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  1. Retrovirus Plasmids

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    Collection
    ...Viral Plasmids γ-Retroviral Plasmids γ-Retroviral Plasmids Additional Resources: γ-Retrovirus Guide Biosafety...discuss retrovirus, they typically are referring to a subset of retroviruses called γ-retroviruses. γ-Retroviruses...FAQ) about retrovirus Popular γ-Retrovirus Envelope and Packaging Plasmids Common γ-retroviral envelope ...Popular γ-Retroviral Transfer Plasmids Common γ-retroviral transfer plasmids. Want a retroviral plasmid ...AAV Plasmids Viral Vectors 101 eBook γ-Retrovirus (gamma-retrovirus) is an RNA virus comprised of its genome...mutagenesis). Read our γ-Retrovirus Guide for more information about: γ-Retroviral packaging systems Frequently...MSCV Retroviral construct for miRNA expression Chen 52114 pMSCV-IRES-mCherry FP MSCV A bicistronic IRES-mCherry...
  2. Control AAV Preps

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    Collection
    ...Packaging Service AAV Controls Viral Vector Packaging Service: Control AAV Browse our control AAV that express...options. Browse all Control AAV *AAVrg = retrograde serotype, produced with the AAV retro helper plasmid from... select plasmids in the repository. Browse our control AAV collection, including fluorescent proteins,...serotypes. Serotype AAV1 AAV2 AAV5 AAV8 AAV9 AAVrh10 Retrograde AAV PHP.eB PHP.S PHP.V1 CAP-B10 CAP-B22 MaCPNS1... from the Karpova and Schaffer labs. See the Retrograde AAV Inventory Page . Don’t See What You’re Looking...
  3. Retrograde AAV viral preps

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    Collection
    ...Ready-to-use retrograde AAV available from Addgene's viral service. Retrograde construct from Alla Karpova...Packaging Service AAV Retrograde AAV Viral Vector Packaging Service: Retrograde AAV As part of our Viral... the rAAV2-retro helper plasmid (Addgene #81070) . This serotype permits robust retrograde access and ...viral catalog items. Browse Available Retrograde AAV See our retrograde AAV based on the functional catagories...-GFP CAG GFP Control Boyden 51502 AAV pCAG-FLEX-EGFP-WPRE CAG EGFP, Cre-dependent Control Zeng 50465 pAAV-hSyn-EGFP...pAAV-hSyn-EGFP Syn EGFP Control Roth 50469 pAAV-CaMKIIa-EGFP CamKII EGFP Control Roth 114472 pAAV-hSyn-...pAAV-hSyn-mCherry Syn mCherry Control Deisseroth 114469 pAAV-CaMKIIa-mCherry CamKII mCherry Control Deisseroth 50459...
  4. An Introduction to Adenovirus

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    Blog Post
    ...family of viruses with broad host range and tissue tropism (Borkenhagen et al., 2019). AdVs are non-enveloped...kilobases; compared to only 4.7 kb for AAV, 9 kb for retroviruses). The tradeoff is that adenoviral vector expression...proteins and downstream cellular events, all recruit strong innate and adaptive immune responses that inactivate...and rapidly caused the death of adenoid cells in vitro (Rowe et al., 1953). While overt death of the targeted...than for other vector types, we hope that this introduction gives a sense of what AdVs are about – and we...References and resources References Bauzon, M., Castro, D., Karr, M., Hawkins, L. K., & Hermiston, T. ...Applications Adenoviral Vector Production and Troubleshooting Viral Vectors 101: Types of Viruses...
  5. A Control for All Seasons

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    Blog Post
    ...specificity control, secondary antibody specificity control, label control, and cross-reactivity control when...helpful to focus on something controllable, like experiments (and their controls!). This blog post will discuss... ins and outs of controls for biological experiments, starting with general controls and then moving on... of controls and be able to use this information to design thoughtfully and thoroughly controlled experiments...experiments.  Broadly useful controls Positive and negative controls Most biological experiments require...positive and negative controls to ensure proper interpretation of results. A positive control is generally a ...serum or isotype-specific control (Hewitt 2014, Alexander 2018). In this control, the primary antibody is...
  6. Troubleshooting and Optimizing a Western Blot

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    Blog Post
    ... strip cycle. Troubleshooting the troubleshooting There’s many different troubleshooting and optimization...blot optimization and troubleshooting.   Before you can optimize or troubleshoot a western, you’ll need...time troubleshooting an assay that’s fine with antibodies that are not.   Table 1: Troubleshooting a western...and may require multiple runs to optimize or troubleshoot.   Before you start… Mark your membrane! Membranes...document it.   Did it work? Save yourself time and trouble by confirming your transfer worked with Ponceau...box to visualize proteins after transfer. Troubleshooting The two most common (non-technical) issues ...your membrane size is probably the easiest troubleshooting step to take. If you’re not sure if you need...
  7. Antibodies 101: Introduction to Immunofluorescence

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    Blog Post
    ...include controls to show that your antibodies are binding specifically. For positive controls, aim to ... and isotype controls are also commonly used in IF experiments. Precisely which controls you perform will...for some background on fluorescence, try the Introduction to Fluorescence Microscopy video from iBiology...been manipulated to overexpress it. For negative controls, use samples in which the target is known to be...research antibodies, protocols, and appropriate controls and you will be well on your way to performing... K, Mareninov S, Diaz MFP, Yong WH (2018) An Introduction to Performing Immunofluorescence Staining. In...10.1007/978-1-4939-8935-5_26 Stuurman N. (2012) Introduction to Fluorescence Microscopy. iBiology. https:...
  8. Antibodies 101: Introduction to Antibodies

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    Blog Post
    ...detection. You’ll also want to use the appropriate controls for your experiment to account for potential non-specific...quantitatively if you also load the gel with appropriate controls for normalizing the protein concentration. To ...plasmid encoding GFP. Actin, a common internal control for Western blots, shows that a similar concentration...biosensor biorecognition elements. Biosensors and Bioelectronics 85:32–45 . https://doi.org/10.1016/j.bios.2016.04.091...
  9. Adenoviral Vector Production and Troubleshooting

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    Blog Post
    ... challenge with using AdV is that it triggers a strong immune response after in vivo administration, which...loss of transgene expression (Interestingly, the strong immunogenicity of AdVs is what makes them ideal...reference 3. Production, amplification, and quality control of 2nd generation AdV     AdV Production in HEK293...looking at absorbance at 260nm using UV/Vis spectrophotometry. An absorbance of at 260nm corresponds to...reference material should be used as an internal control (ATCC#VR-1516) until the assay is validated. Infectious...Adenoviral delivery of CRISPR/Cas9 5 tips for troubleshooting viral transductions Resources on Addgene.org...
  10. 5 Tips for Troubleshooting Viral Transductions

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    Blog Post
    ...Many researchers find themselves plagued by troubleshooting a virus-based experiment at one point or another...will share five hail Marys that have helped me troubleshoot some technical obstacles when working with virus... enzyme digest and compare it to some reliable controls. 2. Viral stocks may be sensitive to freeze thaw...instead of storing it briefly at -80 °C. If troubleshooting, the safest option may be to avoid freezing... to a target cell by reducing the repulsive electrostatic forces between these two negatively-charged ...not work if the virus does not have the proper tropism to infect the packaging cells. Another method of..., and Martin L. Yarmush. "Polybrene increases retrovirus gene transfer efficiency by enhancing receptor-independent...
  11. Plasmids 101: Control Plasmids

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    Blog Post
    ...replicate controls.  Transfection controls: empty vector and internal control A transfection control measures...Negative controls: untreated cells, empty vector control, and non-targeting control Negative control conditions...lacking the proper controls are invariably inconclusive and unreliable. Proper controls provide the constant...about the various controls that can be used for plasmid-based experiments. What are control plasmids? Generally...experiment, delineate its requisite control plasmids, and discuss why these control plasmids are critical for the...use of proper control plasmids would address and answer these questions. Types of control plasmids Part...the target cells. One transfection control is an empty vector control; specifically, the plasmid without...
  12. Plasmids 101: Multicistronic Vectors

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    Blog Post
    ...pMSCV-pBabeMCS-IRES-RFP IRES Retroviral pMSCV-IRES-YFP II IRES Retroviral pCMMP-MCS-IRES-Puro IRES Retroviral pEF1a-...bidirectional promoters, or the creation of bicistronic or multicistronic vectors. Unlike promoters which will...upstream cistron will have a few additional residues added to the end, while the downstream cistron will ... Plasmid Name Multicistronic Element Expression Type MSCV-IRES-EGFP IRES Retroviral pMSCV-pBabeMCS-IRES-RFP...transcripts for each gene that is expressed, multicistronic vectors simultaneously express two or more ... so in this blog post we’ll cover basics of multicistronic vectors: why they are useful, how they work... and how to get started with them. Why Use multicistronic vectors? Detecting cells that are expressing...
  13. Antibodies 101: Flow Cytometry Controls

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    Blog Post
    ...familiar with the basics of controls in experimental design, you’ll need a few controls specific to flow as an...nonspecific binding. Flow specific controls The three types of flow cytometry controls we’ll discuss are single ... may need to include additional controls like FMO or isotype controls, which we’ll describe a bit later...color control for every fluorophore you intend to use in your panel. Using single color controls On a ...your single color controls may not be accurate for your samples. The single color controls should be used...plots for a single color control. A) A flow plot showing a single color control for the APC channel. B)...likely to require FMOs. Isotypes Isotype controls are negative controls that allow you to determine what level...
  14. Viral Vectors 101: The Retroviral Lifecycle

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    Blog Post
    ...The Retroviridae (commonly called retrovirus) family — of which HIV is a member — may seem like an unlikely...molecular biology. “Retroviruses” is an umbrella term which encompasses 1.) the gamma-retroviruses — that have...have been developed into retroviral vectors and 2.) a subclass of retroviruses called the lentiviruses (...foundation for producing and using retroviral vectors effectively. Retroviral structure   Figure 2:..., as the viral particle emerges. Retrovirus life cycle Retroviruses go against the grain of the Central...unlikely candidate to use as a viral vector, but retroviruses have been developed into some of the most widely...Lentiviruses are sometimes referred to as “complex retroviruses ” (Cullen, 1992) because of their more complicated...
  15. Viral Vectors 101: An Introduction to AAV

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    Blog Post
    ...work, although it’s less effective in vivo than in vitro. On the other hand, if you’re interested in expressing...and serotype selection. Promoters and serotype control, respectively, cell-specific gene expression and...viral infection. Cre or Flp dependent switches control gene expression via switches placed in your cell...updates from Addgenies Karen Guerin and Susanna Stroik. Didem Goz Ayturk is a Postdoctoral Fellow in Connie...System: Towards Gene Therapy For Duchenne Muscular Dystrophy. InTech. doi: 10.5772/53023 Veldwijk, Marlon R...
  16. Troubleshooting Your Plasmid Cloning Experiment

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    Blog Post
    ...cloning process, there are also many ways to troubleshoot the cloning experiment. Here are some tips that...cloning strategy you followed. While most of the troubleshooting regarding this step has to be strategy specific...software. Now that you've read our plasmid troubleshooting guide, you hopefully have some new strategies...
  17. Fluorescent Proteins 101: Introduction to FRET

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    Blog Post
    ...if the two proteins form a complex. To help troubleshoot your experimental setup as a potential source...FRET measurements and serve as a type of positive control. In the case of an unfavorable donor-acceptor orientation...
  18. Addgene's Tips for Plasmid Quality Control

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    Blog Post
    ...the plasmids we distribute through our quality control processes. Every plasmid we receive is rigorously...repetitive regions (such as LTRs or ITRs), hairpins, or strong terminators. Detailed instructions on other factors...Addgene Blog Get 6 Tips for Analyzing and Troubleshooting DNA Sequence Learn Additional Ways to Verify...  quality control...
  19. A Primer on Optogenetics: Introduction and Opsin Delivery

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    Blog Post
    ...optogenetics: 1) electrophysiology and 2) behavioral experiments. I’m not an electrophysiologist so I cannot...to hook up your lasers and not have the proper controls or a proper behavioral readout that allows you...behavior, you don’t necessarily need to use electrophysiology to prove that your optogenetic manipulations... (though a reviewer will probably ask for electrophysiology data too but that’s what collaborators are...not be an appropriate test.  Of course, proper controls are required and include, for example, confirmation...Carolina) that offer his constructs. Importantly, control viruses (not expressing the opsin) are also available...into the correct direction and expressed (under control of a generic promoter). A DIO opsin allows cell...
  20. Magnetic Control of Proteins: More than a Dream

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    Blog Post
    ...developing a new fluorescent protein tool that responds strongly to magnetic fields (Frank Hayward et al., 2024... of a magnetoresponsive fluorescent protein. Controlling an optogenetic or fluorescent tool with magnets... 2016), and some previous attempts at magnetic control of biological systems (Wheeler et al., 2016; Duret...al., 2019). But the field has sometimes seen controversy, and the interactions of organic molecules like...science in the open The York Lab operates with a strong commitment to exploration and collaboration. Andrew...says, noting that his team regularly shares troubleshooting tips and works with other labs to help them...Avants, B. W., & Robinson, J. T. (2019). Magnetic Entropy as a Proposed Gating Mechanism for Magnetogenetic...
  21. New Optogenetic Tools for Cytoskeleton and Membrane Control

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    Blog Post
    ..., VVDfast to control recruitment of an “anchor” to microtubules and iLID-SspB to control the recruitment...reversibly control Rho GTPase activity on a global to subcellular scale, enabling precise control over vascular...Optogenetics gives you the power to control cells or organisms with the flip of a switch. You may be ...with popular light-sensitive ion channels used to control activities like neuronal signaling — think of a... a few recently-deposited optogenetic tools to control the cytoskeleton and cell membrane dynamics. First...component of the cytoskeleton – actin? Actin dynamics control cell morphology, movement, and more, often driven...specific Rho GTPase/GEF pair (Ridley, 2016). Taking control of a RhoGEF is a great way to stimulate and study...
  22. Viral Vectors 101: The AAV Retrograde Serotype

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    Blog Post
    ...rabies) naturally exhibit retrograde transport and can therefore perform retrograde tracing, but often at ...synapses.)  In this post we’ll describe the AAV retrograde serotype (AAVrg) and how it enables scientists...visualize neurons that send information to a region (retrograde tracer, Fig. 1A), and another to visualize neurons...upstream from the axon terminals to the soma (retrograde tracer) (Fig. 2). Viruses as neuronal scalpels...tracers, but finding a virus that works well for retrograde tracing can be difficult. The in vivo workhorse...adeno associated virus (AAV) shows relatively low retrograde transport efficiency in most serotypes. Some ...directs high-level transgene expression, and has retrograde functionality to monitor and map neurons (Tervo...
  23. Popular Retroviral Vectors and Their Uses in Scientific Research

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    Blog Post
    ... Viruses of the Retroviridae or Retrovirus family, which includes the gamma-retrovirus and lentivirus ...using retroviral vectors in your experiments. Gamma-retroviral vectors Popular gamma-Retroviral Transfer...genome-wide experiments. Gamma-retroviral vs. lentiviral vector systems Gamma-retroviral vectors have a relatively...cells. Find tips on using gamma-retroviral vectors in our Gamma-retrovirus guide and find popular packaging... codon-optimized Cas9 under the control of Murine Stem Cell retrovirus promoter for mammalian gene knockdown...plasmids for transcriptional inhibition. Retroviral Plasmid Kit Retroviral Barcoding Library (Kit # 1000000097...multiple genes via infection with a single ecotropic or amphotropic MuLE lentivirus provides significant genetic...
  24. REPLACR Mutagenesis: Replacing In Vitro Recombination Methods

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    Blog Post
    ...insertions require complicated, often costly in vitro assembly methods. A new method, REPLACR-mutagenesis...mutagenesis methods Current SDM methods rely on in vitro assembly. To make a point mutation, you design two...Trehan et al. wondered if, instead of relying on in vitro assembly, they could make the E. coli do the work...must PCR amplify the whole plasmid, which could introduce mutations. Using commercially available high-fidelity...-mutagenesis also requires making your own electrocompetent cells in which the phage recombination machinery...since you skip the steps of gel purification and in vitro recombination. Across all modifications described...Reactions 1 1 1 per fragment 1 per fragment In vitro Assembly No No Yes Yes DpnI Digestion Yes Yes ...
  25. Treating Muscular Dystrophy with CRISPR Gene Editing

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    Blog Post
    ...mutations in dystrophin, a protein essential for proper muscle function. Without functioning dystrophin, an individual... condition known as Becker muscular dystrophy (BMD). Dystrophin’s size makes it difficult to deliver via...through NHEJ, will restore the dystrophin reading frame. gRNAs bind in intronic regions, so low-frequency ...editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science. 2015 Dec 31... CA. Correction of dystrophin expression in cells from Duchenne muscular dystrophy patients through genomic...editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy. Nat Commun. 2015b Feb...Our Special Collections Pages Duchenne muscular dystrophy: The most common inherited disease The genetic...
  26. 6 Tips for Analyzing and Troubleshooting Sanger Sequencing Results

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    Blog Post
    ...always include proper controls like uncut DNA and the proper ladder. These controls help you properly visualize...sequencing the chromatogram is your visual control. And, like all controls, missing out is a big mistake. Chromatograms...Chromatograms come to the rescue for analyzing and troubleshooting your DNA sequencing results Above (top right...repeated. Guidelines to help with DNA sequencing troubleshooting and analysis 1. You can use any of the following...70-75 of the read (see image below), so I would strongly recommend using a silica spin column. They can...most out of your DNA sequencing results and to troubleshoot any problems that come up. Good luck analyzing...
  27. Extracurricular Activities for a Strong Science Career Path

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    Blog Post
    ... in extracurricular activities (EA). EAs can: Introduce new perspectives in science and engineering Looking...that your project is an innovative solution with a strong impact, or a journalist does not understand the...
  28. Antibodies 101: Normalization and Loading Controls for Western Blots

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    Blog Post
    ...loading controls are equally strong, so the normalized value simply reports which POI band is stronger. The...loading control is not perturbed by the experiment The loading control is supposed to control for, well...western blots benefit from a positive control — a loading control — that you know is present in every lane...transfer. Figure 1: A loading control is a type of positive control necessary to demonstrate a POI’s...loading control: the intensity of each POI band is divided by the intensity of the loading control in the...with loading controls. Under each lane, the fraction of the POI band over the loading control is shown. ...loading control. First, let’s explore why these assumptions are key to using loading controls accurately...
  29. Viral Vectors 101: AAV Serotypes and Tissue Tropism

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    ...choice for targeting astroglia. It has additionally been reported to have tropism for lung, liver, vascular...as in-stock preparations. AAV retrograde (AAVrg, also known as AAV2-retro) We’ve covered AAVrg in a previous...following table focuses on tissue tropism in mice in vivo. Though tropism depends on many variables, we suggest...the opposite: AAV serotypes with varying tissue tropism, allowing them to target particular cell types....commonly used serotypes in this post. However, tissue tropism varies both between species and even between strains...choice for AAV pseudotyping. It has broad tissue tropism, including liver, muscle, lung, CNS, kidney, and...service.  AAV9 AAV9 has relatively broad tissue tropism and high transduction efficiency, and is an excellent...
  30. Viral Vectors 101: Gamma-Retroviral Packaging Systems

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    ...The widespread use of γ-retroviruses (gamma-retroviruses) in cancer and stem cell research has prompted...flexibility to pseudotype γ-retrovirus using different envelopes to modify tropism. Briefly, different envelope...either an ecotropic envelope, Phoenix-ECO, (for infection of mouse and rat cells), or an amphotropic envelope...prototypical gamma-retrovirus.    Frequently asked questions about γ-retroviral vectors How do engineered...MoMLV and MSCV for γ-retrovirus; HIV for lentivirus). Additionally, γ-retroviruses can only transduce dividing... Are γ-retroviruses self-inactivating (SIN)? They can be. Plasmids that encode SIN γ-retrovirus lack most...blog Viral Vector Elements The Retroviral Life Cycle Popular Retroviral Vectors and Their Use in Research...
  31. Optogenetics + CRISPR, Using Light to Control Genome Editing

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    Blog Post
    ...spatial control. Optogenetics uses genetically encoded tools, such as microbial opsins, to control cellular...genomic loci Photochemical Control Hemphill et al., 2015   Nitrobenzyl-caged lysine Cas9 K866PCK mutant...tools allow scientists to use light to externally control the location, timing, and reversibility of the ...process. Read on to learn about the various light-controlled CRISPR tools available to researchers - some ...published in early 2015 focused on using light to control transcription. Two separate labs, Moritoshi Sato...gene expression while imparting spatiotemporal control, reversibility, and repeatability. The system developed...expression of the human ASCL1 gene could be spatially controlled in a reversible and repeatable manner. With their...
  32. Replacing Paper: Tips for Choosing an Electronic lab Notebook

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    Blog Post
    ...and developed the SciNote Electronic Lab Notebook. Implementing an electronic lab notebook starts with ...should have functionalities such as: electronic signatures, electronic witnessing and audit trails. How ...write things down when doing scientific research? Electronic lab notebooks and researcher's needs The familiar...traceability and reproducibility of research. Electronic lab notebooks (ELNs) are here to make lab work...This includes financial outlay, staff hours, troubleshooting, and the fact that long-term use is likely ...traceable. However, switching from paper to an electronic lab notebook requires time and knowledge. In ...recent article, we translated our knowledge of electronic lab notebooks into 9 questions that will help...
  33. Plasmids 101: NGS Quality Control for Pooled Libraries

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    Blog Post
    ...Addgene uses electrocompetent Stbl4 cells when performing amplifications, and we use electroporation to ensure...library, Addgene usually does four to eight electroporation reactions using a total of ~400ng of library...
  34. AAV Vector Quality Control: Going the Extra Mile with NGS

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    ...highest quality, we undertake a rigorous quality control process. In 2016, we launched our viral service...our scientists perform all the standard quality control assays for AAVs. These steps include measuring ...Addgene is also conducting more rigorous quality control, including next generation sequencing (NGS) of ...VGS). Why should you care about viral quality control? AAV vectors are produced by scientists - not robots...Lecomte et al., 2015). This higher level of quality control is recommended but not yet required for clinical-grade...addgene.org to answer your questions about our quality control process! The images in this blog post were created...2011) Vector Characterization Methods for Quality Control Testing of Recombinant Adeno-Associated Viruses...
  35. Interview: Nicola Patron on Plant Synthetic Biology, MoClo, and More

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    Blog Post
    ...Nicola Patron is Head of Synthetic Biology at the Sainsbury Laboratory, where she often feels more like...that go on between plants and their pathogens. As Patron explains it, her work has always been focused on...Modular Cloning (MoClo) Kit. What does it include? Patron: Sainsbury has a mandate to share and educate and...Addgene: Will the MoClo tools work in any plant? Patron: The MoClo kit is entirely for dicots, not grasses...tips for those looking to get started with MoClo? Patron: The parts will be familiar to most plant scientists...there other tools you are working on for plants? Patron: Our lab and others at the Sainsbury are working... of engaging about science in that way for you? Patron: I started my Twitter account recently inspired...
  36. Export Control Regulations Lifted for Plasmids Containing Rabies and VSV-G Genes

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    Blog Post
    ...pUMVC (MuLV retroviral). Bob Weinberg pMD2.G VSV-G envelope expressing plasmid Didier Trono pVSV-G...plasmids. This is because in 2024 certain export control exceptions were made by the US Bureau of Industry...Industry and Security, pursuant to the international controls prescribed by the Australia Group, for these plasmids...Envelope protein for producing lentiviral and MuLV retroviral particles. Use in conjunction with a packaging...researchers in countries with applicable export control exceptions for rabies and VSV-G genes, plasmids...strive to keep our information updated, export controls and regulations are complex and may change suddenly...
  37. Plasmids 101: In-house NGS Plasmid Quality Control Now at Addgene

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    Blog Post
    ...and last updated Apr 6, 2021 by Will Arnold. Introducing NGS in 2017 for all incoming plasmids was a big...Addgene - we had used Sanger sequencing for quality control for over ten years. Now, thanks to a generous donation...quantification of various intermediate steps, a sizing electrophoretic gel, and qPCR quantification of the final libraries...The first step we take when performing quality control of newly deposited plasmids is to align the NGS... by the depositing lab, we call these “quality control (QC) issues.” We then ask the depositing laboratory...
  38. Plasmids 101: Walkthrough of Addgene’s Snapgene-Powered Quality Control Process

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    Blog Post
    ...talked a lot about the quality control process at Addgene by introducing our new sequencing partner seqWell...generation sequencing results to perform quality control on deposited plasmids. We’ve also talked about ...interface. We regularly use Snapgene for our quality control process because of its expansive feature library... main ways we use SnapGene for plasmid quality control are: To identify important plasmid features ...Identifying plasmid features While our quality control process for incoming plasmids can vary based on... quality control... plasmid quality control...
  39. Teaching an Old DOG New Tricks: Controlling Protein Activity with GFP

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    Blog Post
    ... just label cells. If GFP could be co-opted to control gene expression, he could then selectively manipulate...promoters. Moving to an in vivo system, Tang et al. electroporated T-DDOGs, GFP, and a reporter construct into...TdTomato, whose expression was absent without electroporated GFP. They then tested T-DDOGs with two mouse...robust and specific. When tested in retinal electroporation studies, DsRed reporter expression was induced...
  40. A New Optogenetic Tool Based on AraC Controls Gene Expression with Blue Light

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    Blog Post
    ...Their system is based on the AraC protein that controls transcription from the PBAD promoter. “AraC is... promoter. While none of the constructs gave as strong a signal as with the wild-type AraC, it still reached... of interest, under the pBAD promoter, can be controlled with light without the need of any cloning. ...
  41. Controlling for Off-target Effects with a New Genome-wide CRISPR Screen Design

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    Blog Post
    ...an appropriate control? Many CRISPR screens use non-targeting guides as negative controls. Non-targeting...are well characterized and strategies exist to control for these faults. However, it’s still unclear if...screens and how best to design these screens to controls for flaws. Recently the Bassik Lab at Stanford...site in the genome. This means that while they control for many of the effects of gRNA and Cas9 expression...detecting Cas9 cutting. Safe targeting guides better control for non-specific toxicity than non-targeting guides... a coding region. Targeting these sites should control for the effects of guide expression and dsDNA breaks... the genome, and are commonly used as negative controls for genome-wide CRISPR screens; and 3) safe-targeting...
  42. Quick Guide to Working with Drosophila Part 2: Controlling Gene Expression in Flies with Gal4/UAS

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    Blog Post
    ...systematically study gene function with temporal control and cell-type specificity! The Gal4/UAS system...you mate your fly line containing YFG under the control of the UAS with a fly line expressing Gal4 in your...promoter each time a new hypothesis is tested. Controlling multiple genes with Gal4/UAS system One can also...to drive expression of multiple genes under the control of UAS enhancer sequences. For example, if you ...them individually or simultaneously. Do note for control purposes that there are dosage effects. For example...
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