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...new class of sensors for neuropeptides and neurotransmitters. Motivated to develop a biosensor for oxytocin...devised a “Modular fluorescence unit fused with TRansmembrane region-to-IntrAcellular loop linkers” (MTRIA...sensors based on GPCRs recognizing various neurotransmitters, neuropeptides and other ligands - almost ...kinetics and localization for imaging synaptic transmission. bioRxiv 2022.02.13.480251. doi: 10.1101/2022.02.13.480251...2022.02.13.480251 JEDI-2P voltage sensor The François St-Pierre lab and colleagues have developed a new...they observe fluorescence localizes to the plasma membrane only when the RTK is activated and the ITAM phosphorylated...relies on a change in localization to the cell membrane and not a direct change in the fluorescent properties...

...single targeting strand, with the extra strand usually being degraded. The target strand binds to the mRNA...mature siRNA. siRNAs are delivered via transient transfections and are handy for quick in vitro experiments...gene is achieved through the delivery of a double-stranded RNA (dsRNA) that matches the mRNA target sequence...delivered as any type of small RNA and can occur via transfection or viral delivery of a plasmid. RNAi is an umbrella...processing (Figure 1). The shRNA sequence is transcribed to produce hairpin shRNA structures that are ...fact! Endogenous miRNA production starts with transcription of the miRNA to create pri-miRNA, which is then...the mRNA — the bound miRISC can simply block translation machinery. The composition of the RISC varies...

...errors like uneven pipetting or irregular membrane transfer. Figure 1: A loading control is a type...lies the linear detection range. Figure 4: The linear detection range of the assay is between the.... No pipetter is perfect, and proteins may not transfer precisely evenly across the entire blot. So how...directly correlate to protein abundance on your membrane, which would be directly reported by band intensity... in the loading control are due to loading and transfer errors — not to any factors from before the proteins...control points are outside of the linear detection range. As a result, they have almost the same value on...computational quantification. Outside the linear detection range, band intensity measurements become extremely susceptible...

...permeabilize the cell membrane to allow antibodies to pass through the cell membrane. Formaldehyde fixation...dye emits green, yellow and orange photons while PE emits yellow and orange photons. If FITC and PE were...sources that allow users to assess a cell’s size, granularity, and target protein composition. It is used for...target protein expression levels, assessing post-translational modifications, determining cell health, analyzing...a pressurized buffer called sheath fluid and transported through tubes or capillaries to a laser. As the...degree of internal complexity such as extensive membranes produces a greater SSC.  Protein expression, measured...targets, measured by fixing and permeabilizing membranes For intracellular protein targets, users must ...

...produced at high titers, can accommodate large transgenes, transduce quiescent and dividing cells, and do not..., which results in the death of transduced cells and loss of transgene expression (Interestingly, the ...adenoviral genome. The genome itself is a linear double-stranded DNA of ~36kb, with inverted terminal repeats (...E3 regions and can accommodate up to 8.2 kb of transgene DNA. The second generation vectors are deficient...cloning capacity is limited to 8.2 kb, and in vivo transgene expression ceases relatively quickly due to immune... the E1 gene from the packaging cell line is transferred into the AdV by recombination. It is therefore...adenovirus is required to provide the viral proteins in trans. The Gutless AdV have generated high interest for...

...lateral”) gene transfer. Horizontal gene transfer can occur naturally through transformation (cellular uptake...? Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Transfection Resources on Addgene.org...environment), transduction (viral infection by bacteriophages), or conjugation (direct transfer from one cell...one popular method scientists use to promote transformation, but it isn’t just for lab specimens — there... evidence that lightning can also promote gene transfer in soil bacteria! (Demanèche et al., 2001) Beneficial...humans only noticed last century! Nevertheless, the transfer of plasmids carrying these genes between bacteria...plasmid are now used routinely in biotechnology to transform plant cells. Degradative plasmids: microbial hardiness...

...Tethering dCas9 to the histone acetyltransferase p300 or the DNA methyltransferase DNMT3A allows researchers...affect transcription independent of their enzymatic activity. They interact with transcriptional machinery...on DNA methyltransferase 1 (DNMT1) to copy methylation marks from parental to daughter strands of DNA....specifically edit methylation sites.  Step aside, transcriptionally active dioxygenase  When I started to work...by distinct proteins and result in unclear transcriptional consequences (Sprujit 2013). Enzymatic domains...in the genome and directly interacting with transcription machinery.   Well, I’ve got some good news...rounds of cell division, the original methylated strand is diluted to undetectable amounts: ergo, DNA demethylation...

...mouse cell type specific transcription factor binding sites interspaced with random sequences.   Strategy...synthetic promoters. Legend: TSS: transcription start site. TFBS: transcription factor binding site. Image from...particular cell type is important not only for translational research, such as gene therapy, but also basic... using naturally occurring promoters to drive transgene expression, the Roska lab built synthetic promoters...conserved sequences that are upstream of the transcription initiation sites of at least two genes with ...designed using stretches of DNA that were transcriptionally active in retinal cell types in mice. ...synthetic promoter driving expression of the same transgene: an optogenetic reporter, channelrhodopsin, fused...

...BioRender.com. Transformation Grade DNA (low quality/low quantity) – Bacterial transformation protocols require...highest-competency cells you will still struggle to get transformants if the plasmid you are using is completely degraded...important reactions like PCR or restriction digests. Transfection Grade DNA (high quality/high quantity) – Depending...Depending on the experiment, mammalian cell transfections can require quite large amounts of DNA (easily...carry-over into the eluate, while other kits are guaranteed to yield endotoxin-free DNA. Once you know the...kits are designed to accommodate specific volume ranges of culture as their input (Table 1). When you feel...addition to standard purification kits, there’s a range of specialty kits available for plasmid purification...

...prevent efficient transcription from sgRNA targets. CRISPRi is effective in a narrow range around the transcription...two sets of sgRNAs; A, the top 3 ranked sgRNAs, and B, the next 3 ranked sgRNAs. Dolcetto was able to discriminate...Cas9 derivatives. Unmodified Cas9 causes double stranded DNA breaks that commonly result in a complete ... lab selected sgRNAs in this optimal window and ranked them based on their optimized sgRNA rules and potential...gene expression, dCas9 can also be used for transcriptional activation. Activation of specific genes allows... strategies have been developed to recruit transcriptional machinery to dCas9 and its RNA directed binding...include RNA domains that recruit additional transcription factors. The Root and Doench labs utilized tracrRNAs...

...and membrane integrity (Fig. 2). For example, saponin interacts with cholesterol in cell membranes and...can lose membrane-associated proteins, but it is better at permeabilizing organellar membranes. Created...red lines), which maintains structural epitopes (orange region), but it can block epitopes hidden within...proteins that need a little help crossing cell membranes as they are not able to diffuse across natively...Permeabilization essentially punches holes in the cell membranes of your sample to allow antibodies better access...target is already on the extracellular side of the membrane, then this step may not be necessary. In fact,...and leaves many membrane-associated proteins in place. In contrast, Triton X-100 and Tween-20 are examples...

...Biological Material Transfer Agreement (UBMTA) is one of the most commonly used ones for transfers between academic...for more information. Happy transferring!    Many thanks to our Tech Transfer team members Maura Coughlin...Addgene, then you’ve been involved in the material transfer agreement (MTA) process. Even though this process...curious about the hows and whys of the material transfer agreement, read on!  What is the MTA?  An MTA...the creation of a standardized agreement on the transfer of biological materials in 1995: the UBMTA. Because...most academic and nonprofit institutes, the Tech Transfer department is responsible for managing research...plasmid with Addgene, your organization’s tech transfer office is sent a deposit agreement. The One-Time...

...also incorporated LOV-Turbo into TransitID experiments.  Find TransitID plasmids here! These two developments...genetic cargo Optogenetic proximity labeling and TransitID Tracking cellular history with protein ticker ...in vitro and in vivo. Incorporating reverse transcriptase (RT) variants, PE6a and PE6b are 500–800 bp ...be tethered on the viral surface, expanding the range of targetable cell types. DIRECTED is compatible...37612276.   Optogenetic proximity labeling and TransitID By Mike Lacy Proximity labeling can identify protein...plasmids here! Next, Qin and Cheah et al. described TransitID, where two proximity labeling enzymes, TurboID...catalyzing the biotinylation of nearby proteins. B) TransitID uses sequential labeling by TurboID and APEX2 ...

...of Pennsylvania Vector Core. Transfections are performed using the transfer plasmid, a plasmid encoding...PCR with primers targeting the transfer plasmid used in the transfection. PCR products are analyzed by ...cell cultures are inspected for sterility. Transducibility Some viral vectors are tested in vitro and ...images).     Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre (55634...filtration through a 0.45 µm polyethersulfone (PES) membrane. The preparations are then aliquoted, frozen, ...from Wang et al., 2018). Briefly, 293T cells are transduced with serial dilutions of a lentiviral vector,...taken offline and discarded. Confirmation of the transfer plasmid Addgene uses a rigorous barcode matching...

...fused to the N-terminus of LSSmOrange pLSSmOrange-C1 Orange Mammalian Express a gene of interest fused...GFP variant) commonly used with mRuby2 pLSSmOrange-N1 Orange Mammalian Express a gene of interest fused...Bioluminescence Resonance Energy Transfer CRET= Chemiluminescence Resonance Energy Transfer    Interesting Articles...FRET, aka Fluorescence (Förster) Resonance Energy Transfer, technique which allows the detection of molecules...distance-dependent physical process by which energy is transferred non-radiatively from an excited molecular fluorophore... (the acceptor) by means of intermolecular long-range dipole-dipole coupling." The emission of the acceptor...fused to the C-terminus of LSSmOrange mRuby2 Red Mammalian Expresses mRuby2 (a RFP variant) commonly used...

...flow of electric charges across cell membranes and maintain membrane potential in response to visible light...across the axon's membrane: once a certain number of positive ions crosses the cell membrane, a threshold ...rhodopsins enable ions to flow across the axon’s membrane, thereby controlling neural activity (Bernstein...the opsin into the correct orientation to be transcribed (Figure 2) (Sharma and Zhu, 2014). How does FLEx... optogenetic FLEx vectors in combination with transgenic animals or rAAVs expressing Cre under a specific...the mechanisms of sleep and anesthesia. In Cre transgenic mice, they targeted the dopamine (DA) neurons... vectors combined with the use of several Cre transgenic mice targeting different brain areas enabled ...

... technologies have transformed the palette of experiments possible in a wide range of organisms and cell...genomic locus (CRISPR-ChAP-MS) could further transform the range of experiments imaginable in C. elegans. ...Genome Editing with Short Single-Stranded and Long, Partially Single-Stranded DNA Donors in Caenorhabditis...at 25 ºC. This method resulted in efficiencies ranging from 11-100% of F1 animals carrying precise knock-ins...This stringent selection allowed me to optimize a range of editing parameters: oligo repair templates with...with homology arms of 35-80 bp, and DNA double-strand breaks up to 54 bp from the desired insertion site..., they devised guidelines for designing single-stranded repair templates (Farboud et al., 2019).   The...

... pair has focused considerable attention on transcription factors, starting with an established method... relies on antibodies to recognize and bind transcription factors directly, but that’s a slow-going process...ENCODE project is to look at all 1,500 or 1,600 transcription factors and figure out where they bind to the... ChIP-seq, but it’s really hard for lots of transcription factors. Our groups got together to adapt CRISPR...big difference. We can now look at many more transcription factors. Addgene: How does the system work? ...second plasmid to target an epitope tag onto the transcription factors. By the way, you can actually use this... in all kinds of different assays, not just transcription factors. The second vector is a homology directed...

...A double-strand DNA break occurs within the core spacer of each loxP site and the two strands are ligated...maintained).  Translocation: If the sites are on separate DNA molecules, a translocation event is generated... expression – placing a series of transcriptional and translational termination features with loxP sites... pun) specific, targeted DNA modifications in transgenic animals, embryonic stem cells, and/or tissue-...that there is virtually no chance of it occurring randomly. Therefore, inserting loxP sites at deliberate...Inversion: If the loxP sites are on the same DNA strand and are in opposite orientations, recombination...research tool, especially in the field of mouse transgenics. A few of the most common uses are listed below...

...et al., 2018). Unbiased, random insertions can be generated with a transposon-based strategy (Sheridan...fluorescent fusion proteins: random insertion of GFP with an in vitro transposition reaction." BMC neuroscience... This type of interference is known as steric hindrance. For instance, GFP may occlude catalytic sites...sites or motifs that are necessary for post-translational modifications. Despite these potential issues...targeted to the endoplasmid reticulum based on the appearance of OSER whorls. This assay is quantitative and...various fluorescent proteins (Botman et al, 2018; Cranfill et al, 2016; Lambert, 2019). These characteristics...applications, many were concerned with steric hindrance by the fluorescent protein. Thus, scientist began...