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...simplicity of production, and higher titers. However, the risk of RCA formation is higher for first-generation ...rAdV vectors are also referred to as "gutless" or high capacity AdV (HCAdV) vectors. Here, all viral sequences...adenoviral backbone plasmid have matching left and right homology arms which facilitate homologous recombination...the shuttle plasmid, but this method results in a higher background of non-recombinant adenoviral plasmids... used for rAdV vector construction, due to their high gene transfer efficiency. Other serotypes like Ad26...immunogenicity. For example, Ad5/35 and Ad5/26 combine the high transduction efficiency of the Ad5 serotype with... particle. Vaccine Development rAdV vectors are highly immunogenic because they activate both innate and...

...for a given experiment. In the table below, we highlight the most common categories of genetic manipulations...joining (NHEJ) at the cut site Cas9 or Cas9 nickase High-fidelity Cas enzymes increase specificity Dual-nickase...editors Editing efficiency can be low, but generally higher than HDR pegRNA optimization can be challenging...location. Positioning your chosen Cas enzyme at the right spot within the genome relies on proper gRNA design...design a donor DNA to have the appropriate left and right homology arms. See Addgene's HDR blog post for more...guides that target closer to the edit site yield higher editing efficiencies. The template sequences can...specificity, consider using an engineered Cas enzyme with higher fidelity. In addition to off-target activity, it...

...episome which can concatemerize, producing high molecular weight structures that are maintained extrachromosomally...hybrid capsid derived from eight different serotypes. AAV-DJ displays a higher transduction efficiency in...on transgene expression and the expression of neighboring genes. rAAV episomes can develop chromatin-like...in vitro than any wild-type serotype, and a very high infectivity across a broad range of cell types in...Brain Connectivity Atlas and the Janelia Campus MouseLight project . rAAV vectors have also been developed...signals of cells (commonly neurons) in response to light. The short coding sequences of channelrhodopsins...wild-type). Another split AAV method which has shown higher efficiency in delivering large genes makes use ...

...opto-chemogenetic fusion proteins of a light-emitting luciferase and a light-sensing optogenetic element. When...neurotransmission. This, and the fact that GPCR ligands can have high potency, which is potentially important for dosing...off-target effects in vivo and the receptors exhibited high levels of constitutive activity, making them less...luciferin) is added, the luciferase enzyme generates light that activates the opsin. The opsin may excite or...illumination), making LMOs a versatile option. Coupling brighter bioluminescent modules, such as luciferase-fluorescent...example, a study in rats using an hM4Di receptor might consider an alternative DREADD ligand such as DCZ... chemogenetics by using physical and biological light sources for opsin activation. Proc Natl Acad Sci...

...), including overexpression plasmids with up to eight transcription units. Yarrowia lipolytica Golden ... Expression Tom Ellis Plasmids for constructing highly-tunable GPCR-based biosensors in yeast (requires...Bacterial Expression Naomi Nakayama A toolkit with both high cloning capacity and vector simplicity, compatible...in plants. The constructs are made with zCas9i, a high efficiency intron-optimized SpCas9-coding gene. ...TALEN Takashi Yamamoto Plasmids for construction of highly-active Platinum TALENs for use in a wide variety...

..., respectively). The bases are organized into a right-handed, double-stranded helix. To create the double... insert expression, as a strong promoter directs high expression, whereas weaker promoters can direct ...determines the type of plasmid and dictates how it might be used in the lab. Regardless of type, plasmids...particles) that can infect your target cells at a high efficiency. Viral Vectors , Viral Services Molecular...second-generation sequencing”) comes in. NGS is a high-throughput, multi-parallel sequencing platform that...

...on choosing the right cloning method for your research project . This guide highlights several of the ...

...for fast and efficient generation of large-scale, high-titer preps. This is thanks to established retroviral... on a case-by-case basis. Retroviruses carry a slightly elevated risk of integrating into proto-oncogenes...clinical-grade gamma-retroviral vector purification by high-speed centrifugation for CAR T cell manufacturing...

...immune system, antibodies are proteins capable of highly specific binding to other proteins. Antibody-based...technologies developed for optogenetics research utilize light to detect, measure, and control molecular signals...

...polymerase) to bind to a promoter. Transcription is tightly controlled in both bacteria and eukaryotes by various... Cauliflower Mosaic Virus Ubi Constitutive Plant High-expression promoter from maize ubiquitin gene GDS...

...J., Bae, S., & Hur, J. K. (2021). CReVIS-Seq: A highly accurate and multiplexable method for genome-wide...Kochenderfer, J. N., Norberg, S. M., Hinrichs, C., Highfill, S. L., Somerville, R. P., Panch, S. R., Jin, ...

...additional hypothesis-directed experiments. As you might have guessed from their name, pooled libraries are...

...limit of a light microscope. Due to the wave nature of light, an infinitely small point of light will blur... to provide high 3D spatial resolution while also avoiding the toxic effects of high light doses to the...fluorescence microscopy provides a high contrast image in which a bright signal overlays a perfectly black...placed to only allow light from the focal plane to reach the detector and all light from above or below...for example, red light instead of blue to excite a molecule of GFP. Because red light has approximately...approximately half the energy of blue light, two photons of red light are needed to excite GFP compared to only ...objective can a a high enough density of red-photons be establish for this to happen.Using red light has a second...

...UV-Violet Dark/Green N/A (high) High 503/ 518 Padron Monomer Blue Dark/ Green N/A (high) Low 503/ 518 rsCherry...specific wavelength of light. PA-FPs can be activated from low fluorescent states to high fluorescence states...fluorescence, to a brighter fluorescent state by exposure to a specific wavelength of light. The switch or...fluorescence when illuminated with 488nm light. However, when UV-violet light is presented first, it causes irreversible...illuminated with violet light, both variants are photoconverted to forms that emit brighter red fluorescence....when exposed to blue light, which is less phototoxic to live tissue than the UV light used by other PA-FPs...State Activating Light Pre/ Post Color Contrast (Fold Change in Fluorescence) Brightness Excitation/ Emission...

...with exquisite precision using only light. Optogenetics, or light activated tools, can control everything...an experimental optogenetic setup. A light source delivers light pulses to the subject/cells expressing... photoswitches. Light-gated ion channels directly open and close when exposed to light and change the ...changes with light stimulation to either activate or inhibit a specific receptor or pathway.  Light-gated ion...turn on with a light-pulse at one wavelength and stay on until a second pulse of light, with a different...pathways with light. This is achieved by coupling endogenous signaling molecules with light-activated partners... intensities of light sources used to prevent activation of both tools with each light source used.   ...

...dim" you might get the impression of a candlelight vs. a floodlight. For fluorophore brightness levels,...flow-cytometry-fluochrome-brightness-spillover Excitation Laser Color Very Bright Bright Moderate Dim...Photons of lower wavelengths are higher in energy, while photons of higher wavelengths are lower in energy...important to know the brightness of individual fluorophores. Table 1 describes the brightness of a few commonly...the cytometer. Brighter fluorophores have a stronger signal. Table 1: Degrees of brightness for a few commonly...-Cy7 are "very bright" when they are excited by the yellow/green laser but only "bright" for the blue ...leading to different levels of brightness, depending on the laser light's wavelength. Source: https://www.bdbiosciences.com...

... to their brightness and high degree of overlap. Dyes for DNA/RNA staining should have high affinity and...refers to the emission of light by a substance (fluorophore) that has absorbed light or electromagnetic radiation...whereby excitation of blue light will indicate where FITC is bound through green light emission. Key considerations...also vary in their brightness, depending on the respective fluorochrome. Brightness is impacted by both...of findings. If you’re struggling to select the right fluorescent tools, this blog is here to help. Here...ligand to make your selection easier, as well as highlight some recent advancements within this field. Fundamental...wavelength (Heinrichs, 2009). When a fluorophore absorbs light, electrons are elevated from a ground state to an...

...neuroscience. In fact, precise light-based control of biological processes is highly useful in other fields, ...consistent, and controllable pulses of light. Complicated patterns of light intensity and wavelength can be ...to low-intensity light, so off-target effects should be limited. Phytochromes are light-responsive systems...harness the power of light, Christopher Voigt’s lab created the first E. coli light-sensitive two-component...construct senses light, and an obligate chromophore allows the system to respond to various light inputs. In...various light inputs, and an obligate chromophore allows the system to respond. In far-red light or dark...exposure to red light rapidly deactivates the system. Version 1 of this far-red/red light-responsive system...

...LACE system for light-activated CRISPR-Cas9 effector, Polstein and Gersbach fused the light-inducible protein...binding affinity with light stimulation2. The Kuhlman lab created two improved Light Inducible Dimers (iLID...of Arch mutants screening for improved brightness. The brightest mutants were then further mutagenized ...enhanced sdChr construct, named CheRiff, exhibits high light sensitivity and fast kinetics. By combining both... weak or high activities (Bert, et al. 2000). To design pXPG, the authors incorporated a high-copy origin...repository, we can't give all of them attention they righlty deserve, but, in this post, we'll provide a small...2015 Feb 2;33(2):139-42. doi: 10.1038/nbt.3149. Light-inducible CRISPR-Cas9 system for endogenous gene...

...have higher specificity, lower cross-reactivity, and are more consistent lot-to-lot. The highest reproducibility... Tris-tricine 150 V, 1–3 hours Good for higher weight proteins Long running time   It’s important...during long-term storage and has both high binding capacity and high protein retention. It comes in a variety...usually preferred because of their higher specific activity, slightly lower cost, and smaller size. However...block with? It can be so overwhelming that you might just stick with the protocol your labmate handed...can help amplify a weak signal. However, they have high cross-reactivity, vary significantly from lot-to-lot... your assay. The most common options are 4 °C overnight or 1–2 hours at room temperature (RT). Most people...