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We narrowed to 957 results for: Cre

Showing: 341 - 360 of 957 results

  1. Five Popular Model Organisms

    Type
    Blog Post
    ...developing zebrafish pancreas. They first tagged these cells using plasmids. Then using HOTcre, a method that...of quiescent and proliferative pancreatic β-cells identified by HOTcre mediated labeling." Proceedings...these systems check out Gerald Rubin’s lab who created a set of modular vectors that can be used to optimally...unicellular organisms, they can also be used to easily screen multiple therapeutic compounds in one experiment...embryos with DNA or RNA to edit their genomes or to create transgenic animals. Zebrafish are an ideal model...β-cells that originate in different regions of the pancreas and produce different levels of insulin (Hesselson...

  2. Filming Science Videos in the Age of Social Distancing

    Type
    Blog Post
    ...Creating great educational science videos is tough. Is the content accurate? Are we conveying complex...people in tight locations. Conference rooms for on-screen presenters! Labs and benches! Everything was out...videos. It’s really nice to have a scientist on-screen, somebody who really knows the protocol and can...would be to use an iPad to shoot.  Build: Jennifer created a mobile camera area that included iPad and her...where I could focus on camera and audio, and the on-screen talent could just focus on their interview or performance...re-opened very early in the pandemic with some incredibly strict safety protocols to keep Addgenies safe...camera) would remain masked, it freed us up to be creative.  Test: Our first official shoot went off with...

  3. Targeting HIV-1 with CRISPR: Shock and Kill or Cut it Out?

    Type
    Blog Post
    ...Symptoms of AIDS include rapid weight loss and an increased risk of infection, including both common infections...ART-treated patients lack plasma HIV-1, they are at increased risk for other chronic diseases, including dementia...disorders, nerve damage, and cardiac disease. This increased disease risk is attributed to latent HIV reservoirs... used histone deacetylase (HDAC) inhibitors to increase transcription of the integrated HIV-1 genome. ... enhancer region near the NF-KB binding sites increased activation of HIV promoter-luciferase constructs...microglial line, treatment with CRISPR/Cas9 SAM increased the percentage of cells with activated HIV-1, ...cell. ZFNs and CRISPR/Cas9 have also been used to create mutations in genes essential to HIV-1 replication...

  4. Understanding GPCR Signaling Using TRUPATH

    Type
    Blog Post
    ...resulting in decreased BRET2 signal. With increased concentration of drug, the BRET2 signal decreases. ...leads to downstream effects.  Bryan Roth’s lab created a collection of plasmids called TRUPATH to interrogate...the complex will disassociate, resulting in a decreased GFP2 emission. The activation state can be measured...showing the effectiveness of using TRUPATH as a screening technique. Lastly, the activity of both antagonists...plasmids are available as a collection, facilitating screening across the transducerome, to gain a more complete...

  5. SunTag and Fluorescent Imaging

    Type
    Blog Post
    ...scaffold. To increase expression, Tanenbaum et al. modified the GCN4 sequence to increase its alpha-helical...activation. Multiple nuclear localization signals (NLS) increase protein import into the nucleus. In developing... Weissman labs took this biological lesson and created a system to amplify fluorescent signals. Named ...protein is expressed at low levels, you have to increase your imaging time to get enough signal. This workaround...cells without affecting protein function, thereby creating a single-molecule reporter of intracellular processes...alpha-helical structure and stability, creating v4 SunTag. Since the v4 system does not display protein aggregation...

  6. Hot Plasmids - June 2020 - Barcoded CRISPR Library, Sparse Cell Labeling, Calcineurin Reporter, and DNA Staining Dye Alternative

    Type
    Blog Post
    ... Figure 1: Initial steps in the CRISPR-pooled screening using sgRNAiBAR. Image from Zhu et al., 2019.  ... Peking University has developed a new CRISPR screening method that makes use of guide RNAs with internal...false-positive rate discovery rate, conventional CRISPR screens require a low multiplicity of infection (MOI) during... annotated human gene, it enables genome-wide screening with a high MOI, which is beneficial for a low... calcineurin dynamics and can serve as a drug-screening platform to identify new antifungals that target... assay, and are not suited to high throughput screening, calcineurin dynamics measurements, or monitoring... attached fluorescent protein to the DNA. They created plasmids for purification of tTALE-eGFP and tTALE-mCherry...

  7. Multiplexed Overexpression of Regulatory Factors (MORF) collection

    Type
    Blog Post
    ...finding. Transcriptional program changes from this screen successfully differentiated cells resembling all...from Joung et al.)    Putting MORF to work Screens and selections In its current format, the MORF ...library is most immediately useful for TF library screens. Previously engineered TF libraries were not as... library can be used with other selection and screening mechanisms, although the unique barcodes for each...mutation and/or TF dysregulation. A targeted TF screening approach can not only lead to a better understanding...better simulate a disease in a model system. TF screens can be designed to identify custom TF programs ...Cas-9 knockout and transcriptional activation screening. Nat Protoc. 2017 (4):828-863. doi: 10.1038/nprot...

  8. Choosing a CRISPR Nuclease: Site Accessibility, Specificity, and Sensitivity

    Type
    Blog Post
    ...approach, the Anna Cereseto Lab created pX-evoCas9, which they showed also increased specificity. Most recently...broader PAM specificity can target more sites. Created with BioRender.com. Specificity Specificity...multiple PAM sequences (NG, GAA, and GT), greatly increasing the number of genomic loci that can be targeted...developed SpCas9-HF1. Both of the enzymes showed decreased off-target activity relative to Cas9. Since then...accurate Cas9 (HypaCas9). This nuclease showed decreased or similar off-target activity, relative to eSpCas9..., et al. "Directed evolution of CRISPR-Cas9 to increase its specificity." Nature communications 9.1 (2018...

  9. Sharing is Caring: But How to Distribute Open Hardware?

    Type
    Blog Post
    ... complexity of the device increases, as does the price. With this increased complexity comes the need ...adapted and combined with creativity and feedback from the community to create state-of-the-art devices...via workshops, seminars, or hackathons greatly increases accessibility. Through this approach, the Planktoscope...Madagascar. Platforms that allow for on-going creativity with the construction plans. Appropriate open-source...sources within the next few years, with the goal of creating more transparent and reproducible science in the...

  10. Antibodies 101: Polyclonal Antibodies

    Type
    Blog Post
    ...antibodies produced from its serum several weeks later. Created with biorender.com. To generate polyclonal ...additional booster immunizations every 2-3 weeks to increase the antibody titer. The titer is tested following...epitopes of the target protein. While this can increase background staining (see next section), it may...experimental protocol, using a polyclonal antibody increases the likelihood that one of the clones will still...crosslinking (ChIP) or fixation (IHC). Because of their increased sensitivity, polyclonal antibodies are particularly...epitopes of their target protein thereby greatly increasing signal amplification.  When to stay away from...population recognizes many different epitopes, increasing the likelihood of cross-reactivity with other...

  11. Antibodies 101: Affinity Reagents

    Type
    Blog Post
    ...new Antibody Guide and not just because I helped create it. But while antibodies, as the OG (as the kids...reagents we just didn't have the space to give proper credit to. Thankfully, as Addgene's resident blog editor...the majority of these alternative reagents are created in the lab, instead of generated in vivo, they ...Listed in no particular order.) Fab fragments are created through enzymatic cleavage of the variable region...in clinical applications. Fab fragments can be created in lab in situations where a suitable antibody ... fusion proteins similar to a Fab fragment but created through engineering instead of cleavage. They are...cheaper to make than antibodies. Minibodies are created by binding together two scFvs with a CH3 linker...

  12. Adenoviral Vector Production and Troubleshooting

    Type
    Blog Post
    ... Gen AdVs were designed to have increased cloning capacity, decreased  potential for the generation of...E4 are deleted in 2nd generation AdVs, thereby increasing cloning capacity to ~12 Kb. The ever popular ...generated high interest for gene therapy due their increased cloning capacity (up to 36 kb!), long-term transgene...AdEasyTM system is the most popular method for creating adenoviral vector constructs. It consists of 2...can be repeated multiple times (2-4 rounds) at increasing scale over the course of 1-2 weeks. Each round...of amplification should result in a 10-100-fold increase in virus. Purification (2 days) - Purification...

  13. 5 Tips for Troubleshooting Viral Transductions

    Type
    Blog Post
    ... prior to their transduction has been shown to increase AAV transduction efficiency by approximately 15...efficiency depends on viral titer Both of these can be increased by concentrating a viral stock. Different titering... virus-cell contact A variety of reagents can increase transduction efficiency. These reagents typically...reagent that is commonly used and has been shown to increase transduction efficiency by 10-fold (Davis et al...hematopoietic or primary cells), and has been shown to increase transduction efficiency by 1.5-fold (Stockschläder...12705861. 3. Chen, Sifeng, et al. "Freeze-thaw increases adeno-associated virus transduction of cells."... R. Morgan, and Martin L. Yarmush. "Polybrene increases retrovirus gene transfer efficiency by enhancing...

  14. Degrading DNA with Cascade-Cas3

    Type
    Blog Post
    ...different compositions of the Cascade complex. Created with BioRender.com.   The Cascade complex Cascade...Figure 2: Cascade complex from CRISPR type I-E. Created with BioRender.com. Thanks to this complex,...Tomoji Mashimo lab and the Tilmann Weber lab — to increase efficiency of these systems, though consistent...General overview of the Cascade-Cas3 mechanism. Created with BioRender.com.   The reeling mechanism...unwinding of dsDNA occur simultaneously, which creates a loop in the target strand that is cut occasionally...repair without the section between the two gRNAs, creating a deletion. However, there is the chance of a .... Future targets Cas3 has the potential to be incredibly useful in therapeutics. Imagine deleting a problematic...

  15. Pooled Library Amplifications

    Type
    Blog Post
    ...Factors to consider when creating a pooled library amplification protocol. Created with BioRender.com. ...Re-transformation of the SPLINTR Libraries (PDF, 401 KB) Screening Zhang Lab - Multiplexed Overexpression of Regulatory...et al. Nat Protoc. 2017 with modifications Screening pCMV-Rep78/68 Scanning Saturation Mutagenesis...Amplification and Sequencing Protocol (DOCX, 32 KB) Screening Rinehart Lab Iterative Synthetically Phosphorylated...-Cas9 knockout and transcriptional activation screening, a protocol paper by the Zhang lab, and the Broad...More resources on the Addgene blog Genome-Wide Screening Using CRISPR Preparing Pooled Libraries NGS Quality...

  16. CRISPR 101: Mammalian Expression Systems and Delivery Methods

    Type
    Blog Post
    ...expression? Are you trying to create a single point mutation? Do you want to create a fusion with a reporter...include a homology directed repair (HDR) template to create knock-ins, point mutations, or to add a tag, you...vivo. A common choice for conducting genome-wide screens using CRISPR. AAV transduction Only compatible...Transient expression of CRISPR components, expression decreases as RNA is degraded within the cell. Can be used...Transient expression of CRISPR components, expression decreases as gRNA and Cas9 protein are degraded within the...validated tools.  Special thanks to Joel McDade for creating the Expression Systems table. Marcy Patrick contributed...

  17. CRISPR 101: RNA Editing with Cas13

    Type
    Blog Post
    ...and found that 50 nt spacers increase editing efficiency but also increase off-target editing within the...1090), which decreases the REPAIRv1 construct size from 4,473 bp to 4,152 bp without decreasing editing efficiency..., which deaminate adenosine to inosine in RNA, creating a functional A->G change. They fused ADAR deaminase...to dPspCas13b, but observed low RNA editing. To increase A->G editing, they employed hyperactive ADAR constructs... they hoped to destabilize ADAR-RNA binding to decrease off-targets. Of the tested constructs, ADAR2DD...

  18. Droplet Digital PCR for AAV Quantitation

    Type
    Blog Post
    ...generation oil are moved through small channels to create a water-in-oil emulsion containing approximately...the number of positive droplets as the dilutions increase. The ddPCR software uses the ratio of positive...parameters, here are a few things you can try: Increase the number of cycles. After additional rounds ...amplification, your fluorescence amplitude will increase, leading to greater separation of your positive...droplets. No more than 50 cycles is recommended. Decrease your ramp rate. A rate of 2C/s is recommended ... the droplets, but you can go as low as 1C/s. Increasing the elongation time to 2 minutes and the denaturation...denaturation time to 1 minute has been shown to increase droplet separation (Witte et al., 2016). This is...

  19. CRISPR Between the Genes: How to Experiment with Enhancers and Epigenomics

    Type
    Blog Post
    ...CRISPR screen to disrupt these enhancers. The third and final step is to conduct our CRISPR screen. We start... the marvel of DNA compaction, an enhancer can increase the expression of a gene that is millions of base...stem cells, and conclude with a targeted CRISPR screen that can disrupt these enhancers. A real-world ...that have NANOG binding sites 2. Design a CRISPR screen to target and disrupt the NANOG enhancers 3. CRISPR...you’re interested in designing your own CRISPR screens for enhancers, check out the Appendix. Conclusion...precision medicine. We can apply this knowledge to create targeted disease therapies with minimal side-effects...

  20. Sharing Your Lab Protocols: Using Apps to Save Time & Track Your Experiments

    Type
    Blog Post
    ...Website: http://protocols.io/ Protocols.io is the creation of ZappyLabs. Currently in beta testing, the software...courses, as well as a space for research labs to create their own lab or group websites. OWW is managed... cells. If you’re familiar with the process of creating and editing Wikipedia pages, OWW will be relatively...The site allows you to comment on protocols and create lists of your favorite protocols for future reference...of the early protocol sharing websites. It was created in 1999 and allows scientists to contribute protocols...web-based lab management tool that allows labs to create accounts in which their members can share protcols...your lab’s needs. At Addgene, we’ll continue to create new molecular biology educational resources, such...
Showing: 341 - 360 of 957 results