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... efficiency. Detecting certain cellular changes with an intermolecular probe is often either impractical..., detect cellular processes and signaling cascades, quantify mechanical tension (a molecular “spring”)...the possibilities can seem overwhelming given the large assortment of fluorescent proteins available. Fortunately...plasmids will be most useful for constructing intermolecular FRET probes, where the donor and acceptor fluorophores...fused to two separate proteins. (In contrast, intramolecular FRET probes contain the donor and acceptor ...these biosensors later on). Experiments using intermolecular FRET probes are typically used for studying...proximity of the two proteins can be inferred. Intermolecular FRET can be experimentally difficult to achieve...

...studying other systems in which there is large scale cellular movement throughout an organism. Studying...Patrolling monocytes promote intravascular neutrophil activation and glomerular injury in the acutely inflamed...In complex metazoans, rapid cell division and large scale cell mobility are essential processes during...body. In contrast, these dynamic processes are largely absent in adult organisms, where tissues structures...fluorescent proteins can and have been used to monitor cellular movements in the immune system. The techniques...microscopy from PLoS Pathogens - Kamenyeva et al, 2015. Cellular trafficking studies enabled by fluorescent proteins...organs and visualize how cells interact after a particular stimulus. Using an epifluorescence microscope...

... (FUS), intracorporeal (IC), intracerebroventricular (ICV), intramuscular (IM), intrathecal (IT), and ...to transduce the same cell, can accommodate larger, modular genetic payloads. Since most systemic vectors...S., Siverts, L. A., Larsen, R., Gore, B. B., Szelenyi, E. R., Trader, C., Balaram, P., … Tasic, B. (2021...distributing widely throughout the organism to target large or diffuse biological structures. Of primary interest...brain along with high off-target expression, particularly in the liver, when administered intravenously...Hinderer et al, 2018). The optimal dose for each particular experiment is determined by the capsid and promoter... capsids cross the BBB via interactions with a similar protein called LY6C1. This protein is expressed...

...two appear on an agarose gel at notably higher molecular weights than expected, given the size of the plasmid...observed to occur more frequently in plasmids with large inserts (>7 kb)(Berg et al., 1989), direct repeats...just as well as monomers. However, due to their larger size, multimers may exhibit reduced transformation... & Sherratt, 1984; Summers et al., 1993). These larger plasmids are also maintained at lower copy numbers...monomeric version. However, undigested multimers are larger than monomers and will therefore run higher on ...reads (<300 bp) which cannot distinguish between large repeat sequences within a plasmid, it cannot be ...sequencing. These longer reads are able to detect large repeat sequences within a plasmid and, therefore...

...space. Zebrafish have become a popular model organism because their larval stage lends itself well to studies...to be hyperpolarized. The chloride pump NpHR brings in negative chloride ions which hyperpolarize the cell...studies of neuroscience. The larvae of zebrafish are translucent and allow for noninvasive live imaging... Furthermore, during the first 2 weeks of life, larvae already exhibit distinct behaviors such as spontaneous...positive or more negative.  Neuronal stimulation: Depolarization When the inside of the neuron becomes positive...positive over a certain threshold, it becomes depolarized and will fire a signal. The non-specific cation...both cause ion movement that results in cell depolarization. They therefore function as a neuron “on switch...

...flow”) has emerged as a particularly useful tool for those wishing to run larger panels: five laser spectral...This tool is of particular value when designing a panel.     Figure 3: Similarity index across ...and a low similarity index. In figure 3, we can see that PerCP and PE/Cy5 have a similarity index of 0.87...molecules are added during that particular batch. This can present particular difficulty in unmixing if your...detector to detect one fluorophore and therefore one cellular marker. However, emission spectra of certain fluorophores...fluorophores. This allows for the creation of much larger panels, as fluorophores that cannot be distinguished...you want to use in a given experiment. For very large panels (upwards of 30-40 colors), spectral flow ...

...into the genome, and its large DNA packaging capacity is well suited for larger transgenes. Unfortunately...ongoing. Moving forward with ocular gene therapy The pipeline of AAV-based ocular gene therapies is impressive...genetic diseases such as hemophilia, muscular dystrophy, spinal muscular atrophy, neurodegenerative diseases...life-threatening (cystic fibrosis, hemophilia, muscular dystrophy)? AAV gene therapy started in the mid... in the form of Luxturna™. A better vector for ocular gene therapy: Adeno-Associated Virus (AAV) While...cells, AAV quickly became the vector of choice for ocular gene therapy. AdV and LV have been used successfully...successfully in preclinical and clinical settings for ocular conditions. However, AdV was abandoned early on...

...mdx Mice With Large Truncated Dystrophins Generated by Recombination Using rAAV6. Molecular Therapy 19:36...Reassembly of fragmented genomes is the key to fitting large genes into AAV First, some history. In 2008, the...reproducing these results and determined that even when larger vector genomes are packaged, the physical size ... the genomes is still ~4.7kb. But despite this, larger functional transgene products were produced following...in mice. This vector transduced ~10% of cells, similar to the transduction rate of a single AAV. Odom ...deliver the mini-dystrophin gene to a mouse model of muscular dystrophy. While mini-dystrophin expression levels... 6 kb mini-dystrophin gene to a mouse model of muscular dystrophy. Three different splice site donor and...

...classification system relies on a combination of sequence similarity, phylogenetic analysis, neighborhood analysis... the same class and type don’t share sequence similarity but are grouped due to mechanisms of action and...completely different! They are merely linked by a similar function. After analysis, CRISPR classifications...nearly 100% in archaea, though Class 1 remains largely underutilized compared to Class 2. Class 1 Despite...incredible abundance, Class 1 CRISPR systems are less popular in the lab. This can be attributed to the multi-Cas... ensure every unit of the complex expresses at similar levels. It was only recently that researchers began... type of CRISPR overall. Type I systems degrade large sections of DNA thanks to the dual action power ...

...diffusing in the extracellular space; they also have low immunogenicity and cellular toxicity. AAV vectors...brain’s function is knowing its architecture, in particular the connections between different brain regions...brain tissue is injected with compounds that intracellularly spread away from the site of application and...Schmued and Fallon, 1986) are used. To facilitate cellular uptake of these dyes in vivo, microinjection, ...but these methods can have a negative impact on cellular health. Another limitation of traditional tracing...using these viruses to mark neuronal connectivity a large majority of neuronal cells are labeled overtime ...also others including pseudorabies virus type 1, vesicular stomatitis virus (VSV; Beier et al., 2011), adenovirus...