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...the transient cotransfection of the mARG plamsids into HEK293T cells using a chemical transfection reagent... These plasmids can be used directly in transient transfection in rtTA+ cell lines (such as HEK293-TETOn...into the genome by cotransfecting them with a plasmid expressing PiggyBac transposase. In rtTA-cell lines...engineering a cell line, cotransfect the mARG cassettes 2:1 (A:N-V) with PiggyBac transposase and outgrow the ... of replication (p15A) worked best under a wide range of inducer concentrations (0.1-1% L-arabinose) without...proteins. Excess gvpA plasmid added into the transfection mixture in HEK293T cells gave drastic improvement...improvement in expression. Stabilizing the gvpA transcript with WPRE reduced the number of copies of gvpA ...

...through transformation, so the cloned vector must be ligated. But in LIC, longer single-stranded overhangs...T4 polymerase (left branch); by annealing two separate PCR products (middle branch); or by incomplete ...step to retain products with single-stranded overhangs (right branch). The mixed PCR and incomplete PCR...overlapping regions of single-stranded DNA. But as long as the double-stranded region is long enough to hold...nicks easily repairable by the bacteria after transformation. LIC is a reliable cloning method, but it is... sequences (20–60 bp) on either end and single-stranded 5′ overhangs… but it doesn’t really matter if ...recombination intermediate to bacteria, so it can be transformed as-is into cells. The cells then get to work ...

...neurons! Neurons are really cool because they can transmit information, for example, from one of the 180 ...associated virus (AAV) shows relatively low retrograde transport efficiency in most serotypes. Some viruses (e....e.g., rabies) naturally exhibit retrograde transport and can therefore perform retrograde tracing, but...efficiently infects neurons, directs high-level transgene expression, and has retrograde functionality to...genetically controlled function. By using AAVrg in Cre transgenic mouse lines, you can access and manipulate an...from the hippocampus to Broca’s area would be transduced (Fig 3B). These specific neurons could then be...requires high titers for efficient retrograde transport. For recombinase-dependent vectors, this can cause...

...the opposite strand). ABEs make an A to G change (or a T to C change on the opposite strand). Both are ...place on the free strand of DNA and converts a C to U without producing a double strand break. UGI = uracil...Repair CRISPR 101: Cytosine Transversion Editors CRISPR 101: Any Base Transversion Editors Learn about base...repair (HDR), the pathway for resolving DNA double-strand breaks (DSBs) preferred by researchers. Compared...are base transition editors: converting one purine base to the other or one pyrimidine base to the other...other. Figure 1: Base transition editors catalyze purine-to-purine or pyrimidine-to-pyrimidine base... indicate the change introduced to the opposite strand following base editing and DNA repair. Black arrows...

...for broad or narrow range of microbial species. These also contain the RP4 transfer origin as well as a..., the ability to detect mRNA transcripts of toxins and other transcripts of interest is a benefit of this...the organisms present while sequencing of all transcripts gives clues into what the microbes are doing....followed by a reporter gene. The hairpin blocks translation by sequestering the RBS, but binding of the “...microbial community) reveals the RBS and allows translation of the reporter gene resulting in GFP signal....lacks genetic tools is the bee microbiome. Nancy Moran’s and Jeffrey Barrick’s labs at The University of... RSF1010 origin, an IncQ origin with broad host-range (Meyer, 2009). The vector retains the oriT for delivery...

...therapies and in vivo laboratory experiments ranging from transient gene expression in a particular cell type... Figure 2: DNA rescue and transduction of a conventional single-stranded AAV (ssAAV) and a self-complementary...genome conversion in the transduced cell nucleus, the single-to-double stranded conversion of the DNA goes...structure can readily fold into transcriptionally active double-stranded form through intra-molecular annealing...Adeno-associated virus (AAV) is a single stranded, Parvoviridae DNA virus, packaged in a non-enveloped...hijack host cell machinery, and synthesize a second strand of DNA before expression of their cargo can begin...integrate into the host genome and therefore the transduced gene is episomal and does not divide and replicate...

...generally are not transmitted to adjacent neurons via synapses (i.e., transmitted transsynaptically). This is ...retrograde transmission (Etessami et al., 2000, Wickersham et al., 2007). Thus, RABV transsynaptic spread ...replicate. Modified Rabies Virus: Converting a transsynaptic neuronal tracer into a monosynaptic (first order...subsequent cycles of viral replication and retrograde transmission occur, spreading the virus through higher and...higher order neural connections. While this transsynaptic spread is useful for neuronal tracing, it has...-dependent TVA to Cre-expressing neurons in a transgenic mouse line. By doing this, RABV expressing the...necessarily express EnvA unless it is supplied in trans. Monosynaptic RABV spread can be achieved by modifying...

...inducible gene expression. Transgenic and knockin mice As the name suggests, transgenic and knockin mice contain...their genome. While transgenic mice have the new gene construct incorporated randomly into the genome, knockin...Conversely, outbred strains were originally derived from random crosses between mice of uncharacterized backgrounds...several types of genetically engineered mice: transgenic mice, knockout mice, and mice with conditional...construct inserted at a specific site. To generate a transgenic mouse, microinjection of DNA or infection with...Non-homologous recombination incorporates this DNA randomly into the mouse genome. In contrast, knockin strains...animal for gestation. Nucleases that induce double-stranded DNA breaks, such as ZFN and TALEN, can used to...

.... They can be targeted to the plasma membrane to measure membrane potential and can also be directed to... the membrane), the voltage sensing component (i.e., rhodopsin naturally resides in the membrane), or ... of voltage gated ion channels all depend on transmitting and sensing electrical potential. Thankfully... want to monitor the voltage change across the membrane of cardiac cells. Similarly, neuronal signaling...propagated through multiple cells to do things like translate perceived signals to physical actions. Understanding...rhodopsin proteins were initially evolved for ion transport, not fluorescence, they are not nearly as bright...sensors contained a FP that localized to the plasma membrane due to the addition of several targeting domains...

...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...

...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 ...

...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...

...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 ...

...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...