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...selectable, it can be transferred to a different genetic background via P1 transduction. Plasmid Expressing...products: 1) a partially dsDNA duplex with single-stranded 3’ overhangs or 2) if the dsDNA was short enough...enough, a ssDNA whose entire complementary strand was degraded. Beta: Beta protects the ssDNA created by...higher recombination frequency when the lagging strand of DNA is targeted, it’s best to determine the ...design an oligo that is complementary to the lagging strand. In practice, it’s easier to not determine the ...replication and instead design oligos that target both strands. One of the two oligos will recombine with a higher...you’ll find out empirically which is the lagging strand. ssDNA substrate is more efficient than dsDNA with...

...Interest (GOI), and transfect it into your favorite cell line. In parallel, you transfect cells with another...antigen - Recognition of an immunogen does not guarantee recognition of the target antigen. You want to...Antibody-independent methods may include analysis of transcriptomic or proteomic data from several different tissues...non-specific, background signal, so you’ll need a range of expression strengths to be confident in your ... feasible and (2) large-scale proteomic and transcriptomic datasets already exist.   Example: You want...tissues. You know that there is a wealth of transcriptomic data from this sample type, so you use your...need to be aware of any possible alternative translation start sites or alternative splicing that could...

...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..., 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...overexpression of fusion proteins it requires can lead to aberrant subcellular localization of the studied PIPs. ...frame (ORF) library to screen PPIs among 450 transcription factors in live fruit flies. Although BiFC open-reading... high background fluorescence levels due to the random association between FP fragments independent of...

...(single vs. double stranded) all depends on the size of the knock-in. Single strand oligonucleotide donors...knock-out. The goal of a knock-out is to disrupt DNA translation by generating a frameshift mutation. The majority...pathway cells already have for repairing DNA double strand breaks – (HR). HR is the dominant homology-directed... Fig. 1: Early steps in repair of a DNA double strand break by homologous recombination.    Designing...have larger homology arms in the 500 bp to 1 kb range, however, shorter homology tracts have recently ...recognition sequence in the donor DNA, you can guarantee there won’t be re-targeting after HDR. If your...cycle regulatory proteins responsible for the transition out of S phase, so cells stay in an HDR-promoted...

...host range and tissue tropism (Borkenhagen et al., 2019). AdVs are non-enveloped double-stranded DNA viruses... protein translation. Its genome consists of “early” (E) and “late” genes, and the transition between ...sequence (transgene) instead.    Although the late genes are usually present in the transfer plasmid for... (and therefore between early and late gene transcription) is demarcated by viral genome replication (...vectorization strategies include creating an AdV transfer plasmid with the E1 and E3 (Early 1 and Early ...cells that are infected by the vector, only the transgene will be expressed, since the early genes are absent...possible to design a vector that achieves persistent transgene expression in certain cell types (Tatsis et al...

...nickase. Nickases create a single-strand rather than a double-strand break, and when used with two adjacent...the target strand. Conversely, the H840A mutation in the HNH domain creates a non-target strand-cleaving...strand-cleaving nickase. Instead of cutting both strands bluntly with WT Cas9 and one gRNA, you can create a staggered...to each other? The gRNAs must target different strands to create a DSB, but this can be accomplished with... benefit of using nickases for HDR is targeting range: using an individual gRNA with WT Cas9, repair levels...experiment to insert an EcoRI site using a single-stranded oligonucleotide (ssODN) donor. Using a D10A nickase...across the 51 nt region using either a top or bottom strand ssODN donor with 40 bp homology arms. The HDR efficiency...

...increase as the DNA amplicon transitions from single-stranded to double-stranded, and finally, drop once more... by abundant transcripts, leading to falsely lowered levels for less abundant transcripts. However, adding...can even be used to detect a single copy of a transcript. But this sensitivity can sometimes cause as ...signals are produced by the presence of double-stranded DNA, when temperatures are high, signal intensity...reflecting the presence of primarily denatured (single-stranded) DNA. As the temperature drops, single intensity...assumption that there is a smooth, single-step transition between ssDNA and dsDNA. This is not true for...effects, cation concentrations and a temperature range) to predict PCR melting curves and dynamic melting...

...References: Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY (1999) Rett syndrome is caused...collection of molecular biology tools so that both veteran and new Rett researchers can find the reagents ...maturation (Tillotson & Bird, 2019) and is a global transcriptional regulator of thousands of genes. Studies have...have suggested roles in transcriptional repression, activity dependent de-repression, chromatin remodeling...groove of AT-rich DNA sequences to regulate transcription (Baker et al., 2013). Mutations that affect ...gene editing, MECP2 reactivation, RNA editing, RNA trans-splicing, and protein replacement to reverse the...in animal models, Rett syndrome is a strong translational candidate for gene replacement therapy. In 2014...

...the Netherlands. Transformation of HEK cells with adenovirus 5 DNA was postdoc Frank Graham’s 293rd experiment...endothelial, epithelial, or fibroblast. However, Frank Graham showed HEKs display many immature neuron-...with kidney cells (Shaw et al.). Since then, transcriptome studies of the cell line have determined it ...replication. To generate a virus, you need only transfect the ‘helper’ plasmids (viral packaging machinery...cell or organism. This pipeline is utilized to transduce cell lines for basic science research all the ...Protein production  HEKs are extremely easy to transfect in bulk and produce recombinant proteins at high...cell line model for cancer-specific research. Membrane receptors and ion channels are often exogenously...

...three different membrane-targeting flavors – PDGFR, GPI, and SGZ, to improve neuronal membrane trafficking...properties such as kinetics, ∆F/F0, SNR, dynamic range, photostability, quantum yield, and expression. ...segment (PDGFR TMD) that anchors the sensor to the membrane and displays it on the extracellular side. In .... PDGFR is a C-terminal fusion to the PDGFR transmembrane domain in the mammalian expression pMinDisplay...glycosylphostidylinositol anchor. SGZ contains a PDGFR transmembrane domain, followed by a modified form of the cytosolic...fluorescent probe for visualizing glutamate neurotransmission.” Nature Methods (2013) PubMed PMID: 23314171...kinetics and localiation for imaging synaptic transmission” bioRxiv (2022)...

...use of pre-existing transgenic lines, termed BLITZ (Biotin Labeling In Transgenic Zebrafish) (Xiong, et...“single-use” transgenic lines. Instead, researchers can generate and maintain transgenic lines that can...protein by a specific transcription response element (TRE) or kinase translocation reporters (KTRs). Find...interest and, for in vivo work, generate a dedicated transgenic line. Using zebrafish as a model organism, the...protein can then be co-expressed in essentially any transgenic zebrafish line expressing a GFP-tagged version...generational evolution of the AAV capsids (left) and transduction patterns (right).  Both AAV-PHP.eB and the AAV.CAP-B...target. Across all loci tested, error rates overall ranged from 0.3% to 10%, in comparison to the 62-90% error...

...been able to successfully systemically transduce and then retransduce a model organism such as mice, e.g....potential strategy is to use split-inteins to mediate trans-protein splicing. Note, however, that this will ... at a high MOI, often resulting in a drop in transduction coverage compared to just one vector.   Q2: ... Q4: Are there any AAVs that have successful transduction in microglia? I have been using the AAV.PHP....information.   Q6 & Q7: We have been attempting to transduce mouse liver via tail vein injections but would...capsid was reported for specific and potent liver transduction. While we have not tried it, it might be worth...Animal Models Q9: Is there any difference in transfection efficiency when delivering these viruses via...

...-HA frankenbodies include mEGFP, mCherry, and HaloTag. Figure 1: (a) Anti-FLAG frankenbody (FB)...Here's what you'll find in this post: Anti-FLAG Frankenbodies for modular imaging of cellular targets Optimized...mammalian genome regulation and editing Anti-FLAG Frankenbodies for modular imaging of cellular targets by: ...: Mike Lacy Adding to their set of anti-HA frankenbodies (Zhao et al. 2019), Tim Stasevich and Hiroshi... have recently published a set of anti-FLAG frankenbodies, a flexible, modular tool for labeling. Here...variety of applications. This set of new anti-FLAG frankebodies includes mEGFP, mRuby2, iRFP713, SNAP-tag, and...Liu et al. 2021. Anti-FLAG and anti-HA frankenbodies have a variety of applications, including live-cell...

... engineered to detect a wide array of neurotransmitters ranging from dopamine to glutamate, all the way... or animal system. This ‘thing’ could be a neurotransmitter, such as norepinephrine, or an electrolyte...last component of the biosensor is the signal transducer which will quantitatively give a readout for ...bio-recognition event is (fluorescence, pH, etc.). The transducer will generate a numerical value which represents...activated GFP expression (receptor), then the transducer could be a microscope, plate reader, or any other...—keep reading to find out how!   Neurotransmitters Many canonical fluorescent as well as FRET-based...fluorescence-based and have been optimized for a wide-range of uses to monitor the fundamental cellular building...

...one they have a high transduction efficiency and can effectively infect a wide range of cell types that ...used to achieve transient, high expression of the relevant cargo without the fear of random genomic integration... Packaging Capacity (>8kb) High Transduction Efficiency Transient Gene Expression High Immunogenecity...viral envelope, which fuses with the target cell’s membrane, allowing the viral particle access into the cell...that are typically ‘difficult to transfect’, such as primary cells. They can also be directly injected into...Immunogenicity Infects Non-Dividing Cells Transient Gene Expression Low Tropism for Mouse Cells and...out How to Get the Most from Your Lentiviral Transduction Additional Resources on Addgene.org Browse...

... it will proceed to generate a blunt DNA double strand break. Cas9 has been studied for genome engineering...an expanding number of others. Best for: a wide range of DNA edits.   Cas3 – The shredder What sets Cas3...locates its target sequence and initiates single stranded DNA degradation. Cas3 DNA digestion can proceed...convenient for targeting experiments requiring single strand DNA generation, such as homology directed-repair...therapeutic delivery.   Cas14 – Mini, but single strand mighty Unlike many other Cas enzymes, Cas14 targets...This makes it particularly useful in generating transient edits of signaling molecules (RNA) as opposed ...for targeting mRNA and useful as a mechanism of transient knockdown of gene expression. This system could...

... to create a complete transcription factor, activating luciferase transcription. Tang et al. coined this...Connie Cepko’s lab allow researchers to activate transcription or Cre recombinase activity only in the presence...Using nanobodies to create GFP scaffolds for transcriptional activation The project began with a frustration... in their 293 cell culture system. In cells cotransfected with GFP and the GBPs, GFP linked the two GBPs...this system T-DDOG (Transcriptional Devices Dependent On GFP). In testing the specificity of their system... dsRed, mCherry, and TdTomato do not induce transcription. Thus, T-DDOGs can be used in combination with...T-DDOG results showed that GFP could regulate transcription in a cell-type specific manner, and Tang and...

...remove one strand of the siRNA and are directed by complementarity to the remaining single stranded siRNA ...activity or their translation disrupted through physical blocking of the translation machinery. In the...WISH) for visualizing expression of genes. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) to... by T7 promoters to allow for bidirectional transcription to create dsRNA. Students can produce DNA templates...the temporal and spatial localization of mRNA transcripts in tissue. This is useful for labeling specific...mixing with the liver paste, these E. coli are transformed with the appropriate plasmid and dsRNA expression...Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391 (1998...

...into… Transcription units (TUs), which are functional units composed of multiple parts transcribed together...a protein that regulated its own transcription, or the transcription of another gene? Now you have a genetic...As a plasmid repository, we are no strangers to cloning. If you’re reading this, then chances are you...hierarchical levels: individual DNA parts, transcriptional units, and multigene constructs. Let’s start...start by examining individual parts and transcriptional units. Individual parts are defined DNA sequences...assembled into larger functional units which are transcribed together. Image courtesy of Robert Hurt. ... with DNA, and these complexes regulate the transcription of specific genes. What if one of those genes...

...xCas9 for transcriptional regulation and base editing Hu et al. also engineered transcriptional activators...activating transcription. Sites containing NG, GAA, or GAT PAMs averaged 56-91% transcriptional activation... to create xCas9, an enzyme recognizing a broad range of PAMs like NG, GAA, and GAT, but also displaying...subunit ω. This construct is designed to activate transcription from the accessory plasmid (AP) encoding phage...could recognize a library PAM, it would induce transcription of gene III, permitting phage propagation and...spectinomycin resistance. xCas9 3.7 showed the broadest PAM range, recognizing NG, NNG, GAT, and CAA PAMs, while xCas9... xCas9 3.6 displayed the second best PAM range. Characterizing xCas9 in mammalian cells Hu et al. compared...