...downstream applications. The tradeoffs between various cloning methods are listed in Table 2. For example... TagBFP 399nm/ 456nm histone 2B (H2B) Chromatin 2 Cerulean 433nm/ 475nm Lyn-tag (derived from the tyrosine-protein...based (chaining-based) cloning. The modules (Figure 2) are contained in individual plasmids and are each...suitable for combining endogenous sequences. Table 2: High Throughput Cloning Strategies Method Technology...PMID: 20205762. PubMed Central PMCID: PMC2822740. 2. Engler C, Kandzia R, Marillonnet S. A one pot, one...develop diverse fluorescent reporters is a useful application of high-throughput cloning. MXS Chaining is one...plasmids for fluorescence imaging or flow cytometry applications in mammalian cells. The modules used in this...

...Currently available from the Kim lab are 2 presynaptic and 2 postsynaptic targeting mGRASP plasmids. Additionally...vector and gene primers from Table 2 of the associated publication which contain regions of sequence homology... cells were pulsed with 2µg/ml of Doxycycline for 2 hours, and imaged on the Keyence BZ-9000 fluorescence...work! Robinson et al., Dev Cell 2010 Feb 16;18(2):324-31. Cheeseman et al., J Cell Sci 2013 May 1...promotes the formation of an isopeptide bond between 2 peptide tags, SpyTag and KTag. The group demonstrated... of three sets of cloning vectors (Level 0, 1, or 2) which can be utilized in three successive assembly...1 modules can be similarly assembled into a Level 2 vector, thus forming a functional genetic circuit....

...industrial strain. Not only is MUCICAT better, it is faster. Using MUCICAT only needs 2 newly constructed plasmids... transformation in 30 days. Figure 2: (A) MUCICAT plasmids for multicopy integration. (B) Anhydrotetracycline...plasmids are cured from the cell. Steps 1-1 and 1-2 can be done simultaneously. Image from Li et al., ...1) replaced the promoter of gRNA-pMB1 with PrhaB, 2) changed the replicon of pCas into a non-temperature-sensitive... pEcCas/pEcgRNA system in 4 E. coli K-12 strains, 2 E. coli B strains, 1 E. coli W strain, and 1 Tatumella...far, we’ve used the MUCICAT system in two ways: In our initial paper, we used MUCICAT to optimize the gene...integration using CRISPR-associated transposases” or MUCICAT. Here, we’ll describe each of these CRISPR systems...

...either SARS-CoV-2, Adenovirus type 41 (AdV-41), or Adenovirus-Associated Virus type 2 (AAV2) – or some... was either 1) a physiological effect of SARS-CoV-2 toward destabilizing one’s natural immune response...response to Adenovirus (and/or to its partner, AAV) or 2) a sociological effect of the COVID-19 lockdowns causing...… Chiu, C. Y. (2023). Adeno-associated virus type 2 in US children with acute severe hepatitis. Nature...and late gene transcription) is demarcated by viral genome replication (Harrach, 2014). Given that the...encapsidate new virions) are not expressed. Note that replication-competent AdV vectors also exist and are used...the final topic, which is that natural AdVs, replication-competent viruses with a recombinant genome, ...

... this problem? (2-3 sentences) What will be the focus of your first grant? (1-2 sentences) ...The currency of scientific success is publications and grants. 2. Write succinct and easy to understand...consideration. 3. Present an easy to follow seminar See item 2. Attendees will include grad students (that are solicited...people would agree is important to understand. (1-2 sentences). For example: Distinguishing correctly ... do need multiple first/corresponding author publications in quality journals. It helps to come from the...humbling. My conviction that I had a flair for communication was rapidly dispelled. She would ask me, "Is...succinct. Slide decks leads to the opposite and can suffocate an exciting idea in a swamp of unnecessary details...

...of conventional gene targeting is lowered to about 2-3 months. But certain models remain more difficult...included in the final ssDNA template. Figure 2: Overview of ivTRT to generate an ssDNA repair template... floxed conditional allele requires you to target 2 loxP sites to intronic regions 0.5 - 0.8 kb apart....Typically, Easi-CRISPR works best with insertions of 2 kb or less. Luckily, most of the commonly used cassettes... induction systems (ttA or rtTA) are between 1 to 2 kb in length. There are plenty of Addgene plasmid ...create the ssDNA construct as described in Figure 2.  You can also use Addgene plasmids to purify Cas9... allele To design a knock-in allele at a given location, you’ll need to design only a single guide and...

...United States Standard Occupational Classification System  and Wikipedia. 2. Social and computer scientists...international Twitter users were included in the dataset; 2) users not part of a scientific Twitter list were ...scientists on Twitter are male and 40% are female Figure 2. Gender Ratio of Scientists on Twitter. 60% of scientist...mostly because of this article: “A systematic identification and analysis of scientists on Twitter.” This...: Portion of Tweets with Scientific Domains, Categorized by Discipline. Biologists, neuroscientists, and... Y., & Sugimoto, C. R. (2017). A systematic identification and analysis of scientists on Twitter. Plos...Scientists 5 Reasons to Use Reddit for Science Communication 10 Ways to Share Your Science Why Scientists...

...slash, that indicate the ITR and serotype, respectively. For example, AAV2/8 has a type 2 ITR and a type...type 8 serotype. Almost all vectors contain type 2 ITRs, which comes from the AAV2 serotype. Serotype ...differentiated, non-dividing cells.   Figure 2: DNA rescue and transduction of a conventional single-stranded...quantitative PCR-based method for the titration of AAV-2 vector stocks."Molecular Therapy 6.2 (2002): 272. ...experiments. Recombinant AAVs (rAAVs) The primary application of AAV is delivery of genetic cargo to a host...transduced gene is episomal and does not divide and replicate in the same way the host DNA does. Because of ... since you don’t have to wait for the host to replicate the DNA before expression. Now that you’ve got...

...  Figure 2: Prokaryotic chromosomal replication. Adapted from BioRender template. Since DnaA...Microbiology and Molecular Biology Reviews: MMBR, 62(2), 434–464. https://doi.org/10.1128/MMBR.62.2.434-464.1998...plasmids rely on their host cell's replication machinery in order to replicate—but not always to the same extent...described in our previous Origin of Replication post, DNA replication is initiated at the ori and may or...consider the replication mechanism of plasmids under stringent control, such as pSC101. DNA replication and regulation...review how E. coli normally replicate their chromosomal DNA. Chromosomal replication in bacteria is carefully...entire round of replication. Initiation is the primary control point of DNA replication in bacteria, so...

...conditional knock-in cassette. As shown in Figure 2, the cassette should contain a reporter DNA sequence...targeted neuronal population.    Figure 2 (modified from Schnutgen et al. Nat Biotech 2003): In the ...cellular level in the mouse. 2003 PubMed PMID: 12665802. 2. Branda, CS and Dymecki, SM. Talking about a Revolution... SM. 2008. A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range ...sites directs the Cre recombinase to invert, translocate, or excise a DNA fragment. The availability of... the function of various point mutations and truncations in the absence of the endogenous gene. Spatio-temporal..., allowing for clearer characterization of the location, morphology, and circuit mapping of a targeted...

...Nature communications 8 (2017): 15178. Pubmed PMID: 28474669. Pubmed Central PMCID: PMC5424143. 2. Tsai...guide expression and dsDNA breaks. Figure 2: Non-targeting vs. safe-targeting guides. The genome-wide...target all protein-coding genes present in the genome; 2) non-targeting guides (red), which target no sequence...guides that had zero mismatch off-targets. Guides with 2-bp mismatch off-targets were only toxic if they had...tolerated than mismatches more distal to the PAM, and 2) guides with high GC content have greater off-target... potential control, and provide evidence that truncated gRNAs have improved specificity with only a small...Improving CRISPR-Cas nuclease specificity using truncated guide RNAs." Nature biotechnology 32.3 (2014):...

...//doi.org/10.1002/1096-8628(200022)97:2<147::aid-ajmg6>3.0.co;2-o Baker SA, Chen L, Wilkins AD, Yu P, ...Caused by a mutation in the methyl-CpG binding protein 2 (MECP2) gene, Rett Syndrome is a rare, progressive...X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet 23:185–188 . https://doi.org/10.1038/13810... (2000) Rett syndrome: Methyl-CpG-binding protein 2 mutations and phenotype-genotype correlations. Am ...detrimental as too little, resulting in MECP2 duplication syndrome or Rett Syndrome, respectively. One ...Efficient and Precise CRISPR/Cas9-Mediated MECP2 Modifications in Human-Induced Pluripotent Stem Cells. Front..., Hector RD, Cobb SR, Bird A (2017) Radically truncated MeCP2 rescues Rett syndrome-like neurological ...

...spending time with her horse, and wondering what her cat is thinking. Find her on Twitter @JessicaSacher. ... is also gaining a lot of new traction lately (1, 2), depends on knowledge of the structure(s) a given...per plate) across 20 fresh selective plates. Step 2: Express random phage DNA fragments in E. coli Now... 23.4 (2015): 185-191. PubMed PMID: 25708933. 2. Cooper, Callum J., Mohammadali Khan Mirzaei, and Anders... biology 34 (2015): 69-77. PubMed PMID: 26275959. 2. Weigele, Peter, and Elisabeth A. Raleigh. "Biosynthesis... whole genome amplification (by a mechanism known as multiple displacement amplification) even in the ...same location that their host colonies grew. If your cells of interest stick to the same location as a...

...Request review: 1–2 days Prep and QC completed: 6–8 weeks Average shipping time: 1–2 days (USA); 3–4 days... our production process. This process will take 1–2 days, and we anticipate approving the vast majority...request is approved, you will receive an email notification that your request is ready to order. An “Add...subscribe to the Addgene blog for updates and educational resources.  We hope you’re as excited about this...

...imaging of diverse tissues. Nature Protocols, 17(2), Article 2. https://doi.org/10.1038/s41596-021-00644-9 ...directly from the Zenodo dataset.     Figure 2: Screenshot showing reagent entries with associated...something doesn’t work, it’s not a failure. It’s an education.   Figure 4: Zenodo page showing the IBEX... repository for reagents, protocols, panels, publications, software, and datasets. It’s also a great place...

...for retrograde transport has been found for AAV1, -2, -5, -7, -8 (Taymans et al., 2007), AAV1 (Hollis et... tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One 8:e76310...reveal novel hippocampal CA2 circuits. Nat Neurosci 17(2), 269–279. PubMed PMID: 24336151. PubMed Central PMCID... Mechanisms of Motility and Regulation. Neuron 84(2), 292–309. PubMed PMID: 25374356. PubMed Central PMCID...transduction efficiency of recombinant AAV serotypes 1, 2, 5 and 8 in the rat nigrostriatal system. J Neurochem...analysis of adeno-associated viral vector serotypes 1, 2, 5, 7 and 8 in mouse brain. Hum Gene Ther 18, 195–...retrograde access to projection neurons. Neuron 92(2), 372–382. PubMed PMID: 27720486. PubMed Central PMCID...

...testing your AAV.  Figure 2: Comparison of AAV serotypes 1, 2, 5, 6, 8, and 9 in two different ...) your experimental conditions and measurements; (2) the cells you’re targeting; (3) how much expression... Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and 9 in the Mouse Brain. PLOS ONE, 8(9),... Comparison of four promoters. Gene Therapy, 28(1–2), 56–74. https://doi.org/10.1038/s41434-020-0169-1...analyses of adeno-associated viral vector serotypes 1, 2, 5, 8 and 9 in marmoset, mouse and macaque cerebral...site, or when using chemogenetic tools, where modification of cellular activity outside of your experiment.... A. (2023). Various AAV Serotypes and Their Applications in Gene Therapy: An Overview. Cells, 12(5), ...

...detect your targeting vector or gene of interest. 2. Breed your parental mice to generate F1 Once you’...techniques that can be used to introduce genetic modifications into mouse embryos. But once you generate a ...techniques are each useful for introducing one modification into the mouse genome, they are not commonly...parental mice In order to ensure that your genetic modification will be passed along to all of the offspring...environment and will harbor similar epigenetic modifications. Special breeding cases Whether your mouse colony...the mutations that you are trying to cross are located on homologous chromosomes in the parental generation...

...developed around how they replicate (Figure 2). Figure 2: Baltimore replication classes. Image credit: ...safer viral vectors that can be used in BSL-1 or BSL-2 labs. Viral vectors are viruses that have been genetically...methods of replication. In fact, an entire classification scheme, known as the Baltimore replication classes...that in a bit! Viral genomes and replication When it comes to replication, viruses tend to march to the ...an intermediary DNA replication step between DNA and mRNA. DNA genome replication can involve an RNA or...cell. The cell then uses its own machinery to replicate the viral genome and proteins. This is, for the...before being translated into proteins. RNA viruses replicate their genomes using a variety of RNA or DNA intermediaries...

... Opto-Cre-Vvd is fast and can cut in as little as 2 hours.  Sheets, MB, et al., ACS Synth Biol. 2020....they investigated three Casɸ orthologs, Casɸ-1, Casɸ-2, and Casɸ-3. This system is notable for several reasons...a minimal PAM requirement. For instance, for Casɸ-2 the PAM sequence is 5’-TBN-3’ (where B is G, T, or...deposited by Leopold Parts. This library delivers 2 randomly paired guides per construct and allows a ...including excision or insertion, inversion, translocation or cassette exchange. By utilizing recombinases...BioRender.com In their recent ACS Synthetic Biology publication, Mary Dunlop’s lab generated a photosensitive...regions with Vivid (Vvd) photodimers. Upon the application of blue light, Vvd dimerizes bringing the C-terminal...