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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...

...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 ...

.... 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...

...Cell-surface protein labeling tools for neurons and astrocytes Nerve-to-cancer cell mitochondrial transfer tracker...classrooms Cell-surface proteomics for neurons and astrocytes By Mike Lacy Cell-cell interactions underlie ...important in the brain. To better understand how astrocytes and neurons interact, the labs of Baljit Khakh...extracellularly-anchored horseradish peroxidase (HRP) in striatal astrocytes and neurons to label surface proteins with a ...Biotinylated proteins were isolated, identified by mass spectrometry, and classified by source cell based on single-cell...cell type, the cell-surface shared proteome of astrocytes and neurons (CS SPAN) featured extracellular ... for future studies of the molecular bases of astrocyte-neuron interactions. Plus, the tools used here...

...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...

... 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...

...about the tissue tropism of these and other AAVs. Figure 4: AAV6 and AAV11 tropism and applications...ISYmu1 and an ωRNA under the same promoter, they introduced heritable genome edits in a single step with ...clonal dynamics during differentiation after they introduced mutations that drive leukemia (Figure 3). ... Why these serotypes? AAV6 offers broad tissue tropism for gene therapy and ex vivo cell engineering applications...administration. AAV11 can transduce neurons for retrograde labeling, making it a powerful tool for circuit...applications. A) AAV6 is reported to have broad tissue tropism, including skeletal and cardiac muscle, airway ... studies have shown that AAV11 enables potent retrograde labeling of projection neurons. Created with ...

...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...

...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...

...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...

...-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...

...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...

...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...

...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 ...

...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...

...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...

...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...

...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...

...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...

...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, ...

...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 ...

...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 ...

..., 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...

...-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...

...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...

...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...

...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...

...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...

...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  ...

...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...

... 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...

...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...

...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...

...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...

...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...

...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-...

...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...

...delivery include plasmid transfection, RNP electroporation, and viral transduction; but these methods ...gRNAs. Viral envelope proteins To alter the cell tropism of Nanoblades, different viral envelope protein...with a mix of VSV-G and the baboon endogenous retrovirus Rless glycoprotein (BaEVRless) had high rates...gene, while editing was not detected in cells electroporated with RNPs using the same guide. Nanoblades ...influence the level of off-target effects, with electroporation of RNPs generally having lower off-target edits...polymer. Polybrene has long been used to increase retroviral transduction efficiency by neutralizing the charge...the cell surface, and also helps form DNA and retroviral-particle or VLPs complexes. The team found that...

... No spatiotemporal control. Many PPI detection methods lack spatiotemporal control or an “on/off switch...identifying PPIs, such as affinity purification mass spectrometry (AP-MS), lack the ability to specifically identify... by creating Split-BioID, a spatiotemporally controllable version of the proximity-dependent biotinylation...method Enter Split-BioID, a spatiotemporally controllable version of the BioID protocol. The critical ...interaction partners if they are enriched relative to controls using Split-BioID-GFP and BioID run on six unrelated...combinations and pick the conditions that give the strongest biotinylation signal, as measured via Western ...methods, including BioID, is its spatiotemporal controlled labeling of interacting proteins; nearby proteins...

...classification. Lentivirus (a type of retrovirus) and gamma retrovirus require more caution as they are integrating...virus and its infectivity in check. Engineering controls, safety systems that are built into how something...functions, were designed for this purpose. One such control is dividing up the essential components of a virus...virus into different plasmids. For a retroviral system this looks like the genetic cargo in one plasmid... researcher.   Figure 1: Separation of retroviral packaging components onto distinct plasmids for...common types of viruses grown in the lab are gamma retrovirus, lentivirus, adenovirus, and adeno-associated...  Susanna Stroik...

...lab-adapted bacteria because it has been challenging to introduce CRISPRi machinery into diverse bacteria species... gRNAs, and dCas9 proteins. Second, it can be introduced into a broader range of bacteria. Once transferred...., 2019 with permission.   Two methods to introduce Mobile-CRISPRi by bacterial conjugation For gram...gram negative bacteria, Mobile-CRISPRi is introduced using the bacterial Tn7 transposon system. This system...firmicute gram positive bacteria, Mobile-CRISPRi is introduced using the bacterial integrative and conjugative...Mobile-CRISPRi. The Mobile-CRISPRi ICE element is first introduced into the B. subtilis genome. Mobile-CRISPRi is...into the trnS-leu2 locus.   Figure 2: Introducing Mobile-CRISPRi into bacteria. Top: A Tn7 transposon...

...action potentials, muscle contractions, and the control of voltage gated ion channels all depend on transmitting...been successful even at the single neuron level in vitro. In vivo monitoring has also been achieved with ...effective at the single cell level as they are in vitro. Factors like photobleaching, lack of red-shifted...rhodopsin. FP-fusion to rhodopsin resulted in electrochromic FRET capable GEVIs, with rhodopsin quenching...membrane as well. Another flavor of these tools is Voltron, which uses rhodopsin as the voltage sensing component...genetically encoded component is delivered in vivo or in vitro and a small molecule is added exogenously to measure...Optogenetics Fluorescent Proteins 101: Introduction to FRET ...

... APC control, with APC and APC-CY7 plotted against each other. Since it is a single-color control, you...example with single color control. Flow plots showing an APC single-color control sample before (A) and after...recommended to start compensating with single-color controls before moving onto your experimental samples. ...samples without needing to use your single-color controls as a starting point. Applying compensation to ...experimental samples as they did with your single control samples: if the population needs to be shifted ...School.   More resources on the Addgene blog Introduction to Gating in Flow Cytometry Antibodies 101: ...

... Overview of in vitro AAV CRISPR screens. Created with BioRender.com. The in vitro option requires... screens may be AAVantageous!       In vivo, in vitro, ex vivo CRISPR screens can be carried out using...using a few different methods — in vivo, in vitro, and ex vivo. The general process of these screens is the...with transducing cells with the CRISPR library in vitro before transplanting them into mice. For in vivo...when designing screens. Each serotype has its own tropism, or a preference to infect specific cell or tissue...tissue types. Using serotypes that have a known tropism for your cells of interest — for example, AAV8 ... used these psuedotypes to further improve the tropism of AAV vectors, enhancing their affinity for specific...

...AAV PHP.eB Controls Wilson New viral vector pENN.AAV.EF1a.eGFP.WPRE.rBG AAV PHP.eB Controls Wilson New...pAAV-CaMKIIa-EGFP AAV PHP.eB Controls Roth New serotype pAAV-hDlx-Flex-dTomato-Fishell_7 AAV2 Controls Fishell New serotype...  AAV PHP.eB Controls Gradinaru New serotype pAAV-CaMKIIa-mCherry  AAV PHP.eB Controls Deisseroth New...New serotype pAAV-Ef1a-mCherry  AAV PHP.eB Controls Deisseroth New serotype Looking for more viral...

...28b-Cas9-His from Alex Schier’s Lab. gRNAs can be in vitro transcribed from ssDNA, which can be generated by... advantage has allowed scientists to perform in vitro and in vivo CRISPR studies in experimental systems... common techniques for delivery of RNPs is electroporation (A in the figure above), which generates pores...through a process known as CRISPR-EZ (CRISPR RNP Electroporation of Zygotes) (Chen et al. 2016). For CRISPR ...CRISPR experiments that involve HDR, electroporation can be combined with cell-type specific reagents in a ...approach using RNPs has recently been described in vitro, with the hope of eventually using this method for...genome editing by CRISPR ribonucleoprotein electroporation of zygotes." Journal of Biological Chemistry...

...only a few, very strong associations.   Unexpected results Their results showed 96 strongly correlated genetic...may be the result of a technical artifact that introduced an ancestry bias into their experiments instead... prevent Cas9 from cutting, both of which can introduce significant bias into the results. But the reference...mapped to a SNP would be ignored. Working with Lia Petronio and Katherine Huang from Broad’s Pattern team,... of ancestral bias into genome-wide screens, introducing bias into all work based on these and any other...an ancestry group what is actually an artifact introduced by bias within the design of the experiment or...Noorbakhsh, Isabella Boyle, Joshua Dempster, Lia Petronio, Katherine Huang, Alham Saadat, Thomas Green, ...

...unknown sample. To do this, you serially dilute a control sample of known concentration and measure a specific...example, a BCA assay uses a serial dilution of a control protein, incubated alongside an unknown sample ... absorbance of both is then measured on a spectrophotometer and concentration versus absorbance is plotted... a standard curve by serially diluting a known control sample, called a standard. The ideal standard curve...curve accordingly.    Figure 1: A 1 mg/mL control is diluted in a series ranging from 1:2 to 1:32... for each run. To validate, include a positive control of known concentration in the experiment and use...  Include both a standard curve and a positive control in every run.  When setting up your dilution series...

...very weakly, most likely through unstructured electrostatic interactions. It also can’t recognize PAMs, ... present in the sample. Other techniques, like electron microscopy and FRET, can offer more information...well described in the closed conformation, recent electron microscopy studies have also found Cas9:RNA complexes...loops 2 and 3 are not strictly necessary for in vitro cleavage, but they improve cutting efficiency and...pairing in the seed sequence.   Figure 6: Electron microscopy structure of S. pyogenes Cas9 bound...structure (left, PDB ID 4ZT0) with Cas9:RNA:DNA electron microscopy structure (right, PDB ID 7S4X), viewed...conformational state of the HNH domain also allosterically controls RuvC activity through a hinge region between the...

...tobacco. Studying gene function in C. roseus is strongly limited by the availability of methods. Infection...check for GUS-like activity of your tissue. Run a control with Agrobacterium lacking the GUS gene since plants...promoter activity, for example. Useful positive controls could include: pSB96 - pL2_pSB90_2x35S::fLUC-I...days in the dark after the transformation. Use introns Plant promoters can be recognized or leaky in Agrobacterium...Agrobacterium, but not the plant. For example, we have seen strong expression of the GUS gene with the commonly used...Agrobacterium. This can be easily avoided by the use of introns, which will be removed from the transcripts in ...plants, check out this interview with the Nicola Patron, who shared the MoClo plant kit via Addgene. For...