...discovered in positive single-stranded RNA viruses to allow for the efficient translation of multiple genes from...Elements Translation in eukaryotes usually begins at the 5’ cap so that only a single translation event ...scientists use a multitude of techniques including co-transfection of two or more plasmids, the use of multiple...Unlike promoters which will create unique mRNA transcripts for each gene that is expressed, multicistronic...your gene as they are both derived from the same transcript. Of course multicistronic vectors do not have...from a single transcript. The two strategies most widely incorporated into plasmids for research purposes...Entry Site (IRES) to allow for initiation of translation from an internal region of the mRNA. In the ...

...recent paper, "Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds." Adam has...visualize the concepts discussed in the paper. The transcriptional control of multiple loci is deftly coordinated...cerevisiae with few techniques that are easily transferrable across host species or metabolic pathways. A...CRISPR-based RNA scaffolds to mimic natural transcriptional programs on multiple genes. Modifying the ...synthetic RNA scaffolds that can recruit various transcriptional regulators (Zalatan et al. 2015).  These scaffolds...nucleotide sequence then subsequently recruit a transcriptional regulator to modulate expression at that locus...expression levels dependent upon the type of transcriptional regulator or the number of designed scaffold...

...:     Cells must be amenable to transfection or viral transduction     Appropriate promoters must be...expression     The requirement for Cas9 transcription and translation delays editing What are Cas9-gRNA ... studies in cells that are difficult to transfect or transduce. While using Cas9-gRNA RNPs may require...site of interest. This is commonly performed by transfecting cells with a plasmid, such as PX459, which encodes... often be used in cells that are difficult to transfect, such as primary cells. Using RNPs can also alleviate... are detectable at high levels shortly after transfection, and are quickly cleared from the cell via protein...from Alex Schier’s Lab. gRNAs can be in vitro transcribed from ssDNA, which can be generated by commercial...

...contains 11 β-strands and one central ɑ-helix that can be split into 3 parts: (1) β-strands 1-9 (β1-9) and... and the one ɑ-helix, (2) β-strand 10 (β10), and (3) β-strand 11 (β11). Each part contains components ...developed, such as the Forster resonance energy transfer (FRET)-based executioner caspase reporters. In...need a protease reporter that has a large dynamic range and high brightness. The Shu Lab from UCSF designed...However, the lab wanted to increase the fluorescence range to better study the spatio-temporal dynamics of ...GFP (GFP1-9 and GFP10-11) by flipping a single β-strand of GFP, hence the name FlipGFP. FlipGFP increases...from β1-9 they will rapidly recombine with β1-9 strands nearby resulting in fluorescence within tens of...

...humanized wt Cas9, dCas9 (transcriptional repressor), and dCas9-VP64 (transcriptional activator)-containing...Multiplexing from a single transcript You can also multiplex gRNAs via a polycistronic transcript. Rather than being...being transcribed from different promoters, the gRNAs are transcribed together and are flanked by specific...simultaneously. Vectors for both transient expression and Agrobacterium-mediated transformation are available. For ...system for processing a continuous multi-gRNA transcript, each gRNA must be expressed from its own promoter.... But that doesn’t mean you have to clone and transfect multiple promoter-gRNA constructs in order to ... Cas9, D10A nickase mutant (Cas9n), dCas9 (transcriptional repression), and Fok1-dCas9 (dimeric nuclease...

...but also a biological transposon system. Sleeping Beauty is a synthetic transposon system that can be used...reconstructed a transposase system based on the naturally mutated and non-functional transposase gene found...resurrecting this transposon system? Scientists can use Sleeping Beauty transposons as a non-viral system...Addgene recently moved to our brand new location, with more space to grow and help scientists share resources...you can use to study these genes! Groucho, a transcriptional co-repressor The first conference room, that...Addgenies, was named after the protein Groucho, a transcriptional co-repressor important in development. Groucho.... It binds to a specific set of DNA-binding transcription factors to repress the expression of genes primarily...

...opportunity to discover and evolve your transferable skills. Transferable skills are the non-technical skills...employee who's coming directly from grad school. The transition has been a bit rough for her (3 weeks in and ...there are any resources that can help with the transition.” The lab-to-desk-job culture shock is a real...scientist colleagues have as well. Making the transition from the bench can be jarring for the unprepared...resources out there for scientists making this transition. In this installment of the Addgene career guide...brief period of time, the only, person to know a brand new fact is often enough to keep a scientist going...formulate hypotheses and conduct experiments. You transfer liquids between tubes, look through microscopes...

...required for hok translation. Here, Mok and Hok translation is coupled. Translation from the Shine-Dalgarno...more stable than the methyltransferase. When a plasmid is lost, the methyltransferase is degraded and/or...systems have been used as biological tools for a wide range of applications in molecular biology or in vivo ...ccdB gene die off and a higher percentage of transformants contain the ccdB-less construct. Other toxin-antitoxin...composed of restriction endonucleases and methyltransferases. Restriction endonucleases cleave DNA at ...specific nucleotide sequences. To counter this, methyltransferases add methyl groups to bases at specific nucleotide...or diluted as the cell grows resulting in the appearance of unmethylated sites in the chromosome. The ...

...different ligands, including hormones, neurotransmitters, photons or odorants, to elicit downstream signaling...seven-(pass)-transmembrane domain receptors, due to the seven alpha helices that transverse the cellular...cell membrane. Heterotrimeric G proteins are important molecular switches in signal transduction pathways...fusion to a receptor-transcription factor fusion. TEV protease cleave the transcription factor from the receptor...initiating signaling pathways in inflammation, and neurotransmission. These receptors can be activated by an array...cellular membrane. The receptor recognizes the appropriate ligand to activate the G proteins. It is estimated...parallel receptorome expression and screening via transcriptional output - TANGO) (Kroeze et al., 2015). PRESTO-TANGO...

...increases when target DNA strand separation is stabilized (2,3). Stable strand separation is maintained...and therefore destabilize basal strand separation. This decreased strand separation combined with the weak... Figure 1: Strand separation and target DNA binding to the Cas9 nuclease. Stable strand separation is...the non-target strand and the HNH/RuvC groove, and interactions between the target strand and the gRNA....interactions : 1. Interactions between the non-target strand and a positively charged groove formed by the Cas9...nuclease domains 2. Interactions between the target strand and the gRNA (see figure below) Because off-target...by a technique for detecting genome-wide double strand breaks (BLESS), revealed that these mutants do ...

...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...

...quantify RNA transcription by RNA Polymerase III. By expressing Corn under promoters for transcripts uniquely... and orange fluorescent aptamers Corn was described in the same study as Red Broccoli and Orange Broccoli...fluoresce in the red and orange channels, respectively. Both Red Broccoli and Orange Broccoli are derived ...multiple derivatives of thiazole orange (TO) and fluoresces in the orange or red channel, depending on the...aptamers are used to track cellular processes by transforming cells with plasmids encoding aptamer-tagged ...tagging RNA sequences with an aptamer and RNA transcription by placing the aptamer downstream of a known...hydroxybenzylidene imidazolinone (DFHBI). DFHBI is membrane permeable and non toxic to cells. Because it rapidly...

...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...

...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...

...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...

...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...

...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 ...

...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...