the hypothetical situation in Figure 1 where many... (or pre-publish) sound data even if it isn’t considered immediately impactful. Down the line we may see...Scientific Figures into Dashboards and Share Your Insights Who gives a tweet? 9 facts about scientists on...

...industry positions. There were, however, some downsides to working in a large commercial company. In my...sometimes go unnoticed at large organizations. Another downside to large commercial companies which are reliant... treatment outcomes. I would say that the only downside of modern genetics from the perspective of somebody...

...professional training after earning a doctorate Considered a "stepping stone" position to become a professor... lab Undergraduate student Roles and responsibilities can vary "Lab assistants" usually wash glassware...1-2 years) Often a gap-year position taken to transition to the next career stage (ex: grad school, med...

... the discovery of microbial rhodopsins, light-sensitive proteins that absorb photons for energy conversion... neurons in the brain are not naturally light-sensitive, so the selective expression of opsin genes in...neuronal subpopulations, and this represents a major downside in optogenetics (Belzung et al., 2014). This could...

...sequences can be optimized for specificity and sensitivity, for example by changing certain amino acids ... that can pass through each well - the optical density (OD) - of the known and experimental wells is measured...is used to detect proteins on the surface of or inside whole cells. Antibodies - typically, primary antibodies...

...the surface or the inside of your cells. While proteins themselves can emit intrinsic fluorescence when...fixable dye stains that react with free amines on and inside the cells and are thus typically used for distinguishing...

...developed in David Liu’s lab in 2019, has added considerably more precision and flexibility to the CRISPR...engages the prime editor protein. The prime editor consists of a Cas9 nickase fused to a reverse transcriptase...mismatch repair   ✓   ✓ ~ ~ ~ Transiently co-express mismatch repair inhibitor MLH1dn ...

...people will have different roles, titles, and responsibilities during their careers - being aware of this...a prestigious institution: This criterion is consistently under scrutiny. USCIS wants you to prove that...review at the request of your supervisor is not considered peer review. The peer review request must be ...

...gels. They are shared through Addgene without licensing restrictions. Use these instructions to prepare... genome, many applications in biotech, and is considered the most studied Gram-positive bacterium, many...University of Minnesota recently deposited a comprehensive set of plasmids that can be used for either ...

...you plan PEI transfections for AAV packaging, considering using this PEI Calculator. AAV packaging typically...Additional Resources on the Addgene Blog Important Considerations When Using AAVs AAVs Are Great Tools for Gene...

...AAV Packaged on Request, an expansion of our viral vectors service, has officially launched! As of today... excited for all the ways this much-requested expansion will allow us to better serve our community. If...

...developed a platform called SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) to detect single...further to provide multiplexed detection, higher sensitivity, and more. Read the paper in Science Find...

... hard about picking a good mentor, and begin considering career options early. Tune in for great advice...year dip (see graph). Niroshi and Ben give their insights as to what causes this dip. 11:11 - 15:37 : How...

...journals has risen steadily from 1995 to 2013. Considering citations to all articles, Anurag Acharya and...their own libraries and offering another way to consider and quantify the impact of scientific publications...

...ample resources and a positive perspective on the transition to a post-academic career. This training was ... inadequate job of supporting science trainee transition into diverse, successful careers.  We’re not ...

...exemplifies our long-standing commitment to be responsive to the community’s needs and our commitment to...many exciting things planned in 2023, from the expansion of our Data Hub to process improvements in every...

... It is important to identify your audience and consider their background. Social media is a powerful tool...what you are trying to convey without having to extensively use Google search. Be careful about hidden connotations...

...Directors brings human resources experience and insight to the now 13 member board at Addgene. Let’s meet...Addgene blog Learn more about about Addgene in our "Inside Addgene" blog series Subscribe to the Addgene blog...

... another level with the addition of two light-sensitive ingredients - CRY2 and CIB1 – which they borrowed...advantages of the new system: "First, the light-responsive molecules of cryptochrome 2 are the chromophores...

...delivery by lentiviral capsid-based bionanoparticle Transient expression of CRISPR/Cas9 in cells has advantages...for packaging Cas9/gRNA ribonucleoproteins for transient expression. They replaced the tetraloop of the...

...with cells that already have Cas9). Each library consists of approximately 3,000 gRNAs, as opposed to the...still offering the potential for true biological insight. Pooled and individual plasmid formats A first ...

...CRISPR-based technique to generate C->T or G->A transitions in a target sequence in live cells. This technique...endonuclease (dCas9) fused to a deaminase to make the transitions at target sequences specified by gRNAs. Caveats...

...traction. One way to consider where the open access (OA) movement might be going is to consider some of the places...

...sequence variations (e.g. SNPs) exist. Additionally, consider testing multiple gRNAs targeted to the same locus.... As you develop and confirm new gRNAs, please consider submitting their sequences (and plasmids!) so ...

... the spine. For binders of protocols and data, consider using color coded binders and tabs (for different...to protocols in real time. You may also want to consider moving your paper spreadsheets for reagent storage...