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Showing: 81 - 120 of 303 results
  1. Early Career Researcher Toolbox: Social Media for Scientists

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    Blog Post
    ...scientists are using social media professionally, and 2) I needed to start using Twitter. Sharing Chemistry...but my consistent writing for the Addgene blog (~1-2 blog posts per month) and use of Twitter and LinkedIn...PhD 👩🏾‍🔬💜 (@ThePurplePage) February 13, 2014 2. "The Scientist's Top 10 Guide to Twitter" blog post...you social media can positively impact your career, 2) given you tips for using various social media platforms...professional development, and while it’s not challenging to use, it’s hard to know where to begin. To ...
  2. Technologies Enabled by NanoLuc® Luciferase

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    Blog Post
    ...the system outlined in Schmid-Burgk, J.L., et al (2). In this post, I’ll cover two great applications ...molecularly evolved from mNeptune2 through 33 mutations and 2 deletions. CyOFP1 has a higher quantum yield, brightness...luciferin 24 hours later demonstrated a more than 2-fold brighter signal from Antares as compared to Fluc...PMID: 22894855. PubMed Central PMCID: PMC3501149. 2. Schmid, J.L., et al. (2016) CRISPaint allows modular...information. Many of our technology platforms like HaloTag® Fusion Protein, codon-optimized Firefly luciferase...concentrations of biological molecules. FRET sensors face challenges of photobleaching, autofluorescence, and, in ...cyan-excitable donors, phototoxicity. Another challenge to using FRET sensors comes when employing optogenetic...
  3. FLEx Technology and Optogenetics: Flipping the switch on gene expression with high spatial and temporal resolution

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    ...information. References Abdallah, Khaled, et al. "Adeno-associated virus 2/9 delivery of Cre recombinase ...upstream of an opsin gene (e.g., channelrhodopsin-2, ChR2) fused to a reporter gene (e.g., mCherry) to...the correct orientation to be transcribed (Figure 2) (Sharma and Zhu, 2014). How does FLEx enable strong...Deniz, et al. "A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range ...bacteriorhodopsin; a comparative study of strains of Halobacterium halobium differing in pigmentation." Biochemical...Rhodopsin-like protein from the purple membrane of Halobacterium halobium." Nature new biology 233.39 (1971): 149...opsin genes, and include bacteriorhodopsins, halorhodopsins, and channelrhodopsins, the first two found...
  4. Fluorescent Proteins 101: Luciferases

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    ...its abdomen to produce yellow-green light (Figure 2). Despite the title of this post, this reaction is...; no chemical reaction takes place. Figure 2: Simplified chemical reaction catalyzed by luciferase... has a detailed list of luciferase options. Table 2: Properties of luciferase enzymes Name Natural ...Renilla reniformis), click beetle (Pyrophorus plagiophthalamus), deep-sea copepod (Gaussia princeps), Japanese...None No Click Beetle Red (CBR)(Pyrophorus plagiophthalamus) Natural or codon-optimized 613 (luciferin...
  5. Educational Planarian Tools for Teaching Developmental Biology Techniques

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    ...neoblast cells and the gene Prohormone convertase 2 (pc2) labels the central nervous system. Evaluating... characterized in an unrelated worm (C. elegans) [2]. Delivering dsRNA matching a specific mRNA sequence...223). Link: http://abt.ucpress.edu/content/79/3/208 2. Fire, S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver...experiments are either not accessible or too challenging for amateur scientists. Planarians, wormy creatures...available planarians: Girardia sp., Dugesia dorotocephala, Phagocata morgani, and Phagocata gracillis....differ. For example, Girardia sp. and Dugesia dorotocephalacan can be fed dsRNA-expressing E. coli that are...
  6. Adeno Associated Virus (AAV) for Cell and Gene Therapy

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    ...AAVs in the clinic Thus far, AAV vectors AAV1, AAV-2, AAV1-AAV2 hybrids, AAV-6, AAV-7, AAV-8, AAV-9 and...neutralizing antibodies against some AAV types, AAV1, 2, 3 and 5 are thought to be as high as 70% among the... 10.24 (2003): 1999-2004. PubMed PMID: 14566358.  2. Hirsch, M. L., et al. "Self-complementary AAV mediates... cargo (DNA or RNA) to target cells has been challenging. A variety of vector delivery technologies have...treatment appears to be both safe and effective. Challenges for AAV gene therapy Although AAV-based gene ...promising, scientists are working to overcome key challenges to their efficacy:  Pre-existing immunity: Many...
  7. Viral Vectors 101: An Introduction to AAV

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    ..., AAV2/8 has a type 2 ITR and a type 8 serotype. Almost all vectors contain type 2 ITRs, which comes from...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. ...strand synthesis. In contrast, a scAAV vector with half the size of the ssAAV genome has a mutation in the...
  8. An Introduction to Adenovirus

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    ...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... like other viral vectors, though significant challenges remain (Wold & Toth, 2013).  While the pre-existing...
  9. Pairing CombiGEM and CRISPR for Combinatorial Genetic Screening

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    Blog Post
    ...CombiGEM lentiviral vector backbone pAWp12 (Figure 2). Cloning from the pAWp28 storage vector to the pAWp12... before beginning your experiments. Figure 2: Strategy for assembling barcoded combinatorial gRNA...PMID: 25114216. PubMed Central PMCID: PMC4151723. 2. Doench, John G., et al. "Optimized sgRNA design to...2015; Koike-Yusa et al., 2014; Ma et al., 2015; Shalem et al., 2014; Wang et al., 2014), knock down (Gilbert... 26190106. PubMed Central PMCID: PMC4559080. 8. Shalem, Ophir, et al. "Genome-scale CRISPR-Cas9 knockout...
  10. Fluorescent Tagging of Endogenous Genes with SapTrap

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    ...digestion of the destination vector with SapI yields 2 sites- the first site accepts the sgRNA target sequence...marker, and connector to be released (Fig. 1, Step 2-3). Since donor plasmids of the same type will produce... a gRNA vector that targets the gene of interest, 2) a plasmid to specify the reading frame of the insertion...the tag for each insertion. Also, it remains challenging to screen for the genetically modified strains...fluorescent (EGFP, tagRFP, mCherry) and nonfluorescent (Halo, SNAP) tags, a selectable marker (floxed Cbr-unc...small epitope tags (HA, Myc, Strep tag II, AviTag, HaloTag, SpyTag). The Foerstemann lab developed a polymerase...
  11. Hot Plasmids and Viral Preps - January 2021

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    ... 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 ... is only ~70kDa, with a molecular weight roughly half of Cas9 or Cas12a. This is in part due to the fact...
  12. Antibodies 101: Flow Cytometry

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    Blog Post
    ...termed fluorescence activated cell sorting or FACS (2). By analyzing single cells instead of the population...expression. A routine flow cytometry experiment uses 2 or 3 different fluorescent colors each measuring a...distinct populations on a FSC versus SSC plot (Figure 2).  A user can then “gate” around the specific population...cell and T cell into distinct populations (Figure 2). An immunologist who studies B cells, might then ... B cells, plasma cells or others.   Figure 2: Due to differences in their size and complexity, ...then flow cytometry can help you overcome these challenges and make these types of experiments easier and...fluorophores Fluorescent antibodies get around the challenges of fluorescent protein fusions by binding to ...
  13. Evolution of Brainbow: Using Cre-lox for Multicolor Labeling of Neurons

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    ...Brainbow mouse neural labeling system. Brainbow-1 and -2 have been further refined to create Brainbow 3.2. ...M-CFP expression; these deletions are defined using 2 loxP variants (only identical loxP sites can mediate... each fluorophore. Third, Brainbow-1 and Brainbow-2 each contained a “default” state; for example, Brainbow...processes not previously visible with Brainbow-1 and -2. The general structure of Brainbow-1.0 is retained...First, the imaging of Brainbow mouse tissue was challenging due to low fluorescence intensity, caused partially...expression patterns. Hampel S, Chung P, McKellar CE, Hall D, Looger LL, Simpson JH. Nature Methods. 2011 Mar...
  14. Viral Vectors 101: AAV Variables That Matter

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    ...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...Adeno-Associated Viral Vector-Mediated Gene Delivery to the Hypothalamus. Human Gene Therapy, 21(6), 673–682. https:/...
  15. Synthetic promoter AAVs for cell-type specific expression in retinal cells

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    ...retinal cell-type specific genes from mice. Strategy #2 (ProB): Create promoters using phylogenetically conserved...would express Cre-mCherry and GFP.   Figure 2: Synthetic promoters can be paired for targeted expression...1345-1356. https://doi.org/10.1038/s41593-019-0431-2  Neff, E.P. An AAV library for retinal cell expression... at the Institute of Molecular and Clinical Ophthalmology Basel recently took a different approach to ...to test for cell type-specific expression of GFP. Half (113) expressed in retinal cells four weeks post-injection...
  16. No Llamas Required - Synthetic Nanobodies Against Membrane Proteins

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    ...select for optimal sybody candidates. Figure 2: Outline of the process for selection of sybodies ...identify and isolate sybodies targeting the SARS-CoV-2 Receptor Binding Domain found on the membrane-bound...Generation Toolbox kit. Find the Seeger lab's SARS-CoV-2 sybodies! References Geertsma ER (2013) FX Cloning...2020) Synthetic nanobodies targeting the SARS-CoV-2 receptor-binding domain. BioRxiv.  https://doi.org...access to routinely, and it can be especially challenging when trying to develop antibodies against unstable...
  17. Twenty Years of Addgene Sharing: CRISPR

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    ... were depositing and requesting each year (Figure 2). We did a similar analysis with CRISPR pooled libraries...different categories (Figure 2C). Note that in Figure 2, the numbers on the X axis refer to distribution and...rather than a cumulative total.    Figure 2: Addgene CRISPR deposit and distribution by function...rapid adoption of new tools, illustrated in Figure 2, and the longevity of tools seen in Figure 3, indicates... the easy part; using them can be a bit more challenging. Addgene’s free educational resources help scientists...
  18. Truncated gRNAs for Regulating Gene Expression

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    ...the absence of genome editing (Fig. 2 and 3) (5, 3). Figure 2: Gene activation with nuclease-competent...shown to exhibit more limited activity than SpCas9 (2). In many cases this is due both to lower relative...PMID: 24076762. PubMed Central PMCID: PMC3844869. 2. Chari, Raj, et al. "Unraveling CRISPR-Cas9 genome...different perturbations. Consider, for instance, the challenge of attempting to simultaneously and selectively...
  19. Tetbow: Bright Multicolor Labeling for Neuronal Tracing

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    ...Figure 2). We’ve also created constructs with containing chemical tags such as the SNAP-tag, Halo-tag, ...possible when each of the XFP genes is introduced at ~2 copies per cell per color, following a Poisson distribution...AAV titer - The XFP genes have to be introduced at ~2 copies per cell per color to achieve the highest color...
  20. What's Your Organism? Expanding Genomic Tools via the NSF EDGE Program

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    ...presented in a series of quick (10 minute) or very quick (2 minute) talks so we got to hear from everyone. Addgene...evolutionary tree at least 6-7 times and on at least 2 separate continents. Mimulus (Andrea Sweigart) Monkeyflowers...Organisms, Part 1 Five Popular Model Organisms, Part 2 ...share all of these new organisms continues to be challenging. Organism specific viral vector systems would...thus making progress on the NSF “G to P grand challenge.” Creating a productive, new scientific community...proteins, and DNA. Euprymna scolopes (Joshua Rosenthal) The Hawaiian bobtail squid has an extreme number... a different protein. Did you know that most cephalopods die after they lay their 1 clutch of eggs? Propagation...
  21. Cloning Mammalian Cells with the Agarose Method

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    ...3 plates at different densities for selection). A 2% agarose solution is then melted (and thereby sterilized...96-well assay plate. We generally freeze at least 2 ampules each of 4 clones with different levels of ...of agarose, which is then overlaid with the firmer 2% agarose gel solution discussed above for picking....incubator, additional growth medium is added to about halfway up each well. Tips for using the agarose cloning...Time CRISPR User (by a 1st Time CRISPR User) The Challenges of Cell Culture Plasmids 101: Mammalian Vectors...
  22. Simplify Cloning with in vivo Assembly

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    ...procedure to be performed using a simple two-step, 2 hr protocol prior to transformation (Figure 1). As...Primer design Using specific primer design (Figure 2), IVA cloning can be used to perform any plasmid modification... for each type of modification is shown in Figure 2, and is discussed in more detail below. The main requirement...Biotechniques 10.1 (1991): 62-66. PubMed PMID: 2003926. 2. Bubeck, Peter, Monica Winkler, and Wilfried Bautsch... to amplify away from the insertion site. Encode half the new sequence at the 5’ end of each primer, with...
  23. AAVs in Retinal Gene Therapy

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    ...therapy had fallen - plummeted really -  from grace 2 years prior with the death of Jesse Gelsinger in an... the whole field to a grinding stop and only now, 2 decades later, is it finally back in the limelight...the other hand, have broad tropism in the eye. AAV-2, the first identified serotype and the one used in...reading 1. FDA approves gene therapy for blindness 2. Gene Therapy emerges from disgrace to be the next... completing her PhD and medical training in Ophthalmology. Gene therapy was in its infancy, but the eye...
  24. Fluorescent Proteins 101: Aptamer Fluorophores

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    ... to DFHBI in the presence of that metabolite (Fig.2). Spinach eventually evolved into Spinach2, a more...PMID: 25713381. PubMed Central PMCID: PMC4364232. 2. Baker, Monya. “RNA imaging in situ”. Nature Methods...emerging environmental pollutants.” Front Chem. 2014; 2: 41. PubMed PMID: 25019067. PubMed Central PMCID: ...biosensors.” TrAC Trends in Analytical Chemistry 27(2):108-117 · February 2008. 11. Svensen, Nina and Jaffrey...technology has been studied and used in assays for over half a century; the Addgene blog has an excellent primer...
  25. Predicting Adverse Reactions to Monoclonal Antibody Drugs

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    ...various components of the immune system. Figure 2: In-vitro models for immunogenicity prediction of ...drug reaction. This system would somewhat act like a 2-step verification. All approaches have their own limitations...Future Enhancements. Frontiers in drug discovery, 2, 952326. https://doi.org/10.3389/fddsv.2022.952326Joubert...Formation in Oncology: Clinical Relevance and Challenges. The oncologist, 21(10), 1260–1268. https://doi.org...
  26. Crowdfight, a Platform to Boost Scientific Collaboration During COVID-19 and Beyond

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    ...mammal called alpaca could neutralize the SARS-CoV-2 virus. But while the early results were promising,...receiving dozens of requests every day.    Figure 2: When a researcher needs a collaborator for a specific...Crowdfight will: 1) Find an expert with the right skill; 2) Confirm the expert’s suitability and availability...lockdown forced us to put our research projects to a halt. None of us works directly with viruses or transmissible...
  27. Technical Design of a Western Blot

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    .... The most common options are 4 °C overnight or 1–2 hours at room temperature (RT). Most people choose...overnight at 4 °C and their secondary antibody for 1–2 hrs at room temperature, which allows them to reduce...Tris-glycine 6–400 kDa Tris-glycine 100 V, 1–2 hours Easy and cheap to handcast Short shelf-life...–40 kDa Tris-tricine 30 V, 1 hour or 100 V, 1–2 hours Good separation, quality, and stability ...separation of proteins of different sizes.  Table 2: Recommended gel percentages for various protein sizes...are consistent and stable at room temperature for 1–2 years and are available in formulas specific to your...response over a broad dynamic range that typically spans 2–5 orders of magnitude, which includes a wide dynamic...
  28. Isolating a Monoclonal Cell Population by Limiting Dilution

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    ...Day 1: Seed individual cells in a 96-well plate Day 2–14: Monitor cells for growth and expand cells Day ...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...channel pipette 200–1000 µL single channel pipette CO 2 incubator Pipet controller Hazardous waste container...approximately 50–60% confluent. For 293T cells this is about 2 × 10 6 cells in a 10 cm dish. Each 10 cm dish should...the highest or lowest transgene expression ( Figure 2 ). Sample Data Figure 1: Generation of monoclonal ...inferred by the differences in colony size. Figure 2: Cas9 expression in monoclonal cell lines generated...medium at a reduced antibiotic concentration (e.g., half the concentration that was used for the initial ...
  29. Droplet Digital PCR for AAV Quantitation

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    ... qPCR is that the results can vary by a factor of 2. This means that if you set up two identical assays...amplitude measured for each droplet in each well (Figure 2). A clean ddPCR should have a clear separation between...as low as 1C/s. Increasing the elongation time to 2 minutes and the denaturation time to 1 minute has ...generating the standard curve for qPCR can be challenging to get right. Here at Addgene, we use a linearized...droplet digital PCR for vaccine evaluation in challenged monkeys and TB diagnosis." Emerging microbes ...
  30. Plasmids 101: Protein tags

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    ...(you generally don’t want more tag than protein), 2-3 tags is increasingly common. Tandem affinity purification...TEV cleavage site (more on that in a moment), and 2 ProtA IgG-binding domains. TAP has since come to encompass...found in this paper (available at Addgene).   Table 2: Protease recognition sites commonly used with tags...purify proteins can face any number of technical challenges depending on their protein of interest. After...After several decades of trying to address these challenges, researchers have amassed a considerable molecular...
  31. Visualizing Translation at the Single Molecule Level

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    ...Find ribosome profiling plasmids at Addgene. 2. Halstead, James M., et al. “Translation. An RNA biosensor...the reporter mRNA is labeled by PCP-mCherry (Figure 2). The 3’ UTR also contains a CAAX sequence to bind...translation inhibitors cycloheximide and puromycin, Halstead et al. verified that translation is necessary ...translation is known to be important in development, and Halstead et al. used TRICK to examine translation of Drosophila...
  32. OCaMP: A New Calcium Indicator for Neural Imaging

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    ...the fluorescent protein mOrange2 (Figure 2). Figure 2: OCaMP structure and mechanism of action...module_attribute "schema_version" is_json="true" %}{% raw %}2{% endraw %}{% end_module_attribute %}{% module_attribute...developed a new calcium indicator, OCaMP. The challenge: Expanding the fluorescent toolbox Modern neuroscience...
  33. Antibodies 101: Avoiding the Mouse on Mouse Mess in IHC

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    ...recognize Fab fragments from the same species and 2) these fragments will be more likely to bind (and ...epitopes recognized by the secondary.   Figure 2: There are a few common solutions to avoid background...staining can be a challenge in the best of cases. But it can be particularly challenging when your only ...
  34. Important Considerations in Optogenetics Behavioral Experiments

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    ... will repeatedly nose-poke at the active hole [1, 2].    Place preference/place aversion also models reinforcement...spend most of its times in the opposite chamber) [2]. Laser activation is usually controlled automatically...two different ways 1) your hypothesis is wrong or 2) there is some problem with the technical implementation...PMID: 21716290. Pubmed Central PMCID: PMC3775282. 2. McDevitt, Ross A., et al. "Serotonergic versus nonserotonergic...than ICSS. The test chamber is divided into two halves that are distinguished visually through different...
  35. RNA Interference in Plant Biology: New Tools for an Old Favorite

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    ... analogs such as 2-fluoroadenine (2-FA), organisms with functional APT will convert 2-FA to cytotoxic ...selection of plants undergoing RNAi, and LIIbeta F 1-2 RNAi, which allows assembly of intron-spliced hpRNAs...nucleotides, resulting in death. In the presence of 2-FA, potent gene silencing of APT is required for survival...such as in the model flowering plant Arabidopsis thaliana (Li et al., 2013; Zhang et al., 2018) and the ...
  36. Quick Guide to Working with Drosophila Part 2: Controlling Gene Expression in Flies with Gal4/UAS

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    ... you can read more about them in review articles (2–4). You can also take a look at my recent paper to...Development 118, 401–15 (1993). PubMed PMID: 8223268. 2. J. B. Duffy, GAL4 System in Drosophila : A Fly Geneticist...two genes by one Gal4 driver will produce roughly half as much protein as overexpression of each gene individually...384–391 (2004). PubMed PMID: 15262411. 4. T. D. Southall, D. A. Elliott, A. H. Brand, The GAL4 System :...Marka, J. C. Kagan, Environmental Stress Causes Lethal Neuro-Trauma during Asymptomatic Viral Infections...
  37. Viral Vectors 101: Inverted Terminal Repeats

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    ...ITR, which can either be “flip” or “flop” (Figure 2). Additionally, the ITR contains a 4-nucleotide Rep...site for Rep proteins (Savvy et al. 2013). Figure 2: AAV2 wild type ITR in flip and flop orientation. ...Packaging, and Potency of AAV Vectors. Microorganisms, 12(2), 310. https://doi.org/10.3390/microorganisms1202031038399714...10.1089/hgtb.2016.133. PMID: 28967288. Shitik, E. M., Shalik, I. K., & Yudkin, D. V. (2023). AAV- based vector...
  38. Targeted Mutagenesis with EvolvR

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    ...while increasing the standard mutation rate by only ~2-fold over nCas9 levels. Two sets of modifications ...10-3 mutations per nucleotide per generation, and (2) the addition of the thioredoxin-binding domain (TBD...components of EvolvR and its potential applications. Challenges of existing mutagenesis methods Several continuous...drawbacks with these approaches that makes it challenging for users to achieve targeted diversification...PMID: 30253789. PubMed Central PMCID: PMC6154929. Halperin, Shakked O., et al. "CRISPR-guided DNA polymerases...
  39. Click Biology: How to Click and Play to Explore Biology

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    ... engineered components, like protein tags (Figure 2). Through the work of many groups, there are systems... in Addgene's repository (Table 1). Figure 2: Adding function to My Favorite Protein (MFP) through...Exchangeable HaloTag Ligands HaloTag Reversible binding of fluorescent ligands to HaloTag PCV-Cas9, Cas9...Plasmid Collection HaloTag Collection of research tools from Promega, including many HaloTag plasmids Exchangeable... tube or in cells. There are also SNAP-tag and HaloTag modules, which can connect proteins to any kind...SNAP-tags or CLIP-tags Tetbow SNAP-tag, CLIP-tag, HaloTag Based on the Brainbow system, adds Tetracycline...
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