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We narrowed to 727 results for: cat.1

Showing: 241 - 270 of 727 results

  1. Viral Vectors 101: Systemic Capsids

    Type
    Blog Post
    ...vector at 1 x 1013 vg/mL titer), while AAV-PHP.S is typically used at a dose between 3 x 1011 and 1 x 1012...chosen capsid before using it in an application.     Figure 1: AAV toolkit for targeted gene delivery...capsid applications. For example, AAV-PHP.eB is typically administered at a total dose between 1 x 1011...rodents and broad tropism in primates. Nature Communications, 14(1), 3345. https://doi.org/10.1038/s41467-023...easy to produce in high titer, but higher doses (>1 x 1012 vg total in mice) may increase the chances ...decision tree for selecting a capsid, while Table 1 gives a brief overview of published descriptions of...Capsids page to learn more about that collection. Table 1: Systemic capsid plasmids available from Addgene ...

  2. Antibody Data Repositories and Search Engines

    Type
    Blog Post
    ...Table 1.  Table 1: Antibody data repositories and search engines Website Targets Application Purpose...which allow you to search many different antibody catalogs at the same time.  If you're looking for an antibody... specificities, from target-specific ones to application-specific ones to anything goes. We've shared ...

  3. Optogenetics Guide

    Type
    Guide
    .... Channelrhodopsin-1 (ChR1) is excited by blue light and permits nonspecific cation influx into the cell...based on the LOV2 domain of Avena sativa phototropin 1 LOVETRAP reversibly sequester and release proteins...concept of optogenetics. 2012 Prog Brain Res. 196: 1-28. PMID 22341318 Gradinaru V, Zhang F, Ramakrishnan...diversifying and extending optogenetics. Cell. 196:1-28. PMID 20303157 Han X, Boyden ES. 2007 Multiple-...Limitations and Future Developments. Exp Physiol. 96(1): 19–25. PMID 20621963 Mattis J, Tye KM, Ferenczi ...Channelrhodopsins: cation channels from Chlamydomonas reinhardtii ChR2 Widely used light-gated cation channel from...light of the correct wavelength, the pore opens, cations flow into the cell (yellow dots), and the neuron...

  4. Isolating Genomic Regions of Interest with the CRISPR System

    Type
    Blog Post
    ...allowed for the identification of proteins associated with the IRF-1 locus. Figure 1: Schematic of...the promoter region of the transcription factor IRF-1. Immunoprecipitation, using an antibody targeting ...References Campbell, Amy E., et al. "NuRD and CAF-1-mediated silencing of the D4Z4 array is modulated ...affinity purification with an anti-Cas9 antibody. These different tag systems and affinity purification schemes...genomic alterations. These studies utilize a catalytically inactive form of Cas9 known as dCas9 (Jinek ...interest targeted by a gRNA. CRISPR-mediated purification of a specific genomic region In 2013, the Hodaka...steps (Fujita et al., 2013). CRISPR-mediated purification of a specific genomic region alleviates these...

  5. Minigenomes - a Safe Way to Study Dangerous Viruses Like the Ebola Virus

    Type
    Blog Post
    ... genome which are required for replication and transcription (Figure 1). All viral genes are removed and...interested in genome replication and transcription of negative strand RNA viruses.   References 1. Buchholz, Ursula...cells that stably express T7 (e.g. BSR T7/5 cells) (1). We hope that minigenomes will greatly enhance our...reverse genetics. Springer Berlin Heidelberg, 2004. 1-41. PubMed PMID:15298166. 3. Hoenen, Thomas, et al...Technical Tips). Minigenome replication and transcription In order to replicate the minigenome, the viral...transcription and replication strategies of Marburg virus and Ebola virus by using artificial replication systems...allowing researchers to study aspects of the EBOV replication cycle under BSL-2 conditions (4). What is a ...

  6. Tips for a 1st time AAV user (by a Rookie AAV user)

    Type
    Blog Post
    ...plasmids to produce AAV: 1) the RepCap plasmid which provides the AAV replication (rep) and capsid (cap)... teaching me about AAV production and purification! References 1. Aurnhammer, C., Haase, M., Muether, ...plasmid. This whole process takes ~3 hours to complete: 1 hours hands-on time and 2 hours for the qPCR run and... repeat sequences. Human gene therapy methods, 23 1, 18-28. PubMed PMID: 22428977. 2. Choi, V.W., Asokan...this AAV purification video! It didn’t exist when I did my first iodixanol gradient purification, but I ...In this post, I’ll share some AAV production, purification, and titration tips, while also summarizing ...cap) genes. AAV replication uses the host’s polymerase, but requires Rep proteins to process a double-stranded...

  7. Hot Plasmids: Winter 2025

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    Blog Post
    ...Figure 1), an altered epegRNA delivered with an enhanced pU6cm promoter, viral replicon amplification of ...of cargo, and heat treatment.  Figure 1: Left: recombined prime editing tools PE2max-NC and ePEmax3...conformational dynamics of the motor protein kinesin-1, the Twelvetrees Lab took advantage of the self-labeling..., Craggs, T.D., & Twelvetrees A.E. (2024) Kinesin-1 is highly flexible and adopts an open conformation...single-molecule biophysics Fast and responsive voltage indicator ONE-GO biosensors kit Antibodies for neurodegeneration...2024.12.20.629623.  A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic...and more responsive genetically-encoded voltage indicator (GEVI) than previous versions. ASAP5 can be imaged...

  8. Plasmids 101: Knockout/Knock-In Plasmids

    Type
    Blog Post
    ... the example shown in Figure 1, large sequences corresponding to exons 1 and 3 of the targeted gene have...knock out exon 2 of a given gene.   Figure 1: A knockout targeting vector designed to insert a ... the Neomycin resistance (NeoR) gene between exon 1 and 3 of the targeted gene. Upon recombination, the...in line 4 of Figure 4. Since the loxP sites are located in intronic regions, this gene will still be expressed...

  9. Hot Plasmids: Summer 2024

    Type
    Blog Post
    ...10.1038/s41551-024-01227-1. doi:  https://doi.org/10.1038/s41551-024-01227-1   CHARM: A compact epigenetic...biotinylated SpyTag-SpyCatcher linkers and spacer proteins ending with a GFP nanobody (Figure 1). This enrichment...highly-visible beads on the cryo-EM grid.     Figure 1: MagIC-cryo-EM for sample enrichment and structure...single-particle analysis of PRC2 with RvLEAMshort (1:6 molar ratio) with 10 minutes of glutaraldehyde crosslinking...enrichment streamlines the purification and minimizes sample loss while also supporting imaging, making it...scFvs for other targets or could be used for applications beyond cryo-EM.   Find plasmids for MagIC-Cryo-EM...interact with the air-water interface (AWI) before vitrification, they can be completely denaturated (left) or...

  10. Multifunctional Neural Fibers: Modulation from Gut to Brain

    Type
    Blog Post
    ...Fun fact: A 1 cm block of polymer can generate many meters of fibers!     Fig. 1 - Overview ... and brain neural circuits. Nature Biotechnology, 1–13. https://doi.org/10.1038/s41587-023-01833-5 Resources...channelrhodopsin 2 (ChR2), a blue light sensitive cation channel, fused to mCherry to the brain tissue. ... several months after implantation of fibers, indicating the system was indeed stable for continuous monitoring...interfaces with the entire body, so the obvious next applications are to expand the tool to different brain-organ...

  11. The Developmental Studies Hybridoma Bank: Over 25 Years of Antibody Sharing

    Type
    Blog Post
    ...the Monoclonal Antibody Research Institute dedicated to: 1) developing new ways of generating antibodies... factors such as PAX7, PAX6, AP-2 alpha and ISLET-1. In addition, we distribute monoclonal antibodies ...target proteins of interest for labeling, quantification, purification, chromatin immunoprecipitation and more...Freedom: The Expansion of Data Sharing in Research Publications ...

  12. The AAVantages of AAV in CRISPR Screens

    Type
    Blog Post
    ...integration into CRISPR-induced DNA breaks. Nature Communications, 10(1). https://doi.org/10.1038/s41467-019-12449...NGS) to identify genes of interest. Figure 1: Overview of in vitro AAV CRISPR screens. Created ...certain experiments. AAV can be safely handled at BSL-1, while lentivirus requires a BSL-2 setup. This requires...diseases. Signal Transduction and Targeted Therapy, 9(1). https://doi.org/10.1038/s41392-024-01780-w Additional...This reduces the chance of integration at random locations that could cause deleterious effects. It is important...multi-plasmid system, where you have one AAV vector dedicated to expressing Cas9, while the other houses the...

  13. AAVs in Retinal Gene Therapy

    Type
    Blog Post
    ...and Therapeutic Purposes. Annu Rev Virol. 2014 Nov;1(1):427-51. DOI: 10.1146/annurev-virology-031413-085355...personalized medicine. References and further reading 1. FDA approves gene therapy for blindness 2. Gene Therapy... Med Biol. 2014; 801: 687–693.  doi:  10.1007/978-1-4614-3209-8_86. PubMed PMID: 24664759. PubMed Central... time advances in human genetics led to the identification of several disease-causing genes, including... But it is the hard work and persistence of a dedicated team of scientists led by Dr. Jean Bennett (UPenn...-1250015778 7. Yu W and Wu Z., 2018. In vivo Applications of CRISPR-Based Genome Editing in the Retina...

  14. Viral Vectors 101: Inverted Terminal Repeats

    Type
    Blog Post
    ...) vector (Figure 1). Everything else outside the ITRs gets left behind.    Figure 1: Recombinant AAV ...productivity but increases transgene expression. Sci Rep, 7(1), 5432. https://doi.org/10.1038/s41598-017-04054-4... ITRs serve as the origin of replication and are required for replication, packaging, and vector persistence...viral rep (replication) and cap (capsid) proteins, which bind to the ITRs and initiate replication. ITRs are...structures, ITRs are prone to replication errors and disruptive mutations and truncations that can make vector ...binding site for Rep78 and Rep68 to initiate replication and a terminal resolution site that serves as...are noted. Created with biorender.com. ITR modifications and types  Since ITRs are one of the most important...

  15. Antibodies 101: ChIP

    Type
    Blog Post
    ...DNA with a PCR purification kit or with a phenol-chloroform extraction.    Fig. 1: Schematic overview...reserve a small aliquot, split into two parts to (1) run a DNA gel to confirm fragment size and (2)  incubate... let’s do a quick review. At this point you have (1) crosslinked your proteins of interest to any DNA ...next-gen sequencing. ChIP is an incredibly useful application for exploring these interactions, and can give... protein interactions, including epigenetic modifications. But it's not an approach for the faint of heart...background information. Your options for this step are sonication, which uses high-frequency sound waves to break...break up the DNA, or enzymatic digestion. Sonication provides a truly random digestion, but is harsher ...

  16. Podcast: A Malawian Professor's Path to Biotech Research

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

  17. Flow Cytometry Readouts: Yes, No, and Everything in Between

    Type
    Blog Post
    ...overlap with the negative control population (Figure 1). Based on where the GFP- population ends, you can...total cells present in the gate.   Figure 1: Example of yes-no flow plot of the (negative) control...for a yes-no dot plot, like the one shown in Figure 1.  Figure 3: An example of a histogram plot ...to the right indicates a higher signal, or upregulation, and a peak to the left indicates a lower signal...anything to the right of it, marking it as GFP+. Quantification for such graphs could simply be the percentage...case for you, switching to a contour plot should indicate where the two populations diverge. The smear ...place your gate based on the dot plot would be complicated.  Figure 2: (A) a smeary dot plot showing...

  18. Multiplexed Overexpression of Regulatory Factors (MORF) collection

    Type
    Blog Post
    ...the viral TF library, with each cell receiving only 1 TF expression construct. Cells were cultured following...states was generated (Joung et al., 2023). Fig. 1: Barcoded TF library was packaged in lentiviral particles...atlas of directed differentiation. Cell. 2023. 186 (1) 209-229. doi: 10.1016/j.cell.2022.11.026 Joung J....unique barcodes for each TF help streamline identification. While the Zhang lab controlled their multiplicity...single TF was delivered to each cell, future applications could deliver multiple TFs to each cell, allowing...processes. For help with library transformation and amplification, refer to the Zhang lab’s previously published...published protocol and its relevant modifications on the pooled library webpage. Disease models Good disease...

  19. Synthesized by Ginkgo Bioworks, Shared by Addgene: SARS-CoV-2 Plasmids for Many Expression Systems

    Type
    Blog Post
    ...give your research more visibility.    Figure 1: Illustration of SARS-CoV-2 from Philippa Steinberg...constructs include proteins like nonstructural proteins 1 to 16, spike proteins, envelope small proteins, and...Humana Press, pp 331–338 https://doi.org/10.1007/978-1-59745-181-9_23 Additional resources on the Addgene...other proteins that require post-translational modification may not be easily expressed in non-mammalian...constructs contain protein coding genes, tags, truncations, and codon optimizations similar to those found...are intended for the high level expression and purification of SARS-CoV-2 proteins for downstream experiments...wild type and codon optimized, full length and truncated versions of SARS-CoV-2 S (spike protein) cloned...

  20. Plasmids 101: Dimers and Multimers

    Type
    Blog Post
    ...plasmid. This phenomenon indicates the presence of a multimer.   Fig. 1: The monomer of Plasmid...and catenanes by agarose gel electrophoresis. Methods Mol Biol. 2009;582:11-25. doi: 10.1007/978-1-60761...plasmid sequencing services using long-read sequencing (1-25 kb), such as Oxford Nanopore sequencing or PacBio...10017-26. doi: 10.1074/jbc.M110.176636. Epub 2011 Feb 1. PMID: 21285356; PMCID: PMC3060453. Bazaral M, Helinski...multiple circular DNA forms of colicinogenic factor E-1 from Proteus mirabilis. Biochemistry. 1968 Oct;7(10...additional origins of replication compared to monomers, they are also able to replicate at a higher frequency...the right, with the entire monomeric sequence duplicated in tandem, resulting in a plasmid that is twice...

  21. A Practical Guide to Optimizing AAV DIO and FLEx Vector Expression

    Type
    Blog Post
    ...between 2 loxp sites and is excised by Cre (Figure 1). This results in one loxp site and one lox2272 site...cause of this spurious expression?   Figure 1.  Cre-recombinase binds to either pair of lox sites...recombination and can exhibit recombination levels as high as 1-3%. In light of this finding, we now include guidance...Efficiency, Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and 9 in the Mouse Brain. PLoS ONE 8:e76310...Vectors Pseudotyped with Viral Capsids from Serotypes 1, 2, and 5 Display Differential Efficiency and Cell...promoter, such as the truncated tyrosine hydroxylase (TH) promoter for catecholamine neurons or glial fibrillary... Flp), to either flip or excise a gene that is located between them. Early recombinase-dependent vector...

  22. Hot Plasmids Spring 2024

    Type
    Blog Post
    ...or nuclear expression of BFP2.     Figure 1: CROPseq-multi uses two sgRNAs with internal barcodes...transcript by endogenous processing enzymes (Figure 1). To ensure this tRNA processing only occurs on the...served as positive control (cis-ctrl). v1, vector 1; v2, vector 2; BD, binding domain; SDS, splice donor...)genome editing and gene therapy. Nat Commun., 14(1), 6578. https://doi.org/10.1038/s41467-023-42386-0...mice. B) Quantifications showing high microglia specificity of GFP expression for the indicated ssAAV5s...ssAAV5s. C) Quantifications showing high microglia transduction efficiency for the indicated ssAAV5s. Figure...3’ long terminal repeats (LTR). The 3’ LTR is duplicated during lentiviral integration, producing a second...

  23. The PAM Requirement and Expanding CRISPR Beyond SpCas9

    Type
    Blog Post
    ...that (1) the sequence is unique compared to the rest of the genome and (2) the sequence is located just...will cover three ways to circumvent this limitation: 1) the use of novel S. pyogenes Cas9 variants with varying...Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase smaller than SpCas9, so it can be packaged...Biol 19: . https://doi.org/10.1186/s13059-018-1591-1 Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, ...editing is most efficient when target sites are located in close proximity to the region to be edited. ... that were able to cleave target DNA sequences located upstream of either NGA or NGC PAM sequences (Kleinstiver...variant may increase the specificity of genome modifications at DNA targets adjacent to NGG PAM sequences...

  24. Sequencing Options for CRISPR Genotyping

    Type
    Blog Post
    ...the whole story Figure 1: Schematic representations of the indicated unbiased genotyping options.... and reproducibility of a study’s findings. Table 1: Biased Genotyping Options Technique Sequence Information...types Must be verified with cell-based method IDLV 1% Cell-based Programmable, can detect DSBs in live ...magazine.scienceforthepeople.org/.     References 1. Fu, Yanfang, et al. "High-frequency off-target mutagenesis...next-generation sequencing." BMC genomics 15.1 (2014): 1. PubMed PMID: 25409780. PubMed Central PMCID: PMC4246457...off-target sites across the genome and pinpoint the location of mismatches based on the sequences of the genome...those relating to DNA structure (e.g. epigenetic modification, bulges) that may also impact off-target edits...

  25. Communicating Your Science With Help From ComSciCon

    Type
    Blog Post
    ... skills in science communication will be a positive asset throughout your career? 1. Make scicomm practice... the Communicating Science Conference series for graduate students. I believe that communication is the...skill in science communication over time.  Like most skills, great science communication comes with practice...able to communicate with their colleagues and peers. Scientists also need to be able to communicate to expert...need to communicate with non-scientists, to establish broader impacts for their work in education and policy...with opportunities to practice communicating.  Some fantastic K12 education initiatives like BiteScis (https...careers in science communication The strength of story telling Science communication: 9 strategies to get...

  26. Viral Vectors 101: Optogenetic Tools

    Type
    Blog Post
    ... dual-color control of neurons. Nature Communications, 12(1), 1–20. https://doi.org/10.1038/s41467-021...to consider when choosing one.     Figure 1: Example of an experimental optogenetic setup. A light... 21–51). Springer US. https://doi.org/10.1007/978-1-0716-0755-8_2 Lehtinen, K., Nokia, M. S., & Takala... 21–51). Springer US. https://doi.org/10.1007/978-1-0716-0755-8_2 Boyden, E. S., Zhang, F., Bamberg, E...channelrhodopsin-2 (ChR2), a blue light-activated cation channel, from the archaebacteria Chlamydomonas ...stimulation. GPCR signaling GPCR signaling is a complicated process involving many downstream signaling mechanisms...break your experiments!  First, closely read the publication, including the supplemental data. You’re most...

  27. Antibodies 101: Introduction to Immunofluorescence

    Type
    Blog Post
    ... is conjugated to a fluorophore (Figure 1).   Figure 1: Comparison between how direct immunofluorescence...important considerations are to make sure your secondary 1) will recognize the species of your primary antibody...New York, pp 299–311. https://doi.org/10.1007/978-1-4939-8935-5_26 Stuurman N. (2012) Introduction to ...knowing that where you see fluorescence indicates your target’s location. Some other terms you might see associated...immunofluorescence? Generally speaking, IF is an application in which antibodies that have been joined to ...bind to a single primary, resulting in signal amplification. Another reason is cost effectiveness. Generating...distribute antibodies validate them in a variety of applications, including IF (or IHC or ICC), and will share...

  28. How Dry Ice Affects Viral Vector Sample pH & How to Avoid It

    Type
    Blog Post
    ... the samples stored in our lentivirus tubes (Fig. 1). However, once thawed, there was a substantial reduction...in the pH of samples stored in our AAV tubes (Fig. 1). It’s important to note that the change in pH only...using the thawed virus within 24 hours. References 1. Murphy BM, Swarts S, Mueller BM, van der Geer P, ...improve our shipment processes. After reading a publication describing how short term storage on dry ice ...medium, which is buffered and contains the pH indicator phenol red. We froze samples in a -80°C freezer...closed dry ice storage container for 48 hours (to replicate how our viruses are shipped), where they would...exposed to CO2/dry ice. All samples were tested in duplicate. What we found Not all tubes (or rather, not ...

  29. Hot Plasmids - February 2022

    Type
    Blog Post
    ...include mEGFP, mCherry, and HaloTag. Figure 1: (a) Anti-FLAG frankenbody (FB) construction from ...Comm. 2019. https://doi.org/10.1038/s41467-019-10846-1.   Optimized genetic screening technique using Cas12a... to Cas9’s larger expression requirements (Figure 1), but Cas12a has generally been underutilized in genetic...editing applications.  Figure 4: Overview of CasMINI for genome engineering applications. Image ...stability in cells, and use it in a variety of applications. This set of new anti-FLAG frankebodies includes...to FLAG-tag H2B for nuclear localization. (c) Applications of anti-FLAG-FB-GFP labeling various targets... and anti-HA frankenbodies have a variety of applications, including live-cell imaging, single-molecule...

  30. Viral Vectors 101: Viruses as Biological Tools

    Type
    Blog Post
    ...unleashes its cargo – its own genome.   Fig. 1: Viral particle infection into a host cell. Image ...Vector Resources at Addgene! References and Resources 1. Roe T, et al. “Integration of murine leukemia virus...nuclear import of human immunodeficiency virus type 1 preintegration complexes.” Proc. Natl. Acad. Sci. ...enough to ensure the virus can replicate and spread. Instead, viruses replicate by hijacking the host’s system...low immunogenicity which makes them ideal for applications with whole organisms. Finally, viral delivery...delivery is typically straightforward across applications, while electroporation of a human, for example, isn...people in nature, extra safety measures, like replication incompetent viral particles, are required. We...
Showing: 241 - 270 of 727 results