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We narrowed to 328 results for: hal.1

Showing: 81 - 100 of 328 results

  1. Easi-CRISPR: Generating Knock-In and Conditional Mouse Models

    Type
    Blog Post
    ...much more favorable than previous methods yielding 1-10% knock-in. Read on to learn how you can make CRISPR...one NHEJ-repaired indel, often occur. Figure 1: In conventional gene targeting (A), you must first... drug induction systems (ttA or rtTA) are between 1 to 2 kb in length. There are plenty of Addgene plasmid...CRISPR ribonucleoproteins.” Genome Biol. 18 (2017) (1):92. DOI: 10.1186/s13059-017-1220-4 PubMed PMID: 28511701... using long ssDNA donors.” Nat Protoc. 13 (2018) (1):195-215. DOI: 10.1038/nprot.2017.153 PubMed PMID:...engineering using CRISPR nucleases. Genome Biol. 19 (2018) (1):25. DOI: 10.1186/s13059-018-1400-x PubMed PMID: 29482575...Cre-dependent conditional alleles, has remained a challenge. Enter Easi-CRISPR: a method that harnesses the...

  2. Channelrhodopsins with improved light sensitivity for minimally-invasive optogenetics

    Type
    Blog Post
    ...neuron which generates an action potential (Figure 1). This light-induced excitation can take place in ...channelrhodopsin activation to a small volume of brain tissue (~1 mm3). Current channelrhodopsins also need high-intensity...fiber-optic cables into the brain. Figure 1: With no blue light, the channelrhodopsin remains ...optogenetics.  Challenges of engineering channelrhodopsins There are three main challenges to engineering...overcome channelrhodopsin design challenges To overcome these challenges, the Arnold and Gradinaru Labs ...lower multiplicity of infection (MOI) makes it challenging for ChR2(H134R) to function because it’s lower...

  3. 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...of interest is essential, a true knockout can be lethal, and you'd instead want to create a conditional...depositing with Addgene! Figures were adapted from: Hall B, A Limaye, & AB Kulkarni. (2009) "Overview: Generation...

  4. Quickest Way to Deposit Plasmids: The Deposit Spreadsheet

    Type
    Blog Post
    ...how to use our deposit spreadsheet to complete Step 1. Steps 2 and 3 are usually very easy — our tech transfer...scientist, you will have the most involvement with Step 1, and, while data entry can be a chore, we’ve made ...Deposit” and “Download Deposit Spreadsheet” (see Figure 1). Please read our “How to Deposit Your Plasmids with...thorough guide to our online data entry process. Figure 1: Begin the deposit process by clicking “Submit Online...spreadsheet (Figure 2) and are described in Table 1. Some cells have drop-down options for you to choose...and are exceptions to our standard workflow.  Table 1: Guidelines for the data fields in the deposit spreadsheet... (budding yeast), S. pombe (fission yeast), A. thaliana (mustard weed), O. cuniculus (rabbit), synthetic...

  5. Isolating a Monoclonal Cell Population by Limiting Dilution

    Type
    Protocol
    ...Because this is such a small volume, first make 1 mL of a 1:100 dilution of the homogenized cell solution... were transduced with lentiCas9-Blast 1 and then selected with 1 μg/mL blasticidin for 9 days. Single ...with lentiCas9-Blast 1 . A549 cells were transduced (MOI = 37) and selected with 1 μg/mL blasticidin for...conditioned medium (see below for more details) Day 1: Seed individual cells in a 96-well plate Day 2–14...final 5 cell/mL solution, transfer 12.5 µL of the 1:100 dilution to 10 mL of conditioned medium. Transfer...cells will appear as colonies in the well ( Figure 1 ). You will be able to tell if there was more than...transgene expression ( Figure 2 ). Sample Data Figure 1: Generation of monoclonal cell lines from expansion...

  6. Evolution of Brainbow: Using Cre-lox for Multicolor Labeling of Neurons

    Type
    Blog Post
    ...the Brainbow mouse neural labeling system. Brainbow-1 and -2 have been further refined to create Brainbow...subsequent recombinations cannot occur. While the Brainbow-1 system employs Cre-mediated deletion, the design of...antibodies specific to each fluorophore. Third, Brainbow-1 and Brainbow-2 each contained a “default” state; for...dendritic processes not previously visible with Brainbow-1 and -2. The general structure of Brainbow-1.0 is retained...Rheenen J, Simons BD, Clevers H. Cell. 2010 Oct 1;143(1):134-44. PubMed. Drosophila Brainbow: a recombinase-based...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...

  7. A Primer on Optogenetics: Introduction and Opsin Delivery

    Type
    Blog Post
    ...Jacqueline N Crawley  [et al]. 2012;Chapter 1:Unit 1 20 1-18. doi: 10.1002/0471142301.ns0120s59. PMID:...mutually exclusive) commonly used in optogenetics: 1) electrophysiology and 2) behavioral experiments. ...can be broken down broadly into three main steps: 1) Selection and expression of your opsin of choice ...reviews for a discussion of the different variants [1, 2, 4]. And, of course, the best way to learn about...monitor expression at various time points (e.g. every 1-2 weeks over 8 weeks) in order to determine the optimal...follow him on Twitter @derekpsimonphd.    References 1. Tye KM, Deisseroth K. Optogenetic investigation of..., Jacqueline N Crawley  [et al]. 2013;4(435):4 35 1-4  20. doi: 10.1002/0471142301.ns0435s65. PMID: 25429312...

  8. Antibodies 101: ChIP

    Type
    Blog Post
    ...reserve a small aliquot, split into two parts to (1) run a DNA gel to confirm fragment size and (2)  incubate...with a phenol-chloroform extraction.    Fig. 1: Schematic overview of ChIP. Image courtesy of Shengliu... let’s do a quick review. At this point you have (1) crosslinked your proteins of interest to any DNA ...such as sequencing or qPCR. Yay!  Where’s the challenge in that?  So while this method is certainly long...the sample type and size.) It can be quite the challenge, and take a few rounds of adjustments, to get ...

  9. Tips for Getting a Faculty Position

    Type
    Blog Post
    ...Full "Applying for a Faculty Position" eBook Here 1. Have a competitive CV You don't necessarily need ...that people would agree is important to understand. (1-2 sentences). For example: Distinguishing correctly... What will be the focus of your first grant? (1-2 sentences) Simply stated, what are your aims...Talk." 4. The chalk talk, simple in principle The first question I usually get about chalk talks is, what... whether there is chalk. Sometimes. It can also be a slide deck, markers, etc. Chalk or markers force ... Developing your Chalk Talk These items should be part of the beginning of your chalk talk. These should... This edition has substantial expansions on the chalk talks and Zoom interviews sections, along with new...

  10. Pathways Over Time Plasmids Engage Students in Functional Genomics Research

    Type
    Blog Post
    ...to undergraduate students (1, 2), but educators face significant challenges in designing suitable projects...6). Methionine synthesis in S. cerevisiae (Figure 1) occurs through a well-characterized pathway with ...sequences for ten different MET/Met genes (Figure 1) from the two yeast species into the pYES2.1/V5-His...selected from different parts of the Met pathway (Fig. 1). In addition to the complementation experiments that...about Clare at the Biology Dept. site.  References 1. American Association for the Advancement of Science...

  11. Adeno Associated Virus (AAV) for Cell and Gene Therapy

    Type
    Blog Post
    ...a period of 1 to 3 years – the treatment appears to be both safe and effective. Challenges for AAV gene...reconstitution with a normally functioning T cell repertoire (1). In this example, it was possible to transduce the...research and science communication.     References 1. Gaspar, H. B., S. Howe, and A. J. Thrasher. "Gene... cargo (DNA or RNA) to target cells has been challenging. A variety of vector delivery technologies have...promising, scientists are working to overcome key challenges to their efficacy:  Pre-existing immunity: Many...

  12. Twenty Years of Sharing: Expanding Accessibility

    Type
    Blog Post
    ...our repository to an amazing 111 countries (Figure 1)! But while we were making the materials available...accessibility varied country to country.   Figure 1: Number of countries Addgene distributes to per year...date, we have partnered with six distributors (Table 1), in China, Korea, Japan, Brazil, India, and Mexico...material is right for their experiment.     Table 1: Most popular guides, protocols, and collections of...in 2023, from visitors in 218 countries. In Table 1, you can see our most popular educational resources... execute a step can provide needed clarity (Table 1). Our most popular video, however, is “What is a Plasmid...persisted despite our best efforts. With a little over half of our orders coming from researchers outside the...

  13. Reagent Repositories Are Speeding up Science During the Pandemic

    Type
    Blog Post
    ...available from their original sources. Figure 1: Biological repositories that distribute mouse strains... from Reproducibility for Everyone. Case study 1: Cell lines Many of the reagents used to investigate...COVID-19: an observational study. The Lancet Microbe 1:e14–e23 . https://doi.org/10.1016/s2666-5247(20)30004... J, Jia HP, Halabi C, Sigmund CD, Meyerholz DK, Kirby P, Look DC, Perlman S (2006) Lethal Infection of...

  14. Lentiviral Vector Guide

    Type
    Guide
    ...typically ranges from 8–10 kb, encoded on RNA (Figure 1). This RNA is reverse transcribed into the provirus...been removed or mutated for increased safety. Figure 1: Wild-type lentivirus genome. Created with BioRender.com...genes (for example, vif , vpr , vpu , and nef for HIV-1). The LTRs flank all of these genes, and anything ...transgene and LTRs (chimeric 5’ LTR) Packaging plasmid 1 — contains gag and pol Packaging plasmid 2 — contains...and third-generation systems, please refer to Table 1. Figure 4: Third-generation lentiviral plasmids. Created...heterologous enhancer/promoter such as CMV or RSV Table 1. Summary of key differences between second- and third-generation...More Lentiviral Vector topics Addgene Protocols pLKO.1 - TRC Cloning Vector Protocols Lentiviral Vector Production...

  15. Fluorescent Proteins 101: Luciferases

    Type
    Blog Post
    ...available. Table 1 compares some general advantages and disadvantages of each approach. Table 1: Properties...alternatives to fluorescent proteins. Figure 1: Firefly, from Wikimedia Commons. The North American...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...

  16. Top Requested Lentivirus and AAV of 2016

    Type
    Blog Post
    ...visualizing hM4D(Gi)-mCherry in fixed tissue. References 1. Aschauer DF, Kreuz S, Rumpel S. Analysis of transduction...efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One. 2013...PMC3069789. 3. Kügler S, Kilic E, Bähr M. Human synapsin 1 gene promoter confers highly neuron-specific long-...makes generation of high titer Cas9 lentiviruses challenging. This cytotoxicity also causes growth rate variability...):337-47. PubMed PMID: 12595892. 4. Sanjana NE, Shalem O, Zhang F. Improved vectors and genome-wide libraries...

  17. 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...earlier Voltron indicators (a fusion of Ace2N and HaloTag, Figure 4), they determined that an A122D mutation...changing the dye that becomes conjugated with its HaloTag domain.  Find Voltron2 plasmids here! Abdelfattah...

  18. Truncated gRNAs for Regulating Gene Expression

    Type
    Blog Post
    ...while its nucleolytic activity is eliminated (Fig. 1) (1, 3). The gRNA can be further modified by incorporation... Podcast Interview with Alex Chavez Figure 1: Schematic of native (nuclease-competent) Cas9 interacting...which Cas9 protein is directed to which target site (1). Although this is possible, this strategy suffers...sequence or regulating gene expression.     References 1. Esvelt, Kevin M., et al. "Orthogonal Cas9 proteins...different perturbations. Consider, for instance, the challenge of attempting to simultaneously and selectively...

  19. Viral Vectors 101: Optogenetic Tools

    Type
    Blog Post
    ...dual-color control of neurons. Nature Communications, 12(1), 1–20. https://doi.org/10.1038/s41467-021-24759-5 Zhang...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...nn1525 Gunaydin, L. A., Yizhar, O., Berndt, A., Sohal, V. S., Deisseroth, K., & Hegemann, P. (2010). Ultrafast... Augustin, S., Sauter, K., Papagiakoumou, E., Gottschalk, A., Soba, P., Emiliani, V., Engel, A. K., Hegemann...

  20. Mobile-CRISPRi: Bringing CRISPRi to Diverse Bacteria

    Type
    Blog Post
    ...active for greater than 50 generations. Figure 1: Mobile-CRISPRi's modular components. Image from Peters...on a non-selective plate. Most bacteria tested had 1 transfer in every ~100 to 1,000 colonies, but some...including E. faecalis and P. mirabilis, had only ~1 transfer per 1,000,000 colonies. Higher transfer rates...which creates a challenge when studying them since their complete knockdown is lethal. Incomplete or titratable... to lab-adapted bacteria because it has been challenging to introduce CRISPRi machinery into diverse bacteria...
Showing: 81 - 100 of 328 results