...SARS-CoV-2 infection remain poorly understood but are critical for developing therapeutic strategies to prevent...

...editors are created by fusing Cas9 nickase or catalytically inactive “dead” Cas9 (dCas9) to a cytidine deaminase...

...be required by your country for the import of biological research materials. Please be aware that additional...

...termed a shuttle plasmid and an adenoviral (also called backbone) plasmid, are recombined into a DNA molecule...

...Gene/Insert Promoter PI Publication Activate Catalytically dead dCas9 fused to a transcriptional activator...

...and time. An alternative approach is to use synthetically produced recombinant antibodies, created by ...

...C. elegans strains. CRISPR plasmids from John Calarco's lab as described in Heritable genome editing in...

...Tagging Fluorescent Protein Guide Subcellular Localization Plasmids Protocols The Allen Institute for Cell...

...regulators of membrane traffic, they also play critical roles at cilia, centrosomes, the plasma membrane...

... of p53 splice variants in human malignancy: a clinical perspective. Surget S, Khoury MP, Bourdon JC. ...

...pOpen-BovDNTT Bovine DNTT 165555 pOpen-ALPI CIP (calf intestinal phosphatase) Return to top Additional...

...165-76. PMID: 26390090 Other citations include: Scalabrino, et al. 2015. Intravitreal delivery of a novel...

... removal of a phosphate group. Under normal physiological conditions GTPases play prominent roles in signaling...

... domain of a TALE, along with strong nuclear localisation tags and repress transcription initiation when...

...Of mice and models: improved animal models for biomedical research. Bockamp E, Maringer M, Spangenberg ...

... (including DREADDs) Optogenetics Recombinases Calcium Sensors and other Biosensors Controls To look up...

...sgRNA 46170 Worm PCR (see paper) none S. pyogenes Calarco SP6-sgRNA-scaffold 47912 Worm AflII none S. pyogenes...

...example image). Point Scanning Confocal Microscopy Point Scanning Confocal Microscopy was the first fluorescence... microscopy technique to incorporate optical sectioning. Optical sectioning refers to the ability to extract...detector. The pinhole is specifically placed to only allow light from the focal plane to reach the detector...excluded. Parallelized Confocal Microscopy (Spinning Disk) Parallelized Confocal Microscopy (Spinning Disk...drawbacks of widefield and confocal microscopy. First, widefield and confocal microscopy project excitation...multiple focal planes of a sample may also be useful for providing fast imaging along with optical sectioning...respective focal plane. In sufficiently thin samples, deconvolution can outperform optical sectioning ...

...another critical control in biological experiments. Replicates can be technical or biological and are ... inclusion of biological replicates such as multiple mice in each group and C, technical replicates or...recorded would be due to natural biological variation. This information is critical for correctly interpreting... accuracy and therefore the statistical power of an experiment. Technical replicates are repeated measurements...meaningful results.  Biological replicates are distinct samples that are treated identically, used to assess...binds specifically to its target through the antigen binding site but also binds non-specifically to other...binds specifically to its target through the antigen binding site but also binds non-specifically through...

...report physical titer, which is measured by qPCR.   Physical titer is typically calculated by two ...particle typically contains one viral genome. Other than a theoretical maximum value, physical titers don... you? Physical titer: the concentration of viral particles containing viral genomes. Physical titers ...facilities report the physical titer of a viral solution (Figure 2). Because physical titers are used for...that can transduce cells. Infectious titers are typically quantified by cell transduction assays. Wild-type... AAV2 has been reported to have a near-perfect physical-to-infectious particle ratio of 1:1 (Zeltner et...for recombinant AAV2, the same study reported a physical-to-infectious particle ratio of 50:1 (Zeltner ...

... John H. McDonald. "A practical guide to evaluating colocalization in biological microscopy." American...subcellular localization of your protein of interest The first step in analyzing the localization of your ...organs, cells comprise compartments and structures, called organelles. Take a sneak peak inside a cell with... proteins of interest and examine them using biochemical methods thus removing the context of the cell...living cell. Analyzing differences in protein localization and transport between healthy and diseased states...for example, validate that the fusion protein localizes properly by comparing the signal from the fusion...protein and see who it is hanging out with via colocalization - aka “are they close?” After tagging your ...

...and sensing electrical potential. Thankfully, there is a tool to measure these electrical events! Voltage...essential to their use as biological tools. Why Use Voltage Indicators? Electrical signals are integral to...Sensors This system consists of a genetically expressed enzyme, typically on the cell surface, which can ...the chemical factor, of a naturally occurring voltage indicator. To give an example, a genetically encoded... tag, cage, uncage, and trap the chemical fluor with the genetically encoded component of the system to...needs to be cell type specific as well as specifically localize to the membrane, otherwise they aren’t all... all that useful. The cellular localization component is typically handled by either the fluorescent component...

...interaction between a bisarsenical dye ligand (e.g., FlAsH, ReAsH) and a short genetically encoded tetracysteine...systems consist of a genetically encoded enzyme variant tag that reacts specifically and irreversibly with... as intact tissue. Some chemical labeling strategies such as the bisarsenical dye (Figure 1a) and FAP ...Ronald T. Raines. "Bright ideas for chemical biology."ACS chemical biology 3.3 (2008): 142-155. PubMed... at the Janelia Research Campus, Howard Hughes Medical Institute. Chemistry is dead, long live chemistry...fluorescent protein (GFP) sparked a renaissance in biological imaging. Suddenly, cell biologists were no longer...synthetic fluorophores. Add a dash of DNA with an electrical jolt and cells become perfectly capable of synthesizing...

...the amount of a given factor within a biological system. Basically, biosensors provide quantifiable readouts...ions, calcium is by far one of the most popular, with dozens of sensors developed for it. Calcium triggers...the presence of calcium. This includes FRET-based sensors such as Twitch. While calcium is king in the ...while neuroscientists would like to monitor calcium specifically in their favorite neurons. This specificity...The calcium is strong here….I can sense it….sound like a plot to an biology scifi movie? Or maybe it’...biosensor?! You can do more than just ‘sense’ biological compounds and reactions; you can quantify them...pretty simply – they are systems that measure biological phenomena by generating an output which is directly...

...colleagues have developed a new genetically-encoded voltage indicator (GEVI) called JEDI-2P (Liu et al. 2022)... Verkhusha’s lab has generated a new genetically encoded calcium indicator using small monomeric NIR fluorescent... response kinetics and photochemical characteristics than previous NIR calcium reporters iGECI and NIR-GECO2G...has higher signal to noise ratio and is better localized for synaptic glutamate response. They generated...lower affinity) and with a variety of tags for localization (PDGFR, GPI, and SGZ). Read more from the authors...indicators with improved activation kinetics and localization for imaging synaptic transmission. bioRxiv 2022.02.13.480251...select variants based on performance metrics specifically for two-photon microscopy. The resulting JEDI...

...discovery–a review of theoretical aspects and recent applications." Current Chemical Genomics and Translational...al. "Designing logical codon reassignment–Expanding the chemistry in biology." Chemical Science 6.1 (2015...the American Chemical Society 123.48 (2001): 12123-12125. Journal of the American Chemical Society. 7. ...laboratory, then FRET experiments are a viable biophysical path to answers. What is FRET and what's it...Fluorescence resonance energy transfer (FRET) is a biophysical interaction between two fluorophores. When the...acceptor) if the two probes are close together (typically 30-100 Å, depending on the probes). In fact, FRET... FRET can be used to derive a precise physical distance between the donor/acceptor fluorophores (Stryer...

...Edward S., et al. "Millisecond-timescale, genetically targeted optical control of neural activity." Nature...one that can be achieved using pharmacological or electrophysiological methods (Deisseroth, 2011; Mei ..., et al. "Next-generation optical technologies for illuminating genetically targeted brain circuits." ...halobium differing in pigmentation." Biochemical and biophysical research communications 78.1 (1977): ...Over the past few years, however, remarkable technological advances have taken researchers to the threshold...led by neurobiologist Karl Deisseroth, combined optical and genetic methods to photo-stimulate mammalian... is reached and the neuron fires, sending the electrical signal down the axon (Lodish et al., 2000). Most...

...types of replicate controls: technical and biological. In general, technical replicates can be thought of...independent variable. Replicate controls: technical and biological Reliable results are reproducible from...NOT independent and are typically derived from one source. Conversely, biological replicates ARE independent... more in depth information. Technical replicates An example of a technical control is the transfection...independent variable. Biological replicates In any given set of experiments, the biological controls are more...the observed phenomenon in your experiment is specifically associated with the independent variable you...plasmids, and discuss why these control plasmids are critical for the correct design of the experiment.  The...

...essential aspect of biological research that has defined our understanding of biological processes and developments...Organism. News-Medical. Retrieved on September 18, 2020 from https://www.news-medical.net/life-sciences...evolving and demanding field, requiring a variety of biological and molecular tools. One such tool is the model...developments. Model organisms often feature genetic, physiological, or developmental characteristics that make ...are five more model organisms that have left a historical impact on the scientific community. (And, check... and biochemistry. Certain strains have been specifically developed to thrive in laboratory settings. ... used to study human cardiovascular disease, specifically stroke and hypertension (Iannaccone et al., ...

.... "A genetically encoded fluorescent probe in mammalian cells." Journal of the American Chemical Society...). GFP is the most popular, most widely used genetically encoded fluorescent probe. Several factors contribute...probe, it is the first choice when selecting a genetically encoded fluorescent tag. There are, however, ...of these strategies works best for a specific biological system under anaerobic conditions needs to be...of GFP may interfere with the activity or the localization of the fusion protein. Also, in cases where ...adding GFP may alter diffusion rates. Smaller genetically encoded probes are based on cofactor binding ...tags are peptides that are engineered to bind specifically and with high affinity to fluorophores. Examples...

...non-bench research career such as law, medical/technical writing, clinical, regulatory, product development,...Further, this post may contain technical inaccuracies or typographical errors.  No action should be taken...industry H-1B There is a numerical quota of 85,000 total H-1B visas approved each fiscal year: 65,000 visas ...a "specialty occupation" as requiring theoretical and practical application of a body of highly specialized...a US Master’s degree or higher degree. These numerically limited visas are referred to as “cap” subject...chemistry, architecture, engineering, mathematics, physical sciences, social sciences, medicine and health...industry such as technology transfer, communications, medical writing, compliance, core facility management, ...

...Neurobiology has been working on genetically encoded indicators of calcium and other small molecules since...transfer (FRET)-based calcium biosensors. Perhaps most notably, the Twitch calcium sensors, which are available...-affinity for calcium binding and developed a version of the protein with as few calcium-binding sites...engineered sensors were designed to replace synthetic calcium dyes, which had been in use since the 1980s. “Synthetic...of interest,” Griesbeck said. Because they are chemical compounds, they have to be applied or injected...they shift from "off" to "on" in the presence of calcium. This color change can be observed under a microscope...Griesbeck and his colleagues began by isolating a calcium-binding protein, troponin C (TnC), from muscle ...

...signaling events occur in a physiological setting. Genetic techniques typically rely on overexpression or...needed for function. The lab validated this vector, called pBAM1, by conducting random mutagenesis in the ... sgRNA delivery and providing a means for large scale functional screens. For more information on these... post,Lentiviral CRISPR Libraries Enable Genome-Scale, Knockout Screening. Also, check out our updated...brighter mutant S65T-GFP, and the subsequent biochemical characterization, has enabled scientists to engineer...observations: Rapamycin is a membrane-permeable pharmacological agent that can rapidly induction heterodimerization...flexible cloning process provides improvements to classical cloning methods, particularly for complex transgenesis...

... and LV have been used successfully in preclinical and clinical settings for ocular conditions. However...used in clinical trials, a variety of engineered ones have shown great promise in preclinical studies ...that I got into the field of gene therapy, more specifically AAV-based retinal gene therapy. The year was...treat an inherited retinal degenerative disease called Retinitis Pigmentosa. I quickly became fascinated... Gelsinger in an adenovirus-based gene therapy clinical trial gone horribly wrong. It brought the whole...scientists, doctors and patients hope are many more clinical successes for gene (and cell) therapies. One may... over 25 years ago when completing her PhD and medical training in Ophthalmology. Gene therapy was in ...

...cells and statistically analyze cell movements. By using cells labelled by fluorescent chemicals or by other... steady state or under physio-pathological challenge. However technical specificities have to be taken...complex metazoans, rapid cell division and large scale cell mobility are essential processes during embryonic...organisms, where tissues structures are more stable and local movements predominate (e.g. a basal progenitor cell...stage, only cells from the immune system show wide scale mobility with movement from the bone marrow and ... studying other systems in which there is large scale cellular movement throughout an organism. Studying...mobility than the static microscopy images of chemically coloured lymphoid organs did in the past. How...

...illumination on biochemical processes that are crucial in the world of biological research, medical therapy, ...components, typically a protein of interest. Fluorescent dyes and ligands vary in their chemical structure...both organic tissues and cell cultures, opening biological interiors to many inquisitive scientists. Seeing...the dye and the endogenous target, and thus no genetically encoded tag is required. These dyes can also ...be SNAP-tag® and HaloTag®. These consist of a genetically encoded enzyme variant tag that covalently reacts...more flexibility due to the affinity tag they typically bind to. Stability of fluorescence: For applications...thorough removal of unconjugated dye molecules are critical for accurate and reliable imaging and assay results...

...SR, Bird A (2017) Radically truncated MeCP2 rescues Rett syndrome-like neurological defects. Nature 550...awareness and published his observations in a German medical journal. Decades later and after years of painstaking...silenced by a Lox-Stop cassette, resulting in the neurological deficits observed in Rett Syndrome. Restoring... tamoxifen-inducible Cre not only halted the neurological damage but completely reversed it, regardless...this interaction to varying degrees determine the clinical severity of different MECP2 mutations. More recently...the MECP2 protein is necessary to rescue the neurological defects of Rett syndrome (Tillotson et al., ...amount of protein to each brain cell to restore physiological levels. Much research is now focused on therapeutic...

...Escherichia coli, like the construction of a so-called “genomically recoded organism” (3) but its portability...mutant library generation in several biotechnologically and clinically relevant bacterial species. pORTMAGE...Pathology have developed an alternative method, called STARR-seq (self-transcribing active regulatory ...new ligands for orphan GPCRs is of significant medical interest, however efforts to do so have been hindered...human cell line expression. In a twist on the classical TANGO assay, Kroeze et al. developed a new approach...Ras oncogenes have been difficult to target therapeutically due in no small part to the central role Ras... least three independent gRNAs, each chosen strategically to maximize the likelihood of creating a functional...

...generated from this research. At both a practical and ethical level, scientists arguably have a duty to...). Advances in genetic technologies and other biomedical innovations promise an improved understanding...best way to shepherd technologies through thorny ethical issues, ensure that everyone will have the possibility... fruits of scientific research, and prevent technological advances from exacerbating existing inequalities...based at the Department of Genetics of Harvard Medical School, has a mission of raising awareness and ...the benefits as well as the personal, social and ethical implications of genetics. In these conversations...sensitive topics and uncomfortable places, from sticky ethical dilemmas to dark episodes in the history of eugenics...

...for every application. Plus, the photophysical properties of chemical dyes provide many advantages over...depletion (STED) nanoscopy. Scale bar, 2 μm. Inset shows comparison with standard confocal microscopy. Image adapted... W. (2011). Chemical Tags for Labeling Proteins Inside Living Cells. Accounts of Chemical Research, 44...If you take a look in a typical lab freezer, you’ll probably find lots of different plasmids containing...substrate. If the substrate was linked to, say, a chemical dye or other functional ligand, your protein of...self-labeling tools: Tetracysteine: The first reported chemical labeling tag, from Roger Tsien’s lab in 1998 (...any non-cysteine amino acid) that reacts with biarsenical dyes such as FlAsH (green, based on fluorescein...