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  1. Mouse Modeling, Part 2: Breeding and Crossing Mice

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    ... for introducing one modification into the mouse genome, they are not commonly used to introduce multiple...opposed to ordering control mice from a stock center. This serves as a better control since the experimental...series, we covered techniques that can be used to introduce genetic modifications into mouse embryos. But ...knockout lines and Cre-lox lines, and how to properly control for genetically engineered mice in your experiment...mutations. This is because as more mutations are introduced into a single embryo, the likelihood that a mouse...genetic engineering should be used to separately introduce each mutation independently into different mice...not a waste. Best practice is to use littermate controls in your experiment - use mice that were bred in...
  2. Plasmids 101: Repressible Promoters

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    ...Promoters control the binding of RNA polymerase and transcription factors. Since the promoter region ...promoters can be regulated via positive or negative control. Positive Repressible: An activator protein is ...pJKR-H-tetR from the Church lab uses this system to control expression of GFP.)  To turn this inducible system...system, Gossen and Bujard created the tetracycline-controlled transactivator (tTA). They fused TetR with the...displays low promoter activity (~20% activity of the strong yeast promoter pTEF). As ethanol accumulates, it...interest to be expressed. These permit exquisite control of gene expression and tracing of gene expression... and Potter labs has refined the Q system and introduced the second generation QF2 activators. These activators...
  3. Antibodies 101: Conventional vs Spectral Flow Cytometry

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    ...markers. Compensation/reference control(s) Compensation or “reference” controls are samples stained with a ...is not the same as your single color control. Reference controls: Beads or cells? Compensation beads are...mathematical algorithm and single stained reference controls to distinguish one fluorophore from another within...autofluorescence (you can see this in your unstained flow controls). Spectral flow cytometers include a tool called... or spectral flow. In spectral flow, reference controls tell the software to look for that exact signature...using conjugated dyes, it is critical that your controls and your sample antibodies are from the same batch... are a convenient tool to obtain a strong positive signal for a given fluorophore, as they contain a mixture...
  4. Don’t FRET: Bimolecular Fluorescence Complementation Makes Visualizing Protein-Protein Interactions Easy

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    ...right negative control is critical to the success of BiFC experiments. The ideal control is a mutated variant... work. You will also need to generate negative control constructs — more on this later. For more on constructing... measure the fluorescence of a single FP. Your control construct may generate a modest level of fluorescence...intensity of your BiFC constructs to your negative control to determine whether your PIPs actually do interact...has been compromised (Kodama and Hu, 2012). Such controls are necessary because split FPs can associate,...fragments themselves cannot be used as negative controls because their expression level, stability, and...structural information, constructing appropriate controls may requires that you empirically test a number...
  5. Using Addgene's New Viral Service for Your Research

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    ...delivering control shRNAs, GFP, and rtTA from the Tet-on system. The first two can serve as controls in a variety...virus, and quality control data for each of the materials we distribute. Quality control info can be found...The viral preparations undergo rigorous quality control testing at Addgene meaning they come ready made...used to activate expression of a gene under the control of the tet operator. AAV AAVs are the workhorses...are already available allowing you to chemically control neuronal activity while studying your phenotype...of interest. Optogenetic tools for similarly controlling neuronal activity with light will be available...determined by the serotype. You can use these as controls to determine if your AAVs are getting to the right...
  6. Plasmids 101: Plasmid Incompatibility

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    ...generally it consists of a centromere-like region on the plasmid, a centromere-like region binding protein... the origin of replication and the replication control machinery (Need a refresher on the Origin of Replication... because they compete for the same replication control machinery within the cell. Most plasmids encode...two models for iteron-based plasmid replication control that are not necessarily mutually exclusive. In...plasmids, different negative regulation systems control plasmid replication of each plasmid using unique... complex than simply competing for replication control factors and tends to be more of a concern with ...2012).  During cell division, CBPs bind to the centromere-like region on each plasmid and pair the plasmids...
  7. Predicting Adverse Reactions to Monoclonal Antibody Drugs

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    ...between recipients.   In vitro prediction of drug immunogenicity  The goal of in vitro prediction of immunogenicity..., 2020).  Evaluating a drug’s immunogenicity in-vitro helps researchers understand a drug’s safety and...depending on the person. However, in silico and in vitro assessment of immunogenicity can help evaluate risk...silico prediction algorithms fail to capture. In vitro evaluation of ‌therapeutic immunogenicity is typically...induction (Groell et.al, 2018). Thus, microscopic in-vitro models can be built that contain various components...components of the immune system. Figure 2: In-vitro models for immunogenicity prediction of therapeutic...efficacy.  Better together Combining in silico and in vitro approaches enables researchers to develop safer ...
  8. Hassle-free 96-well Format Epitope Tagging Using Cas9 Ribonucleoprotein

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    ...positive control in our knock-in experiments as it works quite efficiently and serves as a good control for...pull-down assays include immunoprecipitation mass spectrometry (IP/MS) and chromatin immunoprecipitation (ChIP... protein complexes are then analysed by mass spectrometry to identify the interacting proteins. Similarly...the field, it has recently become possible to introduce DSBs and concomitant insertion of epitope tags...of plasmid vectors and/or rely on cumbersome in vitro synthesis of single guide RNAs (sgRNAs), are time-consuming...tracrRNA system is much more potent than the in vitro-transcribed single guide RNA (IVT sgRNA) approach...Direct comparison of Cas9 RNPs prepared with an in vitro-transcribed (IVT) sgRNA versus synthetic cr:tracrRNA...
  9. The Breast Cancer Microenvironment: A Tumor’s Backstage Team

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    ...spread of cancer cells. Besides these stromal cell modifications, stromal cells are also constantly communicating...communicator at the Portuguese science museum "Fábrica Centro Ciência Viva" in Aveiro, Portugal. If cancer was...inflammatory, angiogenic vascular, endothelial, and stromal cells - a rich soup of life encircling the tumor...caveolin 1 in breast cancer also induces changes of stromal cells in the microenvironment. This results in ...microenvironment is the immune cells. The contradiction is strong in this one. After all, the system built to protect...decrease the expression of TAK 1, an enzyme that controls apoptosis and regulates transcription. Without... cannot undergo apoptosis, thus multiplying uncontrollably and building up tumors. “Friendly” microenvironment...
  10. Starter Guide to induced Pluripotent Stem Cells (iPSCs) Part 2:  Reprogramming and Transdifferentiation

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    ...Transdifferentiation in vivo or in vitro in vitro Both in vitro and in vivo Epigenetic marks Complete...include retrovirus [2], lentivirus [4], adenovirus [5], Sendai virus [6], plasmid electroporation (episomal...redifferentiation into the cell type of choice in vitro is known as reprogramming [1]. The process can be...small molecules, and growth factors are used to control the cell fate of iPSCs and differentiate them into...the earliest examples of transdifferentiation in vitro where treatment of mouse fibroblasts with 5-azacytidine...original somatic cells [41]. The in vivo and in vitro methods are similar except that the vectors carrying...reprogramming process as they are propagated in vitro as part of the process. This opens up a wide range...
  11. Corralling and Cornering the Goldilocks Antibody

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    ...use our negative control to compare exposure times of our samples. A prominent, strong signal has a shorter...negative control, and a weak signal has a longer exposure time closer to the negative control. Reproducibility...specificity and exposure time. We first have a negative control which does not have primary and only contains secondary...background noise. We then compare the negative control to a sample with the primary included to confirm...If the sample images similarly to the negative control, that informs us that the primary antibody is very...binding. If the sample differs from the negative control, such as showing a very bright signal only in the...that this antibody has specific binding and has a strong signal. We call the unique pattern of the antibody...
  12. CRISPR 101: Mammalian Expression Systems and Delivery Methods

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    ...appropriate for generating knockouts or knock-ins, or introducing mutations and tags, while a catalytically dead...conjunction with activator or repressor domains to control gene expression. Base editors can help you make...mismatch repair assay? Or is PCR followed by gel electrophoresis and/or next-generation sequencing more appropriate...system, you can then choose the best method for introducing those CRISPR components into your target cells...Plasmids containing gRNA and Cas9 are used in in vitro transcription reactions to generate mature Cas9 ...delivered to target cells using microinjection or electroporation. Transient expression of CRISPR components,...ribonucleoprotein complexes Purified Cas9 protein and in vitro transcribed gRNA are combined to form a Cas9-gRNA...
  13. Deep Dive: Statistical Tests (Comparisons)

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    ...high false positives. Control vs. all samples Oftentimes there exists a control group and multiple experimental...significantly greater or lesser than the mean of a control group. If you only care if one group is different... want to measure if a sample is greater than a control), then a one-tailed test is suitable. Of course...subjects or populations (a treatment group vs. a control group), on the other hand, should be analyzed with...group being compared to a standard value (not a control group). This situation is best served by a one-...can test three or more groups (two samples and a control, for example) simultaneously, with up to two independent...these tests from ANOVA/MANOVAs. Quantifying and controlling for any covariance between manipulated independent...
  14. Tips for CRISPR Gene Editing in Mice

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    ...(4; brighter than control, 3; same as control (Cetn1/sgRNA1), 2; darker than control, 1; very dark). Brighter...your in vivo work is to screen you gRNA in vitro first. In vitro screening is a simple and fast method for...selecting the guide RNA, validating its efficacy in vitro, and using it in mouse embryos to generate gene ... many previously validated gRNAs at Addgene. In vitro screening for the best gRNA Figure 1: Validation...Validated gRNAs to Target Mouse Genes To begin the in vitro screening process, you should first develop primers...in vivo. Find Plasmids for Testing Your gRNAs in vitro at Addgene CRISPR/Cas9 gene editing in mice Now ...mammalian cells was being explored and it allowed control over each individual component of the system. However...
  15. Multifunctional Neural Fibers: Modulation from Gut to Brain

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    ... fibers are drawn with microelectronics included in them for downstream control – this is important later...neural fibers developed by the Anikeeva lab are microelectronic fibers made from thermoplastic polymers that...optogenetic purposes, measure temperature, study electrophysiology, and deliver small molecules.   Fun fact:...(Orguc et al., 2020). Using a programmable microcontroller, NeuroStack coordinates optical stimulation...fibers as previously described, then measured electrophysiological potential afterwards. This measurement trended..., P., & Chandrakasan, A. P. (2020). Modular Optoelectronic System for Wireless, Programmable Neuromodulation... Anikeeva, P. (2019). Flexible fiber-based optoelectronics for neural interfaces. Chemical Society Reviews...
  16. Tetbow: Bright Multicolor Labeling for Neuronal Tracing

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    ...can introduce the four Tetbow plasmids (Addgene #104102 - #104105) using in utero electroporation or with...#104108) (Figure 3) that can be introduced using in utero electroporation. Then at your preferred time points...multiple copies of the transgene cassette are introduced, stochasticity will result in a combinatorial...stochastic expression of XFPs is possible by simply introducing a mixture of XFP genes. Stochastic multicolor...labeling is possible when each of the XFP genes is introduced at ~2 copies per cell per color, following a ...Brainbow) (Sakaguchi et al., 2018) (Figure 1). Once introduced, fluorescent proteins were best imaged after ...procedure, find the protocol here.   Tips and troubleshooting your Tetbow experiment Plasmid concentrations...
  17. New and Upcoming Viral Vectors - June 2019

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    ...intersectional studies And more! Control AAV We’re always excited to add new control AAVs to our inventory since...popular control AAV encoding GFP. Check out our inventory of serotype testing AAV New control AAVs ...our entire AAV inventory. Our new AAVs include: Control AAVs for serotype testing jGCaMP7c for calcium ...scientists, we now offer trial size 20 µL samples of control AAV that scientists can use for serotype testing...Plasmid Serotype Name 119036  AAV1  pAAV-hsyn-flex-Voltron-ST DREADDs / Chemogenetics AAV We have also ...used in combination with Cre for intersectional control of specific neuronal populations. We now offer ... Addgene in the next 6 months, pending quality control. For an estimate on availability date or to be ...
  18. New and Upcoming Viral Vectors - May 2020

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    .... Find the Dolcetto lentiviral prep here Control AAV Control AAV allow researchers to not only interpret...many of our control plasmids in new serotypes, as well as a new Cre dependent GFP control plasmid for ...mediated neuronal control. The advantages of optogenetics lie in the spatiotemporal control offered by the...for their own experiments. To this end, we offer control AAV in smaller aliquots of 20ul for serotype testing...also offered as AAV1 (along with AAV PHP.eB, AAV Retrograde, AAV2, AAV5, AAV8, and AAV9). Once activated,... Addgene in the next 6 months, pending quality control. For an estimate on availability date or to be ... AAVrg pAAV-hDlx-GqDREADD-dTomato-Fishell-4 Controls Plasmid Serotype Name 114471 AAV2 pAAV-Ef1a-fDIO...
  19. 15 Hot Plasmids from 2017

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    ...type SOX2 with either a SOX2-VP16 (strong activator) or SOX2-HP1 (strong repressor) fusion protein, the authors...elavl3/HuC), microglial/macrophage (mpeg1.1), and astrocytic (gfap).This toolbox adds new neuronal tools to...to express the transcription factors from a polycistronic cassette. However, it was unknown whether variations...variations in gene expression from these polycistronic cassettes could lead to comparable experimental...Woltjen Lab compared different variants of the polycistronic cassettes and discovered reprogramming discrepancies...variation in the Klf4 isoform (KLF4s or KLF4L). Polycistronic cassettes that contained KLF4s had overall diminished...reprogramming and global gene expression patterns. Polycistronic cassettes that contained the KLF4L isoform had...
  20. Plasmids for Endogenous Gene Tagging in Human Cells

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    ... and destroyed by CRISPR/Cas9 during transfection. Making the double strand break and introducing the ...individual proteins under endogenous regulatory control. But even with the advent of CRISPR/Cas9 technology... in human cell lines. Furthermore, the quality control necessary to ensure the edited cells are behaving...the tag through homology directed repair To introduce the fluorescent tag to the cells, we used CRISPR/Cas9...with the cut, we trick the cell into using the introduced donor plasmid as a repair template. This results...and the donor plasmid into the cells we use electroporation, a technique that briefly destabilizes the ...Figure 3: Inducing the double strand break and introducing the tag. Created with BioRender.com. Plasmids...
  21. Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

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    ...system to modify your target DNA, you first electroporate a linear donor DNA substrate (either dsDNA or...and SbcCD nucleases from digesting linear DNA introduced into the E. coli. Exo: Exo is a 5’→3’ dsDNA-dependent...Expression of lambda red recombination genes. Electroporation of substrate DNA and outgrowth of bacteria....dsDNA recombineering substrates to avoid errors introduced during PCR. ssDNA Substrate ssDNA substrates ...that avoid MMR activation.  The first way is to introduce a C/C mismatch at or within 6 base pairs of the...1% or 25-50% when activation of MMR is avoided Introduce C/C mismatches or silent changes in wobble codons...these will be discussed in more detail below).  Controlling expression of Red proteins is critical for minimizing...
  22. CRISPR 101: Making a Knock-In Cell Line

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    ...would like to introduce the edit. Finally, you must stipulate what sequence to introduce, which is done...guide’s recognition sequence is destroyed via mutation. By introducing a mutation in either the PAM or...Identify where in the genome you would like to introduce your knock-in. Then choose which Cas enzyme to...region of a gene, make sure that the PAM edit you introduce is a silent mutation, so you don’t accidentally...to your culture media prior to and after Cas9 introduction increases HDR, just make sure to remove the ...homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLIFE. (2014)...efficient homology-directed repair and termperature-controlled genome editing. Nat. Com., 8(2024), (2017). 10.1038...
  23. New Viral Vectors - Fall 2024

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    ...-GFP AAV9-X1.1 Controls Edward Boyden New serotype pAAV-hSyn-EGFP AAV PHP.eB Controls Bryan Roth New...CN3905) AAV PHP.eB Controls Jonathan Ting New viral prep pAAV-CaMKIIa-EGFP AAV1 Controls Bryan Roth New ...minBG-SYFP2-WPRE3-BGHpA (Alias: CN3044) AAV PHP.eB Controls Jonathan Ting New viral prep AiP13038 - pAAV-AiE0743m...minBG-SYFP2-WPRE3-BGHpA (Alias: CN3038) AAV PHP.eB Controls Jonathan Ting New viral prep AiP12408 - pAAV-AiE0600m-minBG-SYFP2...pAAV-AiE0600m-minBG-SYFP2-WPRE3-BGHpA (Alias: CN2408) AAV PHP.eB Controls Jonathan Ting New viral prep AiP13863 - pAAV-AiE0888m_C4...minBG-SYFP2-WPRE3-BGHpA (Alias: CN3863) AAV PHP.eB Controls Jonathan Ting New viral prep AiP13905 - pAAV-AiE0441h...New serotype pAAV-Ef1a-DIO mScarlet AAV5 Controls Karl Deisseroth New serotype pAAV_hSyn-PdCO-mScarlet-WPRE...
  24. Antibodies 101: Designing Your First Flow Panel

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    ...emission Before we design your first panel, we will introduce some background on the color spectrum and the ... photons are absorbed by matter, they promote electrons within atoms to a higher energy state. This process... called excitation. After a short period, the electrons revert to a lower energy state, whereby a photon...those of absorbed photons. When photons excite electrons of a fluorophore, they can do so at a range of...by the cytometer. Brighter fluorophores have a stronger signal. Table 1: Degrees of brightness for a few...absorbed and then emitted, which creates a relatively stronger — or brighter — signal.   Figure 2: Excitation... real-life advantage of a brighter fluorophore? Strong brightness makes it easier to distinguish a signal...
  25. Harnessing Bacterial Toxins for Allelic Exchange

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    ...commercial isothermal assembly master mix. Introduction of the strong transcriptional terminator BBa_B1002 ...decide which path to take. A quick and easy way to introduce a mutation in the chromosome is to disrupt expression...called allelic exchange. Here, a mutated allele introduced from a conjugative plasmid replaces the chromosomal...frequently requires considerable optimization to ensure strong counter-selection. Anecdotally, there is also a... evolved specifically for toxicity would allow stronger counter-selection than one merely co-opted toward...rhamnose- and arabinose-inducible promoters. Tight control of toxin expression is absolutely necessary, because...Proteobacteria. We placed the toxin genes under control of a rhamnose-inducible promoter, though Khetrapal...
  26. Four Ways to Package Transgenes That Exceed the Size Limit of Adeno-associated Virus

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    ...deliver the mini-dystrophin gene to a mouse model of muscular dystrophy. While mini-dystrophin expression levels... to deliver a 6 kb mini-dystrophin gene to a mouse model of muscular dystrophy. Three different splice... and expression of the full-length transgene in vitro and in vivo (Allocca et al., 2008). Shortly after...levels were lower than expression of wild-type dystrophin in normal muscles, mice treated with the overlapping...achieved ~80% of muscle cells expressing mini-dystrophin.  3. Hybrid  Overview: The hybrid approach combines... can be from one of the transgene’s endogenous introns or comprised of non-endogenous sequences, such ...Gene Therapy of mdx Mice With Large Truncated Dystrophins Generated by Recombination Using rAAV6. Molecular...
  27. Antibodies 101: Multiplex Immunofluorescence

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    ...colors you like. Controls and validation Can’t forget the controls! The necessary control experiments will...from Addgene), or be prepared to run some extra controls and validate the antibody yourself. If you can... we’ll walk you through the necessary types of controls for a multiplex experiment.  Check for non-specific...using a different antibody.  Prepare a positive control Prepare and label a sample that expresses your ...naturally or by overexpression, for a positive control. You can also confirm the secondary works as expected...S., Diaz, M. F. P., & Yong, W. H. (2019). An Introduction to Performing Immunofluorescence Staining. In....2018488117 Additional Resources on the Addgene blog Introduction to Antibodies Which Fluorescence Microscopy ...
  28. Deep Dive: qPCR

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    ...If you're just getting started, check out our Introduction to PCR.) But if you're ready to go beyond the...tips on probe chemistries, primer optimization, controls, and analysis.  As a rule of thumb, you can assume...experimental parameters (neighbor stacking energies, loop entropy effects, cation concentrations and a temperature...side and a 5% negative error on the other will introduce unwanted variation into your data. Try different...protocol may have to be re-optimized.  While troublesome, multiplexing your reference gene set can save...the fold change of each sample compared to your control. But as we said previously, just because it’s used...assumes the amplification kinetics of the target and control genes are fairly equal, an assumption that is not...
  29. Common Injection Routes in Mice

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    ...tail vein or retro-orbitally Gauge: 27–30 Volume: < 0.2 ml Skill required: beginner (retro-orbital) to ...restrainer or anesthesia Tail vein or retro-orbital 27–30 < 0.2 mL Beginner (retro-orbital) to intermediate (tail...2020). A retrospective analysis for different routes of administration in Mice-Percutaneous Retro-Orbital...vein injections.” However, they can also be given retro-orbitally, behind the eye. IV injections are used...needle in the correct vein without collapsing it. Retro-orbital injections are also thought to be less stressful...decision Hopefully this post has provided a good introduction to common injection routes in mice! When considering...
  30. CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

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    ...recombineering machinery are first electroporated with pCas9. Then, pCRISPR is introduced along with an oligonucleotide... in a bacterial strain introduced to the population as methods of introduction. Engineering the bee gut... of knockdown and developed new, more tightly controlled, Tet responsive promoters to avoid leaky activation...systems differ on how exactly the CRISPRi locus is introduced (Tn7 in Gram-positives and ICEbs1 in Gram-negatives...CRISPR system targets these genomic locations, and introduces double strand breaks, the bacteria are unable...This approach provides a more granular level of control where individual bases can be modified. Cytidine... that could be fused to dCas9 and efficiently introduce cytosine-to-thymine substitutions in target sequences...
  31. Getting to Know Expansion Microscopy

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    ...super-resolution microscopy or electron microscopy. Since its introduction in 2015 by the Boyden Lab, ExM...expand evenly in all directions, aka isotropically. But achieving isotropic expansion depends on how well the...2010). How do you overcome this resolution limit? Electron Microscopy (EM) is one option, but the equipment...much to be done about that.  Second, expansion introduces distortions. The goal in expansion is for your...variation, your samples can tear or expand unevenly, introducing distortions in your final images. It is possible...a deeper understanding of neuronal synapses, centrosomes, parasites, and more (Bertiaux, et al. 2021; ...resources on the Addgene blog Antibodies 101: Introduction to Immunofluorescence  Antibodies 101: Multiplex...
  32. Plasmids 101: Broad Host Range Plasmids

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    ...1998). Regulatory elements within the ori also control copy number and stability, which influence the ...which consist of a plasmid’s ori and all of its control elements, from broad host range plasmids can be...Hypomycrobium X, Legionella pneumophila, Methylophilus methyltrophus, Methylococcus methanolicus, Methylosinus trichosporium...hydrophila, Agrobacterium tumefaciens, Alcaligenes eutrophus, Azotobacter vinelandii, Brevibacterium methylicum...Klebsiella aerogenes, K. pneumoniae, Methylophilus methylotrophus, Moraxella spp., Mycobacterium aurum, M. smegmatis...Agrobacterium tumefaciens, A. rhizogenes, Alcaligenes eutrophus, Enterobacter sp., Erwinia amylovora, E. carotovora...spp., Legionella pneumophila, Methylophilus methylotrophus, Myxococcus virescens, M. xanthus, Proteus ...
  33. CRISPR 101: Which Cas9 Do I Choose for My CRISPR Experiment?

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    ...type of edit you need to introduce highly specific? Perhaps you need to introduce a single nucleotide modification...McDade and significantly updated in 2022 by Susanna Stroik.  The advent of CRISPR/Cas9 has made it easier ...right for your unique experiment. Here, we will introduce you to the wide array of Cas9s (many available...Knock-out cells or animals are engineered by introducing Cas9-mediated frameshift mutations in the gene...interest. The genomic target for Cas9 and the introduction of this mutation can be any site early in the...and knockouts Some experiments may require the introduction of a mutation at a specific site e.g., deleting...Synergistic Activation Mediator complex, generally the strongest activator, consists of a dCas9-VP64 fusion and...
  34. Antibodies 101: Immunoprecipitation

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    ...protein. With this control set, you should see your target protein IP’d in the positive control sample but not... Basics of Western Immunoblotting.) For mass spectrometry, elute in a urea-containing lysis buffer. Alternatively...permanently altering the structure of the protein.  Controls As with any experiment, it is critical to include...include proper positive and negative controls for an IP. When possible, perform the IP in parallel with ...interest and a sample that does not. Common negative controls include knockout cell lines or tissues that do... not in the negative control, confirming that the capture antibody is functioning as expected. In order...perform parallel IPs with beads only and an isotype control antibody. Alternative applications Oftentimes, ...
  35. Three Key Considerations for Precise Neuronal Targeting Using AAV Technologies

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    ...your cell of interest: AAV tropism and route of administration AAV tropism is defined by the viral capsid...FRT, with AAV vectors are common approaches to control transgene expression by restricting expression ...regulatory elements such as promoters and enhancers, multicistronic vectors, and post-translational regulatory ...Combinatorial strategies can also be used for retrograde tracing: visualizing neurons that received information...region. In the example shown in Figure 1, injected retrograde AAVs at one location result in opsin expression...viral vector related blog posts Learn more about retrograde AAVs Resources on Addgene.org Read our AAV ...guide Learn more about our viral service Find retrograde AAVs at Addgene ...
  36. CRISPR 101: Validating Your Genome Edit

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    ...updated Nov 3, 2022 by Susanna Stroik. You’ve designed your gRNA and introduced it into your target cells ... of your target site is. If you are trying to introduce a homozygous mutation, it’s helpful to know how...knockout cell line is desired, any mutation that introduces a frameshift in the coding region of interest... sequence clones to verify that the deletions introduced are out of frame but finding clones with deletions....   Validating knock-in mutations You’ve introduced a donor template along with your gRNA and Cas9...your desired edit is. Large knock-ins Are you introducing a large insertion, something 20 base pairs or...restriction enzyme sites? Never fear! You can introduce a silent, passenger edit to serve as a proxy for...
  37. Proximity Labeling: A Powerful Tool for Protein Complex Purification and Proteomic Mapping

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    ..., that strongly binds the glycoprotein, avidin or a similar protein, streptavidin. This strong interaction...antibody based affinity purification and mass spectrometry approaches. Recently however, scientists have...classic antibody based affinity purification/mass spectrometry methods.  BioID: a promiscuous biotin ligase...ascorbate peroxidase (APEX), used as a genetic tag for electron microscopy, could be used for efficient proximity...of hydrogen peroxide. This radical reacts with electron-rich amino acids such as tyrosine on neighboring...exogenous biotin allowing for more precise temporal control.  Find plasmids for TurboID and miniturbo Split-BioID...Stephanie S., et al. "Directed evolution of APEX2 for electron microscopy and proximity labeling." Nature methods...
  38. Filming Science Videos in the Age of Social Distancing

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    ...video making.   Cycle One: The Ironing Board Control Center With the pandemic in full swing and everyone...Ironing boards. Good as a WFH desk, and as a video control center.   Cycle 2: Zooming Forward Design: ...me to set up a camera in a room, but still have control over focus, zoom, stop/start record, and allow ...focus system. Follow focus systems allow you to control the focus or zoom on a camera lens without having...door (connected via bluetooth to the camera, to control some basic functions, including starting and stopping... complete with wireless video receiver, camera control, and extra hand sanitizer.    Cycle 4: Venturing...mount a battery to it if necessary. I could still control the camera from a distance if I needed to be close...
  39. Using Phosphoserine to Study Protein Phosphorylation

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    ...-phosphorylated or ‘wild type’ control protein. The ‘wild type’ control can be made one of two ways. First... model systems coupled with advances in mass spectrometry suggest that phosphorylation is far more prevalent...non-phosphorylated recombinant protein and perform in vitro kinase reactions. Often, however, researchers lack...or have difficulties with kinase specificity in vitro. The establishment of a system in which purified...-containing GFP that can be used as a positive control during optimization experiments. This version of... pSer incorporation can also done using mass spectrometry to identify a mass shift proportional to a single...expression plasmid. This is a particularly important control when creating a phosphoserine-incorporation mutant...
  40. Targeting HIV-1 with CRISPR: Shock and Kill or Cut it Out?

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    ...with the lentivirus HIV-1. Today, HIV-1 can be controlled with antiviral therapies such that the virus ... dCas9-SAM to activate HIV-1 transcription and destroy infected cells, the other using wild-type Cas9 ...Read on to learn how CRISPR can take on HIV-1 in vitro, and what obstacles must be overcome for clinical...infections not usually seen in healthy individuals. Antiretroviral therapy (ART) can virtually eliminate plasma...Why can’t the body mount an immune response to destroy these reservoirs? Essentially, the immune system...out of the genome with CRISPR/Cas9 Before the introduction of CRISPR, HIV-1 genome editing with ZFNs and...infection period rather than the ART-induced viral control found in most HIV-1 patients. Obstacles for CRISPR...
  41. Summer SciComm: Science Storytelling

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    ...creative commons license.    The introduction The introduction serves two purposes. First, it should... to have conflict. There are a lot of ways to introduce conflict into your work, but one of my favorite... two other things to complete your story: an introduction, and a resolution.   Fig. 2: The structure...ever thought about how X works?”, or even by introducing a person that the audience can relate to. It ...read (or listen or watch) further.  Second, the introduction needs to provide enough information so that ...thing applies for science storytelling. You can introduce your results right at the beginning of your communication...Addgene blog SSCS: Cognitive Load SSCS: Preprints Intro to our Summer SciComm Series  ...
  42. New and Upcoming Viral Vectors - December 2019

    Type
    Blog Post
    ...26973 AAVrg pAAV-hSyn-hChR2(H134R)-EYFP   Control AAV Control AAV allow researchers to not only interpret...inventory. Our new AAVs include: Optogenetics AAV Control AAV Biosensor AAV And more! Optogenetics Optogenetics...more efficient tools for light mediated neuronal control and we are expanding our viral service to make ...own experiments. To this end, we offer specific control AAV in smaller aliquots of 20 ul for serotype testing... service has now expanded to offer many of our control plasmids in new serotypes. See our Serotype Testing...response to action potentials, is now offered under control of the CAG promoter for ubiquitous expression. ... 44362  AAV1 pAAV-hSyn-DIO-hM4D(Gi)-mCherry Controls Plasmid Serotype Name 50459 AAV1 pAAV-hSyn-DIO-mCherry...
  43. Optimizing Donor DNA for Enhanced CRISPR Genome Editing

    Type
    Blog Post
    ...which introduces functional Cas9 protein and the targeting guide RNA into cells by electroporation. Results...of the nuclease dissociates very slowly after introducing a cut, with residence times on DNA of approximately...free to anneal to complementary ssDNA molecules in vitro. Figure 1: Cas9 binding to the cleaved DNA... if we could leverage this model (built from in vitro studies) to boost the efficiency of sequence replacement...enables sequence replacement without cutting Our in vitro results also demonstrated that catalytically inactive...advantage but error-prone repair of the gene would be disastrous (i.e. if breaking the gene is worse than leaving...homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery." Elife 3 (2015...
  44. Easi-CRISPR: Generating Knock-In and Conditional Mouse Models

    Type
    Blog Post
    ...oviductal lumen, tweezer-type electrodes are placed to mediate electroporation. Ohtsuka et al. compared traditional... homology arms and a selection marker, then electroporate it into mouse embryonic stem cells. After selecting...from Yang et al. requires two gRNAs targeting two intronic regions and two repair templates in order to insert...promoter upstream of the left homology arm, perform in vitro transcription, and use the IVT product as a template...compatible with standard microinjection and electroporation methods using in mouse transgenics. Designing... allele requires you to target 2 loxP sites to intronic regions 0.5 - 0.8 kb apart. In the example below...between the ampulla and infundibulum. In vivo electroporation is performed immediately after injection. Image...
  45. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    ... of biogenesis and mitochondrial degradation in vitro and in vivo. Merged green and red channel fluorescence...Technology Austria, decided to take a novel approach to control for these effects, drawing on the rapidly growing...1):39-48. APEX2 for proteomic mapping and electron microscopy A thorough understanding of complex...enabled progress on both fronts – as a genetic electron microscopy (EM) tag providing superior subcellular...also available at Addgene and can be used as a control for your experiments. For more information, please... allowing labs to precisely and non-invasively control neuronal signaling. For more information on DREADDs...Chen and colleagues for genome editing inXenopus tropicalis. (Guo et al., Development. 2014..) A new, higher...
  46. Flow Cytometry Readouts: Yes, No, and Everything in Between

    Type
    Blog Post
    ...is the best-case result. For example, you have introduced a GFP protein into your cells (visualized via...104–105 signal intensity. If your samples and controls were processed the same way, the GFP- population...population should overlap with the negative control population (Figure 1). Based on where the GFP- population... Example of yes-no flow plot of the (negative) control (left) or with the sample (right). Negative and...biorender.com. Pro tip! If your samples and control underwent different treatment processes, you may... population, so it no longer overlaps with the control. In tricky experiments, these populations may overlap...positions to the base expression. When compared to the control — which should be base expression — a peak to the...
  47. Viral Vectors 101: Viral Applications

    Type
    Blog Post
    ...one type of virus to choose from (lentivirus, retrovirus, AAV, etc.). When you’ve decided on an application...experiment. Research applications Gene expression control Researchers are always wanting to manipulate gene...complementary mRNA sequence for degradation. Viral introduction of shRNA plasmids can induce gene-specific silencing...Viral CRISPR-Cas9 tools have been engineered to introduce frameshift mutations in genes of interest, turning...depending on the virus used. All of these expression control mechanisms require a specific modification of the...that the copies of library cargo delivered are controllable. For many screening applications, it is essential...attenuated, nef-deleted HIV-1 strains in vivo. Retrovirology. 2007 Sep 23;4:66. doi: 10.1186/1742-4690-4-...
  48. Targeted Mutagenesis with EvolvR

    Type
    Blog Post
    ...Lab and the Chang Lab have created methods that introduce diversification by targeting a site of interest...which limits the level of variation that can be introduced.   Diversification is limited due to the integration...use of such libraries means that the diversity introduced by lambda red recombineering and Multiplex Automated...PCR, EvolvR uses an error prone polymerase to introduce mutations. However, by using nCas9 to direct PolI3m...the 3' end of the nick (right), occasionally introducing mutations (red nucleotide). Created with BioRender.com...EvolvR, three additional amino acid changes were introduced to nCas9. These changes promote nCas9’s dissociation...increased the likelihood that a single gRNA would introduce more than one mutation near the target site. Applications...
  49. Giving gRNAs a Facelift - Synthetic and Beyond

    Type
    Blog Post
    ...guides are generated by introduction of a single photocleavable 2-nitrobenzyl linker in the 20-bp targeting...nucleic acids or bridged nucleic acids can also be introduced in the gRNA. These modifications result in conformationally... innate immune response associated with gRNA introduction (Allen, et al., Frontiers in Genome Editing)...experiments in which viability is being compromised by introduction of foreign RNAs or immunity itself is being ...technologies utilize chemical modifications to the RNA to control the entire editing system with a simple LED light...al. Photoswitchable gRNAs for Spatiotemporally Controlled CRSIPR-Cas-based Genomic Regulation. ACS 6, 5... Susanna Stroik...
  50. Lighting Up Cell Signaling with Photoswitchable Kinases

    Type
    Blog Post
    ...Photoactivatable Fluorescent Proteins Control Protein Expression with Cre-lox Control Protein Activity with GFP ...transmitted by intermediate messengers. Optical control of kinase activity can provide greater spatiotemporal...There is no common method for creating optically controlled kinases. The Solution: In Zhou et al, the Lin... the delayed effects because you can precisely control the timing of kinase activity. 2. Studying negative...photoswitchable kinases in vivo not only allows for precise control of a signaling pathway in the context of a whole...addition, it could be possible to use pdDronpa to control other kinases and proteins. Zhou et al suggest ...Pengpeng Li, Kang Shen, and Michael Z. Lin. "Optical control of cell signaling by single-chain photoswitchable...
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