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Showing: 181 - 210 of 727 results

  1. Viral Vectors 101: AAV Serotypes and Tissue Tropism

    Type
    Blog Post
    ...them to target particular cell types. Figure 1: Diagram of AAV components. Created with BioRender.com...depends on your choice of promoter/enhancer. Table 1: AAV capsids and receptors. Capsid Primary receptor...required)2 Human fibroblast growth factor receptor 1 (FGFR1)3 αVβ5 and α5β1 integrins3 Hepatocyte growth...structure of AAV2, so they are formally called rAAV2/1, rAAV2/2, and so on, although we often use abbreviated...efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PloS One, 8(... N.J.), 807, 141–157. https://doi.org/10.1007/978-1-61779-370-7_6 Tabebordbar, M., Lagerborg, K. A., Stanton... and Transduction by Adeno-Associated Virus Types 1 and 6. Journal of Virology, 80(18), 9093–9103. https...

  2. Simplify Cloning with in vivo Assembly

    Type
    Blog Post
    ...transformation (Figure 1). As this approach requires no special reagents or purification kits, it is low cost...with linearized vector backbone. DpnI digestion - 1 μl DpnI is added to the PCR mix to remove parental...confirm primer design.  Use minimal template DNA Use 1 ng of PCR template DNA to minimize false positive ... by PCR, so we usually add DMSO (3%) and Betaine (1 M) to the PCR mix. When PCR is not possible Some plasmids...Twitter @jakefwatson and @JGarciaNafria.  References 1. Jones, Douglas H., and Bruce H. Howard. "A rapid ...3’ end binds template DNA for PCR amplification, while modifications and homologous sequences are encoded...multiple modifications A key feature of IVA cloning is the ability to combine multiple modifications. Any ...

  3. Cpf1: A New Tool for CRISPR Genome Editing

    Type
    Blog Post
    ...guide for a refresher on CRISPR/Cas9.  Figure 1: Cpf1 is a single RNA-guided endonuclease of a class... than the multi-subunit complex employed by class 1 systems. A putative new class 2 nuclease, Cpf1 (CRISPR...that Cpf1 can tolerate mismatches at gRNA positions 1, 8, 9, and 19-23. Accordingly, the 3’ end of the gRNA...Most gRNAs directed low-frequency Cpf1 cleavage at 1-12 off-target sites; in contrast, SpCas9 may cleave...the diversity of CRISPR-based systems. References 1. Zetsche, Bernd, et al. "Cpf1 is a single RNA-guided...mismatches on modification by AsCpf1 (red) or LbCpf1 (blue). Mismatch positions are indicated below each ... this publication at Addgene. Makarova, Kira S., et al. "An updated evolutionary classification of CRISPR-Cas...

  4. Multiplexed Capture of Promoter-enhancer 3D Chromatin Structures Using CRISPR

    Type
    Blog Post
    ... Table 1: Key differences between original CAPTURE and CAPTURE 2.0 methods.   Applications of CAPTURE...by PCR or next-generation sequencing. Figure 1: Overview of the key steps of the CAPTURE method. ...ligases Expression system Stable cell line expressing 1) FLAG-biotin-tagged dCas9, 2) BirA, and 3) one or ...or more gRNAs Two separate lentiviruses delivering 1) a BioTAP-tagged dCas9 and an eGFP tag, and 2) gRNAs...The Xu lab presented three ways to use CAPTURE 2.0: 1. Multiplex capture of chromatin interactions The Xu... gene’s transcription. Sometimes enhancers are located thousands of base pairs away from the gene they...created a dCas9-based CAPTURE (CRISPR Affinity Purification in situ of Regulatory Elements) method. The ...

  5. Magnetic Control of Proteins: More than a Dream

    Type
    Blog Post
    ...the sample (by less than 1%) and recovered after the magnet was removed (Figure 1). At first, Andrew and...since the initial report in 2023.)     Figure 1: Fluorescence intensity of EGFP-FlavinTag in E. coli...that could be expressed and purified. Although a ~1% change in signal might not make a very powerful tool...Hayward et al. 2024 under CC-BY license.   Video 1: Fluorescence movie of E. coli expressing MagLOV while... tools for familiar applications. But how does one invent a tool for applications that don’t even exist...phenomenon, improve the tool, and develop new applications for magnetoresponsive proteins. Motivated by...organisms) are opaque, which makes most biomedical applications of these tools difficult or impossible. But ...

  6. CRISPR 101: Targeting RNA with Cas13a (C2c2)

    Type
    Blog Post
    ...Subscribe to CRISPR Blog Posts Applications of Cas13a   Figure 1: Using Cas13a as a diagnostic ...C2c1, C2c2 and C2c3 (short for Class 2, candidate 1, 2, or 3). C2c1 and C2c3 are related to Cpf1, but ...lacks the ability to cleave the RNA target.  Table 1: Comparison on Common CRISPR Enzymes Name  Enzymatic... This system, dubbed SHERLOCK (depicted in figure 1) has been used to differentiate strains of Zika virus...contributed to the update of this post. References 1. Abudayyeh, Omar O., et al. "C2c2 is a single-component...use, functional in most species, and has many applications. That said, SpCas9 is not the only game in town... makes this molecule unique, and the various applications of Cas13a. The origins of Cas13a: An RNA cleaving...

  7. CRISPR 101: Cytosine Transversion Editors

    Type
    Blog Post
    ...directed base excision repair proteins. Nature Communications, 12(1), 1384. https://doi.org/10.1038/s41467-021...predictable by machine learning methods. Nature Communications, 12(1), 4902. https://doi.org/10.1038/s41467-021...for more details on how they work!) Figure 1: Base transitions are edits between the purine bases...R-loop structure. In order to bind DNA, Cas9 requires (1) a guide RNA, (2) a complementary DNA sequence, and...given the pithier name CGBE1, for “C → G Base Editor 1,” and had a C → G editing efficiency of up to 68%... the CGBE: DNA polymerase D2 (POLD2), exonuclease 1 (EXO1), and RNA binding motif protein X-linked (RBMX...transversions in human cells. Nature Biotechnology, 39(1), 41–46. https://doi.org/10.1038/s41587-020-0609-x...

  8. Antibodies 101: Monoclonal Antibodies

    Type
    Blog Post
    ...animal, those B cells are fused to myeloma cells at a 1:1 ratio and then cultured in a special medium called...antibodies that recognize proteins. Figure 1: Steps in the generation of hybridomas for monoclonal...Washington (DC): National Academies Press (US); 1999. 1, Generation of Hybridomas: Permanent Cell Lines Secreting...cells and antibody-producing B cells that cannot replicate, meaning that only hybridomas will survive this... Antibodies: Distinguishing Characteristics, Applications, and Information Resources. ILAR Journal 46:... the Addgene blog Learn more about research applications for antibodies in Intro to Antibodies Read about...

  9. Fluorescent Proteins 101: Fluorescent Protein Timers

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    Blog Post
    ...that have now become hallmarks of ideal FP timers: 1. Ratiometric determination of expression time The ...the FT series matures from blue to red (see Table 1 for emission and absorption spectra). Subach et al...times and varied blue-red maturation times (Table 1). These separate proteins should be useful for monitoring...cellular events occurring at varied time scales.  Table 1: Monomeric FP Timers and Associated Plasmids from ... know in the comments section below! References 1. Terskikh, Alexey, et al. ""Fluorescent timer": protein... (green or blue) within a population of timer indicates the age of the pool: the higher the ratio, the...-FT-N1 pTRE-Slow-FT pBAD/HisB-Slow-FT -  Applications of fluorescent protein timers FP timers have...

  10. PRIDICT: Predicting Efficiencies of Prime Editing Guide RNAs

    Type
    Blog Post
    ...evaluate all of the pegRNAs in one experiment (Figure 1). Through this method, we found key determinants of...diverse edit types and cell contexts. Figure 1: High-throughput screening of pegRNA efficiency and...Article 3. https://doi.org/10.1038/s41576-022-00541-1 Kim, H. K., Kim, Y., Lee, S., Min, S., Bae, J. Y.,...DNA repair determinants. Nature Biotechnology 2023, 1–11. https://doi.org/10.1038/s41587-023-01678-y Li,...editing systems in multiple cell types. Cell, 186, 1–17. https://doi.org/10.1016/j.cell.2023.03.034 Resources...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...

  11. CRISPR 101: Making a Knock-In Cell Line

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    Blog Post
    ... arms. Nat Chem Biol., 308(20): 1-9 (2020). 10.1038/s41589-019-0432-1 Mehdi Banan. Recent advances in ...before attempting knock-ins by HDR.   Fig. 1: Early steps in repair of a DNA double strand break...traditionally have larger homology arms in the 500 bp to 1 kb range, however, shorter homology tracts have recently...breaks. J Biol Chem., 293(27): 10524-10535 (2018). 1 10.1074/jbc.TM118.000372 Yang, H., Ren, S., Yu, S....cut site location Position your CRISPR cut site as close as possible to the knock-in location. HDR depends...sequence to be incorporated at a precise genomic location, with little to no room for error. It’s no surprise...nutrients in their media, they will not undergo DNA replication, so make sure you are keeping an eye on those...

  12. We're Updating Our Hold for Publication Policy

    Type
    Blog Post
    ...Updated policy As of July 1, 2025, we are updating our Hold for Publication policy with two changes that...other scientists to use? We take this Hold for Publication policy seriously and appreciate the trust you...hold if they are clearly described in a journal publication. Plasmids will be released 3 years after the ...keep things moving internally. And remember, a publication isn’t required to share your plasmids! You can...can make your deposit available before publication and we can update the plasmids’ citation when the paper...

  13. Quick Guide to Working with Drosophila Part 2: Controlling Gene Expression in Flies with Gal4/UAS

    Type
    Blog Post
    ...transcription activation system co-opted from yeast (1). It is a Drosophila geneticist’s main workhorse to...he still reads “#” as a “pound symbol”. References 1. A. H. Brand, N. Perrimon, Targeted gene expression... A Fly Geneticist’s Swiss Army Knife, Genesis 34, 1–15 (2002).PubMed PMID: 12324939. 3. S. E. McGuire,...Versatile Toolkit for Gene Expression in Drosophila, 3, 1–10 (2015). PubMed PMID: 21356876. 5. J. Chow, Z. Marka...as overexpression of each gene individually. Applications of the Gal4/UAS system There are a number of...lines. In the meantime, be sure to browse the publication references and web links from my first post and...

  14. Bacterial Expression Systems

    Type
    Collection
    ...expression inducible by red light. pCph8 50552 pLtetO-1 Anhydrotetracycline Jeffrey Tabor Anhydrotetracycline...Genome Engineering Visualization and Tagging Purification Controlled Expression Reporter Plasmids Reporter...Reporter Plasmids Tagging and Visualization Purification Controlled Expression Reporter Plasmids You may...Bacteria? E. coli are commonly used to store and replicate plasmids of all types, but beyond that, researchers...sites within this plasmid. Return to Top Protein Purification Sometimes looking at a protein inside cells ...and, epitope tags can be used to simplify the purification process. Using plasmids in this collection, ...those tags from the purified protein. Protein purification can be challenging, but once you get it right...

  15. Plasmids 101: Secondary Nanobody Toolbox

    Type
    Blog Post
    ...incubation of 1° and 2° Must use different species and/or different IgG subclasses of 1° for multi-color...displacement 2° incubation can be skipped by pre-binding to 1° Monovalent Monoclonal Only anti- rabbit and -...dyes are also commercially available. References 1. Pleiner, T., Bates, M., & Görlich, D. (2017). A toolbox...NIH’s Developmental Studies Hybridoma Bank (DSHB). 1 ml of antibody-containing hybridoma supernatant is...in bacteria and extracted with common protein purification methods. Read on to learn more about nanobodies..., Alexa Fluor 568, or Alexa Fluor 647 before application to the sample. Staining with this multi-color...PubMed PMID: 29263082 Find plasmids from this publication at Addgene. 2. Bates, M., Böhning, M.I., Chug...

  16. Delivery Methods for Generating iPSCs

    Type
    Blog Post
    ...origin of replication (oriP) element and the cis-acting EBNA-1 from Epstein-Barr virus. EBNA-1 binds oriP... be completely removed.  Replicating oriP/Epstein-Barr nuclear antigen-1 (EBNA1) based episomes: These...reprogramming method Most iPSC research falls into two categories: 1) studies focused on better understanding the...Difficult to purify reprogramming proteins. Table 1: Key Features of Different Methods for Generating ...plasmid-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming factors ...iPSCs experiences in the comments below! References  1. Sano, Masayuki, Minoru Iijima, Manami Ohtaka, and...Methods figures were adapted from this reference. 1. Malik, Nasir, and Mahendra S. Rao. "A Review of the...

  17. Clear and Accessible Writing

    Type
    Blog Post
    ...sighted readers.    Figure 1: An image from AAV Purification by Iodixanol Gradient Ultracentrifugation... it? The image in Figure 1 appears on our protocol page for AAV Purification by Iodixanol Gradient Ultracentrifugation...intended.)  Steps to clear writing Let’s use Figure 1 as our example. Remember, our goal is to use words... share with the rest of the workshop.  For Figure 1, Addgene chose this alt text: "The left panel is a...principles we developed to use in our website and educational content conflicted with plain language guidelines...Stupid, overwhelmed, discouraged, tired…words that indicated there were many barriers between our audience ...

  18. Deciphering the Mysteries of Behavior with Viral Vectors

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    Blog Post
    ...production of the pro-inflammatory cytokine interleukin IL-1 beta in the dorsal raphe nucleus (DRN), a region thought... a transgenic mouse harbouring a Cre-dependent IL-1 beta overexpression cassette. Expression of Cre in...in these neurons turns on overexpression of IL-1 beta. They tested the mice in a series of behavioral ...risky), or a closed arm (safe). Mice with higher IL-1 beta production spent more time in the open arm and... part of the apparatus, it turned out that the IL-1 beta overexpressing mice spent more time in the risky...have made great strides to map brain regions and allocate specific functions to them. Electrical recordings...long-lasting antidepressant effects." Nature communications 10.1 (2019): 223. PubMed PMID: 30644390. PubMed...

  19. Cancer and the Immune System: Deciphering the Relationship

    Type
    Blog Post
    ...such as IL-8, IFNγ, and colony stimulating factor 1 (CSF-1). Additionally, tumor cells recruit regulatory...introduced the “Three Es of Immunoediting” nomenclature. 1. Eliminate - Latent tumors are recognized by effector...the tumor, activates hypoxia inducing factor (HIF-1), which promotes the production of VEGF to increase...This study used CRISPR gene editing to knockout PD-1 on immune cells harvested from patients and thereby...exploring new avenues in healthcare.   References 1. Vinay, Dass S., et al. "Immune evasion in cancer:...lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial." The Lancet 385.9967 (2015)...immune response to the development of tumors is a complicated and dynamic phenomenon that is interestingly ...

  20. Plasmids 101: Broad Host Range Plasmids

    Type
    Blog Post
    ... all replicate in E. coli, which is the most commonly used bacteria in lab manipulations. Table 1 has ...https://doi.org/10.1007/978-1-61779-197-0_19  Meyer, R. (2009). Replication and conjugative mobilization...reduced metabolic load (Meyer 2009). Figure 1: Idealized examples of a narrow host range plasmid...species these plasmids are compatible with.  Table 1: Non-exhaustive list of common broad host range plasmids...for DNA cloning in Gram-negative bacteria. Gene, 70(1), 191–197. https://doi.org/10.1016/0378-1119(88)90117...ori2, ori3) and replication initiation proteins (RepA, RepB) necessary for replication in various hosts...the ori and allow for replication. However, if plasmids have their own replication machinery such as Rep...

  21. Three Key Considerations for Precise Neuronal Targeting Using AAV Technologies

    Type
    Blog Post
    ... the example shown in Figure 1, injected retrograde AAVs at one location result in opsin expression in...from the paper for designing experiments with AAV: 1. Getting your viral vector into your cell of interest... administration routes for neuroscience see Table 1 from the paper. 2. Manipulate your cell of interest...experiments! Late last year, we hosted our first AAV Education and Development workshop aimed at brainstorming...even further. For example, injecting AAVs at one location can result in expression of an optogenetic payload...

  22. Luciferase Plasmid Collection

    Type
    Collection
    ... Eric Campeau 21474 pLenti CMV V5-LUC Blast (w567-1) Firefly CMV Lentiviral expression of firefly luciferase...bacteria to aquatic animals to insects. Luciferase catalyzes the oxidation of a substrate, leading to the emission...advantages and disadvantages depending on the application. Firefly luciferase is considerably brighter ...luciferase-based plasmid systems for many different applications. Browse the Collection Highlights section to...respectively, for increased usefulness in in vivo applications. LuxSit-i : An artificial luciferase desined...

  23. Finding and Joining Your Dream Lab

    Type
    Blog Post
    ...choosing direction of a project. If you expect more 1:1 time with the advisor, you should choose accordingly...potential hire visits the lab for a full day, gives a 1 hour talk about his or her interests, and spends one-on-one...that supports both paths – you won't know in year 1 what you will want in year 6. Have lab alumni taken... next.” Connie Cepko looks for people who are dedicated and interested in science. “Postdoc hires are ...

  24. Photosensitizer Induced Cell Ablation with FAP-TAP MG-2I-dL5**

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    Blog Post
    ...photosensitizer used in this work is composed of two parts: 1) an activatable protein (the FAP - fluorogen activating...reaching greater than 75% photobleaching after just 1 minute of exposure. MG-2I-dL5** experienced minor ...photosensitizers in the comments below!   References 1. He, J., Wang, Y., Missinato, M. A., Onuoha, E., Perkins... encoded photosensitizer. Nature Biotechnology,24(1), 95-99. doi:10.1038/nbt1175. PubMed PMID: 16369538...generation when bound by the dL5** FAP.  The iodine-modifications also shift MG-2I’s excitation wavelength to ...FAP-TAP system works. Find the Plasmids from the Publication Here Light-induced cell ablation with MG-2I-dL5...

  25. Treating Muscular Dystrophy with CRISPR Gene Editing

    Type
    Blog Post
    ...X-linked recessive disorder affecting approximately 1 in 5000 males. DMD is caused primarily by frameshift... applicable to 80% of DMD patients. Figure 1: Exon skipping strategies for Duchenne muscular dystrophy...needed. First, CRISPR delivery must be optimized to: 1. Reach a high percentage of muscle cells throughout...also be closer than we might expect.   References 1. Kaiser J. CRISPR helps heal mice with muscular dystrophy...neurogenetic diseases. Nucleic Acid Ther. 2014 Feb;24(1):69-86. PubMed PMID: 24506781. PubMed Central PMCID...);186-91. doi: 10.1038/nature14299. Epub 2015 Apr 1. PubMed PMID: 25830891. PubMed Central PMCID: PMC4393360...setting. CRISPR is often used for animal germline modification, to repair or add in disease-causing mutations...

  26. Viral Vectors 101: Parts of the AAV Packaging Plasmid

    Type
    Blog Post
    ... The three VP proteins assemble into capsid at a 1:1:10 ratio (VP1:VP2:VP3). There are eleven naturally... Figure 1. The RepCap plasmid and its products. Created with BioRender.com   Replication (rep) gene...Rep78/68 play a key role in AAV genome replication. Replication initiates from the AAV genome ITRs on ...contains the proteins needed for the virus to replicate,  and the transfer plasmid which contains the ...aka the RepCap plasmid encodes two genes: the replication (rep) gene and the capsid (cap) gene. In wild-type... proteins that are required for viral genome replication and packaging: Rep78, Rep68, Rep52, and Rep40... generate a 3’ hydroxyl group needed for DNA replication, Rep78/68 binds the Rep binding element (RBE)...

  27. Fluorescent Proteins 101: Photoactivatable Fluorescent Proteins

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    Blog Post
    ...discovered such as Kaede and PS-CFP2 (see Table 1). Table 1: Properties of Selected Irreversible Photoactivatable...) by mutating Threonine 203 to Histidine (T203H) (1). wtGFP normally contains a mixed population of neutral...in fluorescence when illuminated with blue light (1). Similar to PA-GFP, scientists later developed red...fixation conditions to preserve cellular structures (0.5-1% osmium tetroxide). These conditions destroy most ...with extraordinary molecular detail.   References 1.Patterson, George H., and Jennifer Lippincott-Schwartz...is believing." Annu. Rev. Biomed. Eng. 10 (2008): 1-38. PubMed PMID:18647110. Additional Resources on ...up. Applications of photoactivatable fluorescent proteins Some of the most important applications for ...

  28. An Introduction to Adenovirus

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    Blog Post
    ...that the culprit for these unusual cases was either 1) a physiological effect of SARS-CoV-2 toward destabilizing...an AdV transfer plasmid with the E1 and E3 (Early 1 and Early 3) genes deleted. These genes’ functions...certain contexts (Bauzon et al., 2003).        Figure 1: The adenovirus life cycle. Image reused from Kremer... review of the evidence. Emerg Microbes Infect, 8(1), 1679–1687. https://doi.org/10.1080/22221751.2019.1690953...574–580. https://doi.org/10.1038/s41586-023-05949-1. PMID: 36996871. Tatsis, N., Fitzgerald, J. C., Reyes-Sandoval...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...

  29. New Acoustic Reporter Genes: Ultrasound Imaging of Gene Expression

    Type
    Blog Post
    ...origin of replication (p15A) worked best under a wide range of inducer concentrations (0.1-1% L-arabinose...bacteria have colonized (Fig. 1c).     Fig. 1: Expressing bARGSer in E. coli. (a) Plasmid diagram...ultrasound contrast. In all the cell lines we tested, 1 µg/mL doxycycline was sufficient to saturate expression...engineering a cell line, cotransfect the mARG cassettes 2:1 (A:N-V) with PiggyBac transposase and outgrow the ... tumors and tumor-homing bacteria. Nat Biotechnol 1–13 (2023) doi:10.1038/s41587-022-01581-y  ... they are based on light which is very easily scattered by tissue. Ultrasound, on the other hand, is a...protein nanostructures called gas vesicles, which scatter sound waves and thereby produce ultrasound contrast...

  30. Plasmids 101: Modular Cloning

    Type
    Blog Post
    .... Level 1: Assembly of multiple Level 0 parts into TUs. Level 2: Assembly of multiple Level 1 TUs into...: promoter, gene, and terminator.     Fig 1. MoClo enables individual parts to be assembled into... peptide can easily be tested by generating Level 1 TUs with unique promoters, and the ideal phosphatase...most obvious example of an individual part, this category also includes promoters, UTRs, terminators, etc...multi-purpose it can be. Let’s review a list of MoClo applications. Genetic circuits Most things in biology are...performing production and subsequent necessary modification of these chemical products. The flexible nature...genotype and phenotype is best described with “it’s complicated.” Applying MoClo Interested in using MoClo? You...
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