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...kinetics which are illustrated in figures 2 and 3.  Figure 2: Glutamate titration curves for the ... glutamate neurotransmission.” Nature Methods (2013) PubMed PMID: 23314171. PubMed Central PMCID: PMC4469972...accurately differentiating between synaptic vs. extrasynaptic glutamate release. Building on the previous ..., GPI, and SGZ, to improve neuronal membrane trafficking and localization. Improved photophysical properties... Figure 1: 1P and 2P excitation and emission spectra of SF-Venus-iGluSnFR.A184V, iGluSnFR3 v82, and ...Supplementary Figure 3.   Figure 3: pH titration curves and ∆F/F0 for the different iGluSnFR variants...al…, Supplementary Figure 2.   Improved trafficking and localization through different C-terminal...

... system, however, RepZ concentration is controlled at the level of translation. RepZ mRNA must fold into...pseudoknot” held together by intramolecular base pairs for the protein to be translated. An antisense RNA called...the proteins cooperatively can separate the DNA strands. Created with BioRender.com. Once DnaA breaks...it recruits helicases to continue unwinding the strands. This creates two replication forks that move in...elongation phase, a DNA polymerase synthesizes a new DNA strand at each replication fork. Termination sequences...act as a feedback mechanism to reduce further transcription. Replication is also regulated by the balance...newly synthesized DNA, blocking binding sites, or trading out ADP for ATP to re-activate DnaA. These regulatory...

...use them in, is known as your gating strategy.  Gating Strategies   The series of markers you use to define...isolated.  Following a gating strategy Figure 4 shows a gating strategy from a published experiment (Barlow-Anacker... define your population is called your “gating strategy.” This series of markers are set up sequentially...(Barlow-Anacker et al., 2017). This gating strategy is used to identify two types of dendritic cells (...to each other.        Figure 4: Gating strategy used in Barlow-Anacker et al., 2017 to identify...populations on a plot.      Figure 5: The gating strategy from Barlow-Anacker et al., 2017, with quadrants... lot longer than learning how to read a gating strategy.  Other information FACS plots often contain ...

...tRNAs contain internal transcriptional elements that enhance pol III transcription, potentially increasing...protoplasts. Compared to matching traditional gRNA-expressing constructs, transcript levels of PTG1 and PTG2 were...various tRNA expression strategies? No worries, I've summarized the three strategies described in this post...viruses have illustrated the power of polycistronic gene organization, and its translation to CRISPR/Cas9...lower genome editing efficiency. One previous strategy for multiplexing gRNAs used the RNA nuclease Csy4...base Csy4 recognition sites from a polycistronic transcript. Although this method shortens construct size...coexpression of Csy4 with Cas9 and the polycistronic transcript. As nucleases may have off-target effects and...

...SLC6A8 mutations in Creatine Transporter Deficiency Creatine Transporter Deficiency (CTD) is a rare X-... to be transported from the blood and cross the blood brain barrier using creatine transporters. However...However, mutations in the transporter SLC6A8 impede this transport and result in global creatine deficiency... the SLC6A8 gene, also known as the creatine transporter. The Association for Creatine Deficiencies (ACD...metabolic demand that is powered by ATP. A molecule central to the regeneration of ATP from ADP is creatine...the lab observed abnormal cell migration and contraction. The lab hypothesized that this is abnormally...identify whether any rescue cell migration and contraction defects. The lab also plans to use CRISPR plasmids...

...introduce epitope tags and RNA transcription start sites. The lab further demonstrated that PRIME-del can be “...Cas9 fused to the reverse transcriptase (RT), which induces two double-strand breaks and deletes the intervening...engineering the Cas12f scaffold gRNA and making strategic mutations in the DNA binding cavity of Cas12f....Un1Cas12f1 (Cas14). They noted that Cas12f1 had an extra-long gRNA for its small size. They were able to ...editing when delivered in an AAV system, and demonstrated that the new system was able to activate expression...eNcapsidation for cellular Delivery (SEND). They demonstrate SEND as a modular system for packaging, secretion...They further develop a version of SEND for gene transfer with pseudotyped viral particles, expanding options...

...The development of the transient transfection platform for assaying transcriptional activity, for example...origin) that are diffused or actively transported from cells, traverse the body through the bloodstream, ...such as p300 and CBP histone acetyltransferases or CARM1 methyltransferase. This attendant signaling feature...deliberate and compulsory actions instigated by the central nervous system signaling to peripheral effector...ligands NRs represent the largest family of transcription factors found in metazoans. The superfamily ...integration and communication of such signals between central and peripheral organs because of their established...profiling datasets in to our data mining tool, Transcriptomine. We are currently nearing the completion of...

...mitosis, support transport of organelles and vesicles, and more. Although several strategies are available... tool. The authors demonstrated its use to probe cell mechanics, vesicle transport, and cell division,...in mammalian cells used here could be an attractive strategy for engineering other enzymes. Conclusion...like lamellipodia or filopodia formation, cell contraction, or other morphological changes, depending on...tools to control formation of lamellipodia and contractile stress fibers, and recently developed a full ...optogenetic systems (Berlew et al., 2022). Stopping traffic with CIB1-Rabs Instead of using an optogenetic ... blue light to generate CRY2-CIB1 oligomers that trap the fusion protein away from its usual location,...

...vectors have a relatively high transduction efficiency, but in contrast to lentiviral vectors, their infectivity... Viruses are intracellular parasites and natural vehicles for genetic information. Therefore they make...split-vector systems’ by separating viral genes and transgene expression across several plasmids. The most commonly...separate envelope and packaging plasmids as well as transfer plasmids. This concept ensures safe handling and... are very versatile tools as they are able to transduce dividing and non-dividing cells. More detailed...Gamma-retroviral vectors Popular gamma-Retroviral Transfer Vectors   Expressing your gene of interest pMKO...events and is widely used in the field of mouse transgenics. Experimental uses include: Cre-dependent gene...

...behavior. A fact to consider: Rodents can hear ultrasonic frequencies (> 20 kHz, produced from fluorescent...behind from a preceding animal (ex: urine) can be distracting. If it is not cleaned, animals tested towards...experiment series would behave very differently (distracted) from the ones tested at the beginning. Variability...Variability source Standardization parameters Animal Strain, age, sex, housing conditions (individually versus...genes. A popular method uses Cre recombinase on transgenic flox animals to introduce modifications at the...such modification in other areas. An alternative strategy is to knockdown a gene with RNAi. Here, a short...Both AAV and lentiviral vectors are used for intracerebral injections. AAV particles are smaller and may...

...the constraints of antibody labeling. This opens the door to in vivo experiments such as tracking the ... proteins (FPs) traditionally introduced in Caenorhabditis elegans on extrachromosomal arrays can lead... efficient double stranded templates, rather than the more efficient single-stranded oligodeoxynucleotide...3: BaF3 cells were transduced with H2B-FT virus and grown in varying concentrations of IL-3. Cells grown...generally very small, and can contain different neurotransmitters. Using genetically encoded fluorescent proteins...proteins coupled with Fibronectin intrabodies generated with mRNA display (FingRs), Xue Han’s lab has developed... them to localize to the synapse and minimize extraneous fluorescence from other regions of the cell. ...

...biosafety cabinet. For ultracentrifugation, heat sealed tubes can be used for extra security. Finally, when... you’ll need to get trained in each new place (and pay attention to the trainings!), even if you know ...looks like the genetic cargo in one plasmid (the transfer plasmid), the envelope (Env) gene on a separate... plasmid). Viral production then proceeds by transfection of all three plasmids into a producer cell line...HEK-293T, production of viral particles, and transduction of a target cell line with those particles. ...Env, Gag, and Pol genes are not encoded in the transfer plasmid, the component that physically integrates...into the host’s genome. However, some adenovirus strains are known to cause respiratory diseases in humans...

... the transition felt scary. While I knew two people from graduate school who were at AstraZeneca and could...Laura Desrochers, a postdoctoral scientist at AstraZeneca. Why I chose an industry postdoc I’m currently...doing an industry postdoc in neuroscience at AstraZeneca’s Waltham site after doing a brief academic postdoc... industry postdocs have a set time limit. At AstraZeneca, this is three years. This time limit can feel... to enter academia, it is easier to make that transition from an academic setting—especially if job applications... can be helpful. Publishing: As a postdoc at AstraZeneca, you are expected to publish, but this can vary...team as well as their scientific abilities. At AstraZeneca, scientists who get postdocs and do not support...

...products are then transformed directly into chemically competent E. coli and transformants selected with ... for further purification before transformation. After transforming cells, you should perform colony ...foundational elements of biomedical research. Traditional restriction cloning is one of the oldest of these...consisting of digestion, purification, ligation, and transformation. While restriction cloning is still routinely...+ + Gel Purification + - Ligation + - Transformation + + The FastCloning process in brief In ...for this procedure to work properly. Directly transform chemically competent cells with the digested mixture...-cycle PCR leads to the most colonies after transformation. When a 1:1 vector:insert ratio is used in ...

...permeability Bactericidal 10-100 ug/mL Tetracycline tetracyclin Binds 30S ribosomal subunit; inhibits...light-sensitive antibiotic like Tetracycline is used. Keep in mind that some E. coli strains have natural antibiotic...how much easier it makes our lives. Plasmid transformation into E. coli is a fairly inefficient process...reagents can kill bacteria when used at high concentrations (and vice versa). Looking around the lab, you'll....   Name Class Mode of Action*   Working Concentration** Kanamycin aminoglycoside Binds 30S ribosomal...ribosomal subunit; causes mis-translation Bactericidal 50-100 ug/mL Spectinomycin aminoglycoside Binds...Blocks 50S ribosomal subunit; inhibits aminoacyl translocation Bacteriostatic 50-100 ug/mL in EtOH Polymyxin...

...Forster Resonance Energy Transfer (FRET). Through the non-radiative transfer for energy between neighboring...Whether BiFC constructs are expressed transiently by transfection or stably impacts the success of BiFC...a dose-dependent manner. This is straightforward in easy-to-transfect cell lines where plasmid dosage ..., FRET can be used to detect the inter- and intramolecular interactions that underlie protein function..., kinase signaling, integrin signaling, and transcription factor interactions (Kerppola, 2006). One of...interactions between basic leucine zipper and Rel transcription factors (Hu et al., 2002). This would be more...subcellular localization of the studied PIPs. The straightforward nature of BiFC also makes it easier to generate...

...frankenbody can be effectively used to track single mRNA translation in U2OS cells. HA is small, only 9 amino...Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable...based probes the Stasevich lab used an alternative strategy to develop a genetically encoded antibody based...which contains a 10xHA tag. These experiments demonstrated that the HA frankenbody can be used to amplify...minutes in living cells, meaning that scientists can track the movement and localization of single HA-tagged... amino acids, thus it is translated quickly by the ribosome. This feature provides HA frankenbody the ...researchers used the HA frankenbody to successfully track nuclei in a developing zebrafish embryo over an ...

...ready-to-use viral preps   Improved cell-free transcription/translation system to examine biosynthetic pathways... Gabrielle Clouse Recently, cell-free transcription/translation systems (also referred to as TX-TL systems..., the DNA of interest, and cell extract.    Graphical Abstract: Schematic representation of cell-free...the expression of fluorescent proteins and oxytetracycline enzymes. Next, this system was used to recreate...modifications to edit DNA without requiring a double-stranded break. The Xiao Wang Lab has recently deposited.... These plasmids use “Cas9-mediated adenosine transient reporter for editing enrichment”, or XMAS-TREE...modifications and work with them further. The lab demonstrated that XMAS-TREE can be multiplexed and can be...

...C2c3 are related to Cpf1, but they require both a tracrRNA and crRNA to cleave target DNA (Cpf1 requires ... Cas13a binds and cleaves RNA rather than DNA substrates. In terms of structure, Cas13a shares no homology...PAM (PFS) Cleavage Mechanism Cas9 HNH, RuvC TracrRNA, crRNA DNA 5' NGG Specific blunt ended DSB...endogenous CRISPR/Cas9 system, Cas9 uses both a tracrRNA and crRNA to facilitate binding and cleavage of...the crRNA or presence of a PFS. This is in stark contrast to Cas9, which requires that each DNA target have...Recombinase Polymerase Amplification (RPA) or Reverse-transcription RPA (RT-RPA), respectively. Amplified nucleotides...have utilized dCas13 fusion proteins for imaging, tracking, modulating splicing, and regulating expression...

...cytosol (instead of on the yeast extracellular surface) which enables tracking of protease activity with a ...protease allows a transcription factor to enter the nucleus and modulate transcription of the mCitrine ...affinity variants of TEV. The new system has been demonstrated to work for evolution of other target proteins...structures within cells. HeLa cells were transfected with plasmids encoding FPs tagged with the plasma...biotin-4-fluorescein to be dissolved at a higher concentration, and results in uniform NB biotinylation. Once...expression can vary significantly depending on transgene design and delivery. To overcome these variations...reporter. With this system, they could quantify and track cells expressing both dCas9 and gRNAs. The...