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We narrowed to 835 results for: Eras

Showing: 151 - 180 of 835 results

  1. Beyond the Blood Brain Barrier: PHP.B Enters the Central Nervous System Via the LY6A Receptor

    Type
    Blog Post
    Published
    July 23, 2019, 12:46 p.m.
    ...One of the biggest barriers to gene therapy treatment of central nervous system (CNS) diseases is the...because in vivo transduction of AAV serotypes generally tracks between strains of mice. This suggests ... engineered PHP.B. PHP.B was created by first generating a diverse library of AAV9 mutants that had a ...inheritance pattern of PHP.B permissiveness. The first generation of outbred mice (F1) had half the level of CNS...transduction as their C57BL/6J parent. The second generation (F2) of outbred mice also showed a Mendelian ...galactose and AAVR to transduce cells. The Wilson Lab generated results similar to the Deverman Lab when they ...Transport across the Blood-Brain Barrier." Molecular Therapy 27.5 (2019): 912-921. PubMed PMID: 30819613. Hordeaux...

  2. Getting the Most from Your Lentiviral Transduction

    Type
    Blog Post
    Published
    April 7, 2016, 2:30 p.m.
    ...that stably express a gene of interest. While the general approach for using lentivirus, infect and select...consider is the age of your culture post thaw. In general, the higher the passage number of a culture the...routinely test for mycoplasma contamination. There are several commercially available mycoplasma detection kits...parameters. For example, while freeze thaw is generally not recommended, Holic et al. found that lentivirus...your target cells Labs tend to follow the same general procedure for lentiviral infections, produce the...earliest steps in lentiviral transduction is the interaction between the viral envelop and the receptors on... on the target cell surface. The possible interactions and hence possible targets for a lentivirus are...

  3. Plasmids 101: Modular Cloning Applications and Kits

    Type
    Blog Post
    Published
    May 14, 2024, 1:15 p.m.
    ...enhancers, and cross-cassette interactions in gene expression, and to generate multi-gene knockouts. Mammalian...MoClo background MoClo is a clever way to assemble several DNA parts in a specific orientation using Type ...2011). It can be performed at multiple levels to generate anything from individual transcription units (...products without the undesirable side products generated during chemical synthesis of these molecules. ...bacteria. If your yeast experimental goals are more general in nature, or you aren’t planning on using K. phaffii...Multiplex Yeast Toolkit builds on the YTK and adds several additional capabilities, including conditional ...., 2014). This kit is a great starter for your general plant cloning experiments. Addgene also has two...

  4. Hassle-free 96-well Format Epitope Tagging Using Cas9 Ribonucleoprotein

    Type
    Blog Post
    Published
    June 28, 2018, 3:01 p.m.
    ...in Steve Pollard’s lab at the MRC Centre for Regenerative Medicine (CRM), Edinburgh. Most commercial antibodies...therefore, critical to understand how proteins interact with one another (or with DNA) to perform diverse...the most popular approaches to study protein interactions is the pull-down assay, wherein a protein of...analysed by mass spectrometry to identify the interacting proteins. Similarly, in ChIP-seq assays, chromatin...however, to these IP-based assays is to find (or generate in-house) a pre-validated antibody for each target...throughput because numerous cell lines can be generated, each with a different tagged protein of interest...these factors prevent stem cell researchers from generating clonal knock-in lines, especially for ambitious...

  5. Illuminating Choices: A Guide to Selecting Fluorescent Dyes and Ligands

    Type
    Blog Post
    Published
    March 21, 2024, 1:15 p.m.
    ...photobleaching. Interaction with receptors: Understanding how fluorescent dyes interact with specific receptors...intermolecular FRET for ligand-receptor interactions, require specific considerations regarding the choice of fluorescent...crucial in the world of biological research, medical therapy, and even drug development (Grimm & Lavis, 2022...targets through fluorescent labeling. There are several advantages to using these self-labeling proteins...is bound through green light emission. Key considerations in selecting fluorescent dyes/ligands Effects...best practice to determine optimal DOL is through several small-batch experimental labeling. Size is also...under certain conditions, which is an important consideration for assay design (Breen et al., 2016). When ...

  6. Hot Plasmids: Summer 2024

    Type
    Blog Post
    Published
    Aug. 27, 2024, 1:15 p.m.
    ... for Autoinhibition Release of Methyltransferase (CHARM), with several advantages over the earlier system...and stimulate the endogenous methyltransferase. The strategy is generalizable to a variety of DNA-targeting...Arimura et al. used MagIC-cryo-EM to determine several structures (at < 5 Å resolution) of the linker ....    Figure 2: A) When proteins (blue) interact with the air-water interface (AWI) before vitrification... 2024). The best-performing variant yielded an average integration efficiency of 23%, a more than four-fold...editor consisting of dCas9 fused to a DNA methyltransferase — that can drive lasting transcriptional silencing...  Rather than overexpressing an exogenous methyltransferase for epigenetic silencing, which can be toxic...

  7. Four Ways to Package Transgenes That Exceed the Size Limit of Adeno-associated Virus

    Type
    Blog Post
    Published
    Sept. 1, 2020, 1:15 p.m.
    ...Chamberlain JS (2011) Gene Therapy of mdx Mice With Large Truncated Dystrophins Generated by Recombination Using... reassemble fragmented genomes is the basis of several split vector approaches developed in the early ...full-length transgene (Chamberlain et al., 2016). It’s generally accepted that some form of homologous recombination...exact mechanism is not yet known. Drawbacks:  Generally an overlapping split vector has equal or higher...is currently no one ideal split vector system. Overall the efficiency of transgene expression from split... of Adeno-Associated Virus Capsids. Human Gene Therapy Methods 27:1–12 . https://doi.org/10.1089/hgtb.2015.140...Capacity in a Transgene-independent Manner. Molecular Therapy 16:124–130 . https://doi.org/10.1038/sj.mt.6300322...

  8. CRISPR 101: Cytosine and Adenine Base Editors

    Type
    Blog Post
    Published
    Feb. 13, 2025, 2:15 p.m.
    ...mouse inner ear. Fourth-generation cytosine base editors The fourth-generation base editors, BE4, reduce...variety of targets in human cell lines, with an average indel frequency of only 1.1%. These numbers are...over Cas9-mediated HDR for the loci tested, where average HDR-mediated editing frequency was only 0.5%, and...18 bases upstream of the PAM.  David Liu’s lab generated BE3 variants with other deaminases: AID, CDA1,...these three improvements represent the fourth generation of base editors. Compared to BE3, BE4 offers ...ABE7.10 is the most active editor, displaying an average editing efficiency of 53% with an editing window... phage-assisted evolution selection systems to generate ABE8e(TadA-8e V106W), which edits ~590-fold faster...

  9. Viral Vectors 101: An Introduction to AAV

    Type
    Blog Post
    Published
    May 30, 2023, 1:15 p.m.
    ...organism. These features make AAV ideal for human therapies and in vivo laboratory experiments ranging from...terminal repeats – the sequences that flank the deliverable DNA) are removed, resulting in recombinant AAVs... over 4.7 kb. Co-infection rates for AAVs are generally high enough for this approach to work, although...some mammalian tissues has shown expression for several years (Wonjo et al., 2013). Remember that AAVs ...As a starting point, we recommend checking the literature and our AAV data hub to see if others have used...transduction independently of DNA synthesis." Gene therapy 8.16 (2001). PubMed PMID: 11509958. Vandenberghe...adeno-associated virus manufacturing." Human gene therapy 21.10 (2010): 1251-1257. PubMed PMID: 20649475....

  10. dTAG - You're it!

    Type
    Blog Post
    Published
    June 21, 2018, 2:06 p.m.
    .... To overcome this limitation, we created a generalizable tag-based strategy, which we refer to as the... target degradation (Figure 1). Plasmids: We generated a series of plasmids that are available from Addgene...of N- or C-terminally FKBP12F36V-tagged fusion chimeras or locus-specific knock-in of FKBP12F36V. Detailed...or Jay Bradner ([email protected]). Considerations when getting the dTAG system up and running...your lab Confirm the functionality of your fusion chimeras For each protein-of-interest, it is important ...FKBP12F36V tag without disrupting protein function. A literature search can help determine which terminus is favorable... evaluate degradation of the FKBP12F36V-fusion chimera of interest. In the cell lines we have evaluated...

  11. Troubleshooting Your Plasmid Cloning Experiment

    Type
    Blog Post
    Published
    Sept. 24, 2019, 12:55 p.m.
    ...Fragments generation After you’ve designed your cloning scheme, you’re now ready to generate fragments... specific, there are few general parameters that you can adjust: temperature and time of incubation, and...fragments. You can generate the parts of DNA that you want to assemble together in a combination of different...multiple digestion be sure that the buffers and temperature are compatible between the different enzymes....genome organisms like mouse or human. There are several online tools that can help you with that, they ...companies selling PCR reagents. Nowadays commercial polymerases are very efficient, but if you are having difficulties...conditions. Purifying DNA fragments Once you’ve generated your DNA fragments, it is always a good habit ...

  12. Enhancing CRISPR Targeting Specificity with eSpCas9, SpCas9-HF1, & HypaCas9

    Type
    Blog Post
    Published
    Dec. 16, 2015, 3:30 p.m.
    ...separation is maintained by 2 sets of interactions : 1. Interactions between the non-target strand and a... through interactions between the non-target strand and the HNH/RuvC groove, and interactions between ...weakening interaction sbetween the non-target strand and Cas9, Kleinstiver et al disrupted interactions between...by the Cas9 HNH and RuvC nuclease domains 2. Interactions between the target strand and the gRNA (see ... the HNH/RuvC groove, then they could weaken interactions between the groove and the negatively charged...that if they could weaken sequence independent interactions between Cas9 and DNA, then they could diminish...effect on specificity was similar; SpCas9-HF1 generated fewer off-target cuts when compared to WT SpCas9...

  13. CRISPR 101: Cytosine Transversion Editors

    Type
    Blog Post
    Published
    March 25, 2025, 1:15 p.m.
    ...to base transition editing, we can expect several generations of optimization before the field begins to...review how CRISPR-derived base editing works in general. Base editors use either catalytically dead Cas9... Beyond UNG: Optimizing C → G editors Now that several labs had shown that preferential C → G editing ...rXRCC1, or the DNA binding and lyase domain of DNA polymerase β (PB) (Chen et al., 2021). Both of these fusion... Sun and Erwei Zuo labs tested and implemented several of these advances for CGBEs (Yuan et al., 2021)...efficiency and produced fewer indels than first-generation editors. This team also demonstrated that different... proteins whose fusion improved the CGBE: DNA polymerase D2 (POLD2), exonuclease 1 (EXO1), and RNA binding...

  14. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    Published
    Jan. 6, 2015, 5:21 p.m.
    ...cloning. The general cloning strategy described in the associated publication can be used to generate any combination... a modular mini-Tn5 vector that can be used to generate random mutagenesis libraries or to insert heterologous...oxidized. The Timer tool has already been used by several groups to monitor protein and cell ageing, as the...action via sequestration. The strategy relies on several observations: Rapamycin is a membrane-permeable...vectors are used with Multisite Gateway cloning to generate the final construct. Up to eight entry vectors...demonstrated the use of the SpyLigase peptide-peptide interaction to link affibodies or antibodies against common... Despite these advances, the Ting lab’s first-generation APEX suffered from inconsistent activity levels...

  15. Viral Vectors 101: AAV Variables That Matter

    Type
    Blog Post
    Published
    June 1, 2023, 1:15 p.m.
    ...your serotypes, titer, and promoter. Note that several steps in testing are effected by multiple AAV variables... as proxies.  Finally, the size and receptor interactions of a serotype can affect spread from the injection...and are useful if you're going to be working in several different systems or want to transduce a large ...Various AAV Serotypes and Their Applications in Gene Therapy: An Overview. Cells, 12(5), 1–41. https://doi.org... Gene Delivery to the Hypothalamus. Human Gene Therapy, 21(6), 673–682. https://doi.org/10.1089/hum.2009.169...Carroll, S. J. (2019). Astrocyte-selective AAV gene therapy through the endogenous GFAP promoter results in... in the rat spinal cord following injury. Gene Therapy, 26(5), Article 5. https://doi.org/10.1038/s41434...

  16. Simple CRISPR-based Epigenetic Editing: dCas9-directed DNA Demethylation

    Type
    Blog Post
    Published
    Dec. 14, 2021, 2:15 p.m.
    ...Tethering dCas9 to the histone acetyltransferase p300 or the DNA methyltransferase DNMT3A allows researchers...2013). Enzymatic domains tethered to dCas9 often interact with DNA independently of dCas9 targeting (Galonska... independent of their enzymatic activity. They interact with transcriptional machinery to modify local...modifying other sites in the genome and directly interacting with transcription machinery.   Well, I’...demethylation?  Dividing cells depend on DNA methyltransferase 1 (DNMT1) to copy methylation marks from parental...   Let me leave you with a few practical considerations: Design your gRNA to overlap the CpG of interest...that unmethylated CpG so that nothing else can interact with it. Remember, the goal of this experiment...

  17. Hot Plasmids May 2018 - Optogenetics, Decaffeination, Biosensors, and Fluorescent Protein Tools

    Type
    Blog Post
    Published
    May 15, 2018, 12:43 p.m.
    ...demonstrated that the caffeine concentrations in several beverages could be estimated by measuring growth of ...permits it to bind to the CIB1 (cryptochrome-interacting basic-helix-loop-helix) protein. After fusing...proteins of interest, a researcher can cause them to interact by stimulation with blue light.  Similarly, light...work including the soCoChR-GFP plasmid as well as several derivatives encoding alternative promoters and/...as starting material for the bioproduction of therapeutic methylxanthine drugs like theophylline. To facilitate... required enzymes, including a glutathione S-transferase homolog from a Janthinobacterium sp. Moreover...minimal media supplemented with various caffeinated beverages, but not with caffeine-free drinks. Finally, they...

  18. Popular Retroviral Vectors and Their Uses in Scientific Research

    Type
    Blog Post
    Published
    July 24, 2018, 12:51 p.m.
    ...things) gene delivery, generating pluripotent stem cells, and developing gene therapies for monogenic diseases...excellent tools for genetic engineering. There are several different viral vectors to choose from, for example...which includes the gamma-retrovirus and lentivirus genera, have the unique ability to integrate permanently...separating viral genes and transgene expression across several plasmids. The most commonly used viral vector systems...of the lentiviral elements and the 2nd and 3rd Generation Lentiviral Systems can be found in our Lentiviral...gamma-retroviral packaging plasmids are not interchangeable. General envelope plasmids, such as VSV-G, may be used across...interest into this tetracycline inducible, 3rd generation lentiviral vector backbone from the Shih lab....

  19. BeHeard Award 2018: Diseases of Glycosylation, Arginine Mutagenesis, & Neural Development

    Type
    Blog Post
    Published
    June 26, 2018, 1:33 p.m.
    ...structural determinants of protein-DNA interactions and how these interactions regulation gene expression. Recent...structural determinants of protein-DNA interactions and how these interactions regulation gene expression.     ...are being awarded free plasmids form Addgene to accelerate their research. Read on to learn how these researchers.... These studies are expected to identify new therapeutic targets/solutions and enable scientists to discover...mutations. References: Baldwin, Enoch, et al. "Generation of ligand binding sites in T4 lysozyme by deficiency-creating...in brain development since 2000 and have shown several mechanisms by which they affect brain and muscle...muscle development. The Gronostajski lab has generated mice wherein it's possible to delete the functional...

  20. The Materials Science of Optogenetics Experiments

    Type
    Blog Post
    Published
    Sept. 17, 2015, 2:30 p.m.
    ...needed for optogenetics. There are several important considerations with fabrication of the fiber optics...pulse generator such as the A.M.P.I Master-9 Pulse Stimulator, which can control output of several lasers...equipment, most of which is relatively cheap). Several protocols have been written outlining the different...stimulation. The scatter effect is also an important consideration when preparing the implant itself, which brings...least 85% of the initial laser output. The same considerations apply to the polishing and preparation of the... channel, Channelrhodopsin 2 (ChR2), exists in several states: closed/light receptive, open, closed/light...manipulation of the laser power source itself. Pulse generation (TTL pulse) is a standard electronics protocol...

  21. No Llamas Required - Synthetic Nanobodies Against Membrane Proteins

    Type
    Blog Post
    Published
    June 18, 2020, 1:15 p.m.
    ... plasmids for generating your own libraries are also available in the Sybody Generation Toolbox plasmid... two different coronaviruses into a llama over several days and isolated special camelid antibodies that...proteins that may unfold due to camelid high body temperature (37.2 - 39.2°C). The Seeger lab found a way around... around this by using plasmids to generate sybodies, synthetic nanobodies against the spike protein (Walter...blog post.  A rapid selection platform for generating sybodies against membrane proteins Developing...membrane protein of interest using the Sybody Generation Toolbox and commercially available reagents in...libraries (Zimmermann et al., 2018). Find the Sybody Generation Toolbox! Starting with libraries and a biotinylated...

  22. Plasmids 101: Protein Expression

    Type
    Blog Post
    Published
    June 7, 2018, 1:17 p.m.
    ...yeast and cell free expression systems. Overall the general strategy for protein expression consists ...or purify proteins to study their structure, interactions and functions. Proteins can also be purified...purified for use in molecular biology research (eg. polymerases and other enzymes might be purified and used ...derived from cells or using live cells. There are several types of expression systems used for protein production...Deciding which expression system to use depends on several factors: The protein you are trying to express...most popular hosts for protein expression with several strains that are specialized for protein expression...be purified from the cellular debris. There are several popular DNA vectors that can be used to produce...

  23. When is a Monomer not a Monomer? The Top Three Ways Your Favorite Fluorescent Protein Oligomerizes in Cells

    Type
    Blog Post
    Published
    April 19, 2016, 2:30 p.m.
    ...GFP/EGFP) for a transcriptional reporter or as a general cytoplasmic label of cells, there's no problem....to not produce artifacts in your experiments. Considerable effort has gone into making FPs monomeric, yet...ignorant of FP dimerization. Equally problematic, several reportedly monomeric FPs are not actually monomeric...more likely molecules of a particular type will interact. A protein with a nanomolar Kd will exist primarily... of the FP. The FP is confined to a plane and generally only rotates in 360°, increasing the probability...2]. 2. Fusion to an obligate dimer or oligomer Several cell proteins normally self-associate into homodimers... and increasing the signal from the second FP. Several early FRET biosensors were made with CFP and YFP...

  24. Deep Dive: Statistical Tests (Comparisons)

    Type
    Blog Post
    Published
    March 7, 2023, 2:15 p.m.
    ...Newman-Keuls method – More powerful. Moderate false negatives, moderate false positives. Duncan’s multiple...is being experimentally manipulated. Below are several core factors of statistics relevant for many downstream...Parametric tests  Statistical tests fall into two general categories: parametric and non-parametric. In this...variable. A two-way ANOVA could test the effect of temperature and humidity on an outcome such as total crop... one-way ANOVA can test either the effect of temperature or humidity on crop production.   MANOVA The ...example, a one-way MANOVA could test the effect of temperature on total crop production and the production window...A two-way MANOVA could measure the effect of temperature and humidity on total crop production and the...

  25. Plasmids 101: Using Transposons in the Lab

    Type
    Blog Post
    Published
    July 6, 2021, 1:28 p.m.
    ...We often think of DNA as inert. It generally stays put, which makes it easy to locate in a genome. But...mobile DNA, called a transposon, that’s a bit hyperactive and likes to jump around from one location in... at rates similar to retroviral vectors. The hyperactive version of the SB transposase, SB100X, is ~100...100-fold more efficient compared to first generation SB transposase (Mátés et al., 2009). hySB100x improves...human cells similar to retroviral vectors. The hyperactive PB transposase (hyPB) is ~10-fold more active...transposon insertion sites are detected with next generation sequencing, and analyzed to identify which insertions...Transgenic animals Transgenic animals are often generated by directly injecting DNA into the pronuclei of...

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

    Type
    Blog Post
    Published
    Dec. 22, 2022, 2:15 p.m.
    ...translation by generating a frameshift mutation. The majority of cellular repair events will generate these types... a less frequent DNA repair mechanism in general. On average, it accounts for less than 10% of DNA repair...family member which generates sticky ends upon cutting. Cas12a may promote HDR by generating ssDNA overhangs...Taking the road less traveled and generating a knock-in cell line instead of a knock-out? We’ve got you...editing Targeted genome editing events fall into two general classes: knock-in and knock-out. The goal of a ...exogenous DNA can be used as well. Below are some considerations for designing donor DNA for successful HDR ...viability of your cells. Enrich S-phase cells HR operates predominantly in S and G2 phase of the cell cycle...

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

    Type
    Blog Post
    Published
    Aug. 9, 2016, 2:30 p.m.
    ... the notation works? The + operator denotes intersection and the – operator denotes set difference. That... ChiRP-seq RNA Hi-C DNA (genome-to-genome interactions) DNase-seq DNA (regions that are accessible... the foundation of modern drug discovery and therapeutics. We’ll have more to say about clinical applications...Poisson statistics, each sub-population has, on average, one distinct enhancer disrupted. As our ESCs die...differentiate over time, we periodically use next-generation sequencing to measure the relative proportion...apply this knowledge to create targeted disease therapies with minimal side-effects on healthy cells and...Hypersensitive Sites and Intergenic CRISPRs Yours truly generated the DHS data set by starting with all of the DHS...

  28. CRISPR/Cas9 FAQs Answered!

    Type
    Blog Post
    Published
    March 13, 2014, 4:08 p.m.
    ...A7: A few notes below are considerations for designing HR donor. Generally if off-target (non-specific... and surveyor. Q15: I used DNA polymerase Takara Ex Taq™ DNA Polymerase for my genomic PCR, but couldn't...In our hands, Herculase II Fusion polymerase or Kapa Hifi Polymerase work very well. Some groups have ...concentration of Cas9 and guide RNA (gRNA). These considerations only matter if the PAM sequence is immediately...additional PAM sequences should not be sufficient to generate off-target DSBs; there needs to be extensive homology...most efficient and easiest way is to co-transfect several plasmid together, with each plasmid having a targeting...purified oligo is fine based on new data). For general transfection, you could use 1 µl of 10 µM oligo...

  29. Viral Vectors 101: Preparing Pooled Libraries

    Type
    Blog Post
    Published
    July 13, 2023, 1:15 p.m.
    ... are typically DNA preps, but we do distribute several of our most popular CRISPR pooled libraries and... A) Amplification, validation, and lentiviral generation of a pooled library. An aliquot of the pooled...experiments. Pooled library lentiviral preps are generally aliquoted to reduce the number of freeze-thaw ...libraries we amplify in house, but these are not general amplification protocols. Rather, each protocol ...amplified, you'll need to verify your library by next-generation sequencing (NGS). The representation of your ...our protocol on Lentivirus Production provides a general approach for producing lentivirus in Lenti-X 293T...you can transduce your cells.  Titering After generating your lentivirus, you’ll want to infect your cells...

  30. Career Insights: Technical Support Specialist

    Type
    Blog Post
    Published
    Nov. 9, 2017, 2 p.m.
    ...including tips, advantages, and disadvantages of this overall rewarding role. Learn about Other Addgenies and...role in technical support? It takes some time (several years) to build up comprehensive lab experience...You will also help troubleshoot products, give general experimental guidance, and resolve complaints about...willingness to help others with their problems, a general positive attitude, and an open-minded personality...any incoming requests or problems - there is considerable diversity in the types of questions customers...will do well in a technical support role. What general characteristics do jobs in technical support have...covered by the lab based team as part of their general job duties. Bigger companies have dedicated technical...
Showing: 151 - 180 of 835 results