...mutations in RPE65 (n=3), showed significant improvements in vision over a period of 1 to 3 years – the treatment...neutralizing antibodies against some AAV types, AAV1, 2, 3 and 5 are thought to be as high as 70% among the human...PMID: 20463753. PubMed Central PMCID: PMC3152950. 3. Jacobson, Samuel G., et al. "Improvement and decline...passionate about HIV cure research and science communication.     References 1. Gaspar, H. B., S. Howe, and...affinity-purified receptor-targeted viral vectors." Nature communications 6 (2015): ncomms7246. PubMed PMID: 25665714...our AAV Production Protocol Check out our AAV Purification Protocol ...

...yeast as an alternative source of essential omega-3 fatty acids complementing vegetable oil, a somewhat...PMID: 27984002. 2. Bilan, Dmitry S. et al. “HyPer-3: A Genetically Encoded H2O2 Probe with Improved Performance...Lifetime Imaging” ACS Chemical Biology 2013 Mar 15;8(3):535-42. PubMed PMID: 23256573. Additional Resources...scientists work on projects spanning from the identification of plant compounds and insecticidal proteins...bacteria. Learn more about the different CRISPR applications in plants.     Transgenic yeast as source of... yeast capable of desaturating fatty acids. Identification of Insecticidal Proteins Linh Bao Ton (M. Sc...Arturo Rivera Solís, Universidad Autónoma de Yucatán, Mexico Dr. Rodrigo Arturo Rivera Solís, Associate...

...in plants. When expressed, the construct produces 3 enzymes from a single mRNA using 2a sites. These convert...Betacyanin is produced from tyrosine by the activity of 3 enzymes and a spontaneous step. (B) RUBY is a reporter...collect seed for further experiments.     Fig. 3: (Top right) A RUBY-expressing Arabidopsis seed following...markers for plant transformation fall into two main categories: antibiotic/herbicide resistance and fluorescent...culture), which is great for a wide variety of applications. But some techniques require you to select against...these mutant plants. Using RUBY to simplify the identification of these mutants will speed up my work and ...student studying plant development and cell-cell communication in the Nimchuk Lab in the Biology Department...

...deciding which one to buy. Table 3: Comparison of popular plasmid purification kit options Manufacturer...it easier to compare kits at a quick glance, Table 3 below contains details regarding kits from a variety...of some commonly used kits, please note that Table 3 is certainly not comprehensive — there are many other...column-based DNA purification methods perform well in terms of quality and yield. Bead-based purifications tend to...standard purification kits, there’s a range of specialty kits available for plasmid purification. Kits can...optimized for purification of these large plasmids, while others have suggested modifications to their normal...you’re new to using purification kits, or if you’re struggling with a purification, reading the full instruction...

...imaged the mouse brains, they found many more than 3-4 colors due to the tandem integration of multiple...colorful technology. Optimizing the system: Brainbow-3 Although Brainbow provided neuroscientists with the...Looger LL, Simpson JH. Nature Methods. 2011 Mar;8(3):253-9. doi: 10.1038/nmeth.1566. Epub 2011 Feb 6. ...Dickson BJ, Salecker I. Nature Methods. 2011 Mar;8(3):260-6. doi: 10.1038/nmeth.1567. Epub 2011 Feb 6. ...Beaurepaire E, Morin X, Livet J. Neuron. 2014 Feb 5;81(3):505-20. PubMed.   ...of farnesylated derivatives enables labeling of delicate axonal and dendritic processes not previously ...Between Brainbow Systems   Brainbow variants and applications beyond the mouse brain In addition to improving...

...DNA primers that bind upstream (5’) and downstream (3’) of the DNA region of interest. Primers are crucial... the dNTPs.  Let’s jump into the specifics of the 3 basic steps of a PCR reaction:  Denaturation- To...extension is repeated 25-35 times to exponentially replicate the target DNA of interest. This entire reaction...have been developed for different scientific applications. These PCR methods all use the same basic PCR...the final cycles which would slow or stop PCR amplification. Other times, the PCR reaction may not be 100%...quantified by qPCR. qPCR is used in a variety of applications including gene expression profiling, studying...concentration, unlike qPCR where results can vary across replicates. ddPCR can be used to quantify DNA sequences ...

...organs. Restraint: scruffing Location: peritoneal cavity Gauge: 25–27 Volume: < 2–3 ml Skill required: intermediate...or mechanical restraint Location: back or hind legs Gauge: 25–27 Volume: < 2–3 ml (~1 ml per site) Skill...Intraperitoneal Scruffing Peritoneal cavity 25–27 < 2–3 mL Intermediate Intramuscular Mechanical restrainer...Back or hind legs; between skin and muscle 25–27 < 2–3 mL (~1 mL per site) Beginner Intradermal Anesthesia.... It sounds complicated but has a pretty low learning curve! Restraint: scruffing Location: throat Gauge... Restraint: mechanical restraint or anesthesia Location: tail vein or retro-orbitally Gauge: 27–30 Volume...practice to ensure the proper injection site has been located and to avoid potential off-target pokes to nearby...

...we look closely at Figure 3, which shows requested CRISPR plasmids categorized by “age” or year they were...    Figure 3: Percentage of requests per year for CRISPR plasmids categorized by year of deposit...2, and the longevity of tools seen in Figure 3, indicates that the usefulness of popular CRISPR tools ...plasmids requested each year by year of deposit (Figure 3), we saw that once CRISPR plasmids become popular,...estimated scatterplot smoothing. (C) Number of requests per year for pooled libraries, categorized by function...insertion via a chemical modifier. We used these categories to see what kinds of CRISPR tools people were... pooled libraries, although we used different categories (Figure 2C). Note that in Figure 2, the numbers...

...sequence (Zong et al., 2018) (see CBE-3 in Figure 3). Figure 3: Activity windows of plant base editors...position 4-8 counting the ‘NGG’ PAM as 21-23 (Figure 3). If the target ‘A’ is closer to the PAM, i.e., positioned... 353.6305 (2016): aaf8729. PubMed PMID: 27492474. 3. Gaudelli, Nicole M., et al. "Programmable base editing...          The artificially evolved adenosine deaminase catalyzes the conversion of a target ‘A’ to ‘I’ (inosine... optimize base editing efficiency based on the location of the ‘A’ relative to the protospacer adjacent...Choose a base editing platform depending on the location of your target base in the protospacer sequence...

... point for a new generation of ABEs), consists of 3 components:  a deaminase (TadA, originally from E.coli...al., 2020). The generated ABE8s needed to produce 3 concurrent base edits to withstand antibiotic selection...protospacer adjacent motif (PAM) made up of 5′-NGG-3′ (“N” can be any nucleotide base, followed by 2 guanines...ABE8.20-m was the winner. It could simultaneously edit 3 genes (98.1%, 98.3%, or 98.6% efficiency, respectively... E.coli, named TadA7.10 in ABE7.10)  a catalytically impaired Cas protein (dCas or Cas nickase)  a guide...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...

... Figure 3: Overview of library generation and screening that led to identification and characterization...: 38036853. Preprint: https://doi.org/10.21203/rs.3.rs-2941917/v1.  Ivorra-Molla, E., et al. (2023). A...: 38081970. Preprint: https://doi.org/10.21203/rs.3.rs-2684100/v1.  Piatkevich, K., et al. (2023). Bright...ResearchSquare (Preprint). https://doi.org/10.21203/rs.3.rs-3188559/v1.  Neurogenetic alchemy: CAP-Mac unleashes..., its dimeric nature can interfere with some applications. Several labs have been working to produce monomeric... for molecular fusion and membrane-targeting applications. Nature Methods, 1–9. https://doi.org/10.1038...rodents and broad tropism in primates. Nature Communications, 14(1), 3345. https://doi.org/10.1038/s41467...

...Figure 1, large sequences corresponding to exons 1 and 3 of the targeted gene have been cloned into the vector...Neomycin resistance (NeoR) gene between exon 1 and 3 of the targeted gene. Upon recombination, the GFP/...-PGK-Puro from the Jaenisch lab.   Figure 3: A knockout/knock-in targeting vector designed to ...resistance marker using Cre recombinase. Since there are 3 loxP sites, recombination can occur in multiple ways...in line 4 of Figure 4. Since the loxP sites are located in intronic regions, this gene will still be expressed...

...molecular targets under research include TIM-3, LAG-3, STAT-3, EGFR, CD20 and CD73 on specific solid as ...cell-surface antigen production to evade the immune system. 3. Escape - Tumor cells that survive this phase are ... situ before being injected back into the body in 3 doses intravenously. B cells and T cells activated...medicine 10.9 (2004): 942-949. PubMed PMID: 15322536. 3. Dunn, Gavin P., et al. "Cancer immunoediting: from...immune response to the development of tumors is a complicated and dynamic phenomenon that is interestingly ...effects, damage healthy cells, and often cannot eradicate metastasis or eliminate the probability of a relapse...based on their level of progression, anatomical location, characteristic (solid localized tumors or systemic...

... writing for the Addgene blog, sharing Chemistry Cat memes was how I used social media as a scientist....needed to start using Twitter. Sharing Chemistry Cat memes use to be how I used social media as a scientist...can also find Dr. Knoepfler on Twitter: @pknoepfle 3. "Social Media as a Scientist: A Very Quick Guide"...tips for using various social media platforms, and 3) helped you fit social media into your busy schedule...remotely as an Addgene Science Communication Intern. Developing science communication skills, while also learning... on a panel about “Social Media for Science Communication” at the American Society for Cell Biology (ASCB...Twitter Learn about using Reddit for science communication ...

... et al. created the 3rd generation system (Figure 3), which differs from the 2nd generation in two key...SIN) lentiviral vectors contain a deletion in the 3’ LTR that prevents aberrant activation of nearby genes...PubMed PMID: 9765382. PubMed Central PMCID: PMC110254. 3. In-vivo RGB marking and multicolour single-cell tracking...RA, Berinstein EM, Medin JA. Immunotherapy. 2015;7(3):271-84. PubMed PMID: 25804479. 9. Development of ...many CRISPR applications, and they’ve also had success in clinical gene therapy applications. Read on to...to learn more about the current (and future) applications of lentiviral vectors! Lentiviral vector history...original system, the possibility of creating a replication-competent virus via recombination between the...

...incubating the columns with 1M phosphoric acid for 3 minutes followed by a short centrifugation for a total...ethanol. After regeneration, trace amounts of DNA (3 femtograms/ul) could still be eluted from the column... DNA purification without a kit as well as one way to reuse silica columns from DNA purification kits....consider some ways to purify DNA without a kit. DNA purification kits have advantages: they are convenient and...because you’ve run out of columns. Kit-less DNA purification methods avoid many of the drawbacks of kits ... and then rehydrated. There are some slight modifications depending on the type of DNA you’re purifying...After this, the DNA is ready for use! Plasmid purification and DNA gel extraction with glass syringe filters...

...genome manipulation. Nature Reviews Genetics, 24(3), Article 3. https://doi.org/10.1038/s41576-022-00541-1 ...over a thousand genomic locations. This allowed us to train ePRIDICT (Figure 3), a model that predicts...influence editing results at your target. Figure 3: High-throughput screening of the effect of chromatin...genome editing technology that allows precise modifications of DNA (replacements, small insertions, and ...configurations can be used to introduce specific target modifications. Since this means we could design hundreds to...affects prime editing outcomes across many genomic locations. In our study, we used the TRIP (Thousands of ...

...as a reference band. The uncut DNA (lane 2) shows 3 possible plasmid conformations, with relaxed and nicked...The double digest with both HindIII and BamHI (lane 3) produces bands at 6kb and 1.2kb (red box), matching... and run on an agarose gel, you are likely to see 3 bands. This is due to the fact that the circular DNA... I hope these tips demonstrate that plasmid verification is not just necessary but also an easy process.... Please vist Addgene's resource for plasmid verification to find additional tips and detailed protocols...

...contains two sets of sgRNAs; A, the top 3 ranked sgRNAs, and B, the next 3 ranked sgRNAs. Dolcetto was able ...dsDNA cutting. A Dolcetto library containing only 3 sgRNAs (setA) also outperformed CRISPRi libraries ...PMID: 26780180. PubMed Central PMCID: PMC4744125.  3. Gilbert, Luke A., et al. "Genome-scale CRISPR-mediated...is only effective when sgRNAs target a specific location on the gene. For CRISPRa, sgRNAs were targeted...Calabrese pooled library and lentiviral prep here! Applications for the Root and Doench CRISPR libraries Overall...et al. "A protein-tagging system for signal amplification in gene expression and fluorescence imaging....

...) 0:00-1:00 : Intro 1:01-3:48 : Kingdom Kwapata's background and education 3:49-4:35 : Kingdom on opportunities...Born to a family in Malawi that pushed science education, Kingdom Kwapata grew up to be one of the best...