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... map the turnover of Langerhans cells in the dermis and identify that Langerhans cells are replenished...membrane-tethered CFP (cyan). Brainbow 1.1 adds OFP (orange) (Livet et al., 2007). Brainbow uses a Cre/lox ...respectively, cells will fluoresce either red or orange until recombination occurs. Following the development...incorporates the highly photostable fluorophores mOrange2, EGFP, and mKate2 (red) to solve the problematically... to develop a system for tracking the fate of Langerhans cells. In the Ubow mouse, cells expressing YFP...through the proliferation of specific, and not all, Langerhans cells, to create foci of proliferating cells...., Clément, et al. "Multicolor fate mapping of Langerhans cell homeostasis." Journal of Experimental Medicine...

..., 343-345. PubMed PMID: 19363495. 2. Wang JW, Wang A, Li K, Wang B, Jin S, Reiser M, Lockey RF. CRISPR... the 5’ ends of the fragment, generating long overhangs which allows the single stranded regions with ...described in a recent publication by the Lockey lab (Wang, et al. 2015). In this case, rather than using a... along with CRISPR was also described recently (Jiang et al. 2015), in which very large fragments of the... it a try! References: 1. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO. Enzymatic...BioTechniques 2015; 58:161-170. PubMed PMID: 25861928. 3. Jiang W, Zhao X, Gabrieli T, Lou C, Ebenstein Y, Zhu TF...

...Mi-Yoon Chang, Baek-Soo Han, Sanghyeok Ko, Eungi Yang, Kwang Yul Cha, Robert Lanza, and Kwang-Soo Kim.... 2677165. 3. Jia, Fangjun, Kitchener D. Wilson, Ning Sun, Deepak M. Gupta, Mei Huang, Zongjin Li, Nicholas...includes measles and mumps. Sendai transduces a wide range of cell types and replicates in the cytoplasm independent...using EBNA1 based vectors is that the plasmid can hang around for awhile, even after selection is removed...

...enzyme, or enzymes that have compatible overhangs or no overhangs after digestion, you will need to use ...with restriction enzymes to generate compatible overhangs that allow them to bind. Ligase is used to make...ended up in the direction you want - usually by Sanger sequencing or further restriction digests. Of course...inserts cut out of it and your new insert from any overhangs cut off of it via their different migration speeds...insert in a single tube allowing the compatible overhangs generated by restriction digestion to anneal to...only one enzyme or used enzymes with compatible overhangs for your ligation, then you will need to verify...appropriately sized insert, you should send it for sanger sequencing using primers that will allow you to...

...fluorescent output, which will turn on, change color, or change intensity of fluorescence in the presence...receptor, but readouts could involve fluorescence, pH change, heat, etc. The last component of the biosensor...engineered to detect a wide array of neurotransmitters ranging from dopamine to glutamate, all the way to acetylcholine...fluorescence-based and have been optimized for a wide-range of uses to monitor the fundamental cellular building...actinin will produce different signals based on changes to the structure of actinin in low vs. high tension...real-time cellular reactions to stimuli and as well as changes in cellular dynamics during so-called housekeeping...

...Figure 2: The steps of colony PCR.   Sanger sequencing Sanger sequencing determines the precise order... you'll want to design a custom primer. Because Sanger sequencing can usually sequence just 1 kb of DNA...post for tips for analyzing and troubleshooting Sanger sequencing results. At Addgene, we now use next-generation...that NGS verification is more time-intensive than Sanger sequencing or the other techniques mentioned above...Learn more about Sanger sequencing Read the Molecular Biology Reference ... Jennifer Tsang...

...CRISPR technology has changed how scientists edit and control genes, but according to the Broad Institute's...December, Konermann and her colleagues in Feng Zhang’s lab describe just such a system, which they call...perfect complement for one conducted earlier by the Zhang lab for GeCKO, a lentiviral CRISPR library enabling...enabling genome-scale, knockout screening). As the Zhang lab's Nature paper reports, “Here we have shown ...in the genome for gene control over a 1,000-fold range. Weissman explained that there are two levels of... Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Cell. 2014 Feb 27;156(5):935-49. doi... Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F. Nature. 2015 Jan 29;517(7536):583-8. doi: 10.1038...

...transformed the palette of experiments possible in a wide range of organisms and cell lines. In C. elegans, one ...at 25 ºC. This method resulted in efficiencies ranging from 11-100% of F1 animals carrying precise knock-ins...This stringent selection allowed me to optimize a range of editing parameters: oligo repair templates with...injected animal.  PAM sequence considerations For changes 5’ to the PAM, sequences corresponding to the same...locus (CRISPR-ChAP-MS) could further transform the range of experiments imaginable in C. elegans. Equally... and systems, allowing rapid progress in a wide range of fields.   Thank you to our guest blogger! Jordan...//doi.org/10.1534/genetics.118.301532 Ebbing A, Shang P, Geijsen N, Korswagen HC (2017) Extending the ...

...through filtration methods because of its ability to change shape and the absence of a rigid cell wall. Here.... For testing, the medium should not have been changed for at least two or three days before sampling ...commonly infecting mycoplasmas are used. A broad range of primers encompasses almost all the infecting ...chemical reactions and this induces a noticeable color change. This method has high sensitivity but low specificity...are working without gloves or masks. Make sure to change the gloves and masks regularly instead of keeping...water provided to maintain the humidity should be changed regularly as mycoplasma, bacteria, and fungus can...contamination, easy to use, and effective for wide range of mycoplasma. These methods cannot guarantee complete...

...set-up has been changed (for instance after replacing a broken lightbulb or exchanging excitation filters...fluorescent proteins may display reversible intensity changes (Shaner et al, 2008; Bindels et al, 2017) and photoswitching...fluorescence signal, showing no or little deterioration or change of the signal during the course of the experiment...and by comparing how the fluorescence intensity changes over time, the photostability of the fluorescent...comparable (lower panel of figure 1). To avoid changes in excitation power, it is important to perform...filters). This avoids substantial changes in excitation power and will help to keep the photostability of...

...specific tissues where they can scan for any signs of danger. In this post we’ll focus on how fluorescent proteins...GFP and its derivatives by the late Roger Tsien changed everything. For the first time, fluorescent proteins...Blue-green fluorescent proteins like EGFP Yellow-orange fluorescent proteins like TagRFP, tdTomato, DsRed...tdKatushka2 (Drobizhev et al. 2011) The commonly used orange and red fluorescent proteins are excited by 750...imaging studies with blue or cyan proteins without changing excitation wavelength (Salomonnson et al. 2012...it has been shown that even a small incremental change of the excitation wavelength can significantly ...Ubow mouse strain has been created to fate map Langerhans cells and follicular dendritic cells within the...

...in comparison to protein-coding genes (Rinn and Chang 2012). Scientists have determined the function ...completely disrupt expression of the transcribed ncRNA (Jiang et al. 2014).  Simple knock-out experiments are .... doi:10.3390/ncrna7040079 Horlbeck MA, Liu SJ, Chang HY, Lim DA, Weissman JS. Fitness effects of CRISPR...(5):573. doi:10.1038/s41587-020-0428-0 Rinn JL, Chang HY. Genome Regulation by Long Noncoding RNAs. Annual...10.1146/annurev-biochem-051410-092902 Liu Y, Cao Z, Wang Y, et al. Genome-wide screening for functional long...Nature. 2016;539(7629):452. doi:10.1038/nature20149 Jiang Q, Meng X, Meng L, et al. Small indels induced by...2015;6. doi:10.3389/fpls.2015.01001 Ho TT, Zhou N, Huang J, et al. Targeting non-coding RNAs with the CRISPR...

... By Jennifer Tsang GFP-based Touching Nexus (G-baToN) is a new tool developed by Rui Tang in Monte Winslow...the sender cell and the receiver cell. Image from Tang et al., 2020.   G-baToN can also be applied ...modulating the interactions between cells.      Tang et al., eLife 2020. https://doi.org/10.7554/eLife...conditionally degrade proteins of interest in cells (Zhang et al., 2015). Recently the Ward lab created a tool-kit...genetics/iyab006       The CRISPR corner By Jennifer Tsang Here are a few highlights from recent CRISPR plasmids...of Cas enzymes. CRISPR-ARE is a new system from Jiazhang Lian’s lab that enables simultaneous gene activation...

...Note: Cpf1 is also called Cas12a. In 2015, Feng Zhang’s lab characterized two Cpf1 nucleases, distant cousins... staggered fashion, creating a 5 nucleotide 5’ overhang 18-23 bases away from the PAM. Cpf1’s staggered...gene delivery due to their low immunogenicity and range of serotypes allowing preferential infection of ...regulatory elements. To solve this problem, the Zhang lab has previously used SaCas9 (1053 aa/3.2 kb) ...transgene insertion mediated by Cpf1-generated overhangs. Exploring the efficiency of these two processes...SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F. Cpf1 Is a Single RNA-Guided Endonuclease of a...Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, Barrangou R, Brouns SJ, Charpentier E, Haft DH, Horvath ...

...a cell and understand how protein localization changes over time.Hallmarks of fluorescent protein timers...they predominantly fluoresce a second color. This change is usually hypothesized to be a result of chromophore...embryos. Of course, timers may be sensitive to changes in oxygen, temperature, and pH, but timer activity...and the formation of limbs are correlated with changes in gene expression. In a developing embryo, for...long-lived transcription factors control gross changes in gene expression as new body sections are formed...Alexey, et al. ""Fluorescent timer": protein that changes color with time." Science 290.5496 (2000): 1585...Fedor V., et al. "Monomeric fluorescent timers that change color from blue to red report on cellular trafficking...

...Did you notice the “Filter By…” options changed when we changed from searching for viral preps instead ...assembled a series of screenshots to highlight changes to both the design and functionality of searches...of plasmids? The available filtering options now change dynamically based on the type of material and specific...for details). The following screenshots show the range of different terms we can use to find materials ...exact plasmid you need. The ability to apply a wide range of filters isn’t new to Addgene’s plasmid search...you may find the available categories and filters change as we now only show options relevant to your current...

...the human genome Wang, T., Antonacci-Fulton, L., Howe, K. et al. The Human Pangenome Project: a global...diligent scientist, they sought to understand these strange results by digging deeply into their data.   ...Samples are divided into those of African ancestry(orange) and those of other ancestry groups (grey). ...Cancer Data Science team at Broad, they were able to change their analysis to use a corrected genome map. Now...ignored. Working with Lia Petronio and Katherine Huang from Broad’s Pattern team, they designed a tool ...Boyle, Joshua Dempster, Lia Petronio, Katherine Huang, Alham Saadat, Thomas Green, Adam Brown, John G ...

...SpCas9 variants effectively double the targeting range of CRISPR/Cas9 within the human genome. Another ...guide or leave a comment below. Note: Jennifer Tsang and Mary Gearing contributed to the update of this...Kellner MJ, Regev A, Lander ES, Voytas DF, Ting AY, Zhang F (2017) RNA targeting with CRISPR–Cas13. Nature... Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang F (2017) RNA editing with CRISPR-Cas13. Science ...publication at Addgene. Hu JH, Miller SM, Geurts MH, Tang W, Chen L, Sun N, Zeina CM, Gao X, Rees HA, Lin ...Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F (2015) In vivo genome editing using Staphylococcus...SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F (2015) Cpf1 Is a Single RNA-Guided Endonuclease...

...Transient reporter for editing enrichment (TREE) The Wang and Brafman lab developed a transient reporter for...’ after a C-to-T base editing event. This edit changes the histidine to a tyrosine generating a GFP variant...target specific loci of interest in the genome. The Wang and Brafman lab showed that GFP positive cells isolated...subsequent base editing efficiency. TLDR: TREE from the Wang and Brafman lab uses an engineered BFP variant that...https://doi.org/10.1093/nar/gkaa124 Martin AS, Salamango DJ, Serebrenik AA, Shaban NM, Brown WL, Harris....org/10.1038/s41598-018-36739-9 St. Martin A, Salamango D, Serebrenik A, Shaban N, Brown WL, Donati F,...K, Tekel SJ, Brookhouser N, Schwarz G, Nguyen T, Wang X, Brafman DA (2019) A transient reporter for editing...

...2.7 to Python 3.5. There are significant changes to the language and it took us a total of two years, from...of 40 minutes to 10 minutes. While users can’t tangibly see these improvements, you are benefited with...the flexibility to adapt and easily respond to changing needs of the teams at Addgene. Another advantage...only do the dishes once a month, resulting in a dangerous mountain of dirty dishes or you could choose to...updated in several years) or they make significant changes that make it incompatible with our software. When...you, which brings me to my next analogy. Human languages evolve over time, for example from Ancient Greek...