...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...

...-1 Veres A, Gosis BS, Ding Q, Collins R, Ragavendran A, Brand H, Erdin S, Cowan CA, Talkowski ME, Musunuru... efficacious to target the coding or non-coding strand of DNA. As a rule of thumb, we avoid target sites..., 2020). is not limited to single nucleotide transitions but still requires a nearby PAM, although these... modulating gene expression at the level of transcription – CRISPRa (activation) and CRISPRi (inhibition...most-efficacious to target a ~100nt window upstream of the transcription start site (TSS), while for CRISPRi, a ~100nt...of producing the guide (synthetic, in vitro transcription, or lentiviral delivery) can affect the accuracy...examined whenever possible when using knockout or transcriptional modulation approaches. Avoiding off-target ...

...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...

...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...

..., and deletions) without introducing DNA double-strand breaks (Anzalone et al., 2019; Chen & Liu, 2023...typically a Cas9 H840A nickase fused to a reverse transcriptase) together with a prime editing guide RNA (pegRNA...containing a primer binding site and a reverse transcription template (RTT). The design of the pegRNA has...provides users with different designs of pegRNAs, ranked by their PRIDICT/PRIDICT2.0 score. For the best...editing strategies) based on a DeepSpCas9 score ranking (Kim et al., 2019). PRIDICT saves time and resources...doi.org/10.1016/J.CELL.2013.07.018 Anzalone, A. V., Randolph, P. B., Davis, J. R., Sousa, A. A., Koblan, L....Search-and-replace genome editing without double-strand breaks or donor DNA. Nature, 576(7785), 149–157...

...number of transformants); the greater the number of inserts, the lower the number of transformants. To compensate...containing the transformants onto the selection plate. Figure 2 shows representative transformation plates obtained...high levels of transformants and low backgrounds – so much so that there was little range for detectable...the number of transformants while maintaining high levels of correct assemblies) translate to faster assembly...background by antibiotic selection. Screening of transformants by colony PCR confirmed the insertion of the...volume of the 1 ml outgrowth spread on each transformation plate can be manipulated to result in appropriate...

...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...

...serotype. Additionally, we check viral transgene expression in transduced cells whenever possible. This post... for transgene expression. Once packaged in AAV, this pre-recombined DNA could lead to transgene expression... to these expected hits, we always get hits to “random” genes. Do these hits always mean the sample is...wanted to see how low we could go. We prepared a range of spiked samples from 0.1-20% and discovered that...find the optimal dose that allows for sufficient transgene expression but limits the likelihood of recombinase-independent...pioneering the systematic use of this new QC assay to guarantee that you receive the best AAV vectors for your...Broucque F, Robin C, Hebben M, Merten O-W, Blouin V, François A, Redon R, Moullier P, Léger A (2015) Advanced...

...can be gained by understanding the location and transport of the protein within a living cell. Analyzing...Analyzing differences in protein localization and transport between healthy and diseased states can also provide...endoplasmid reticulum, modified in the golgi and then transported in vesicles via the secretory pathway to their...the end of their life cycle, proteins may be transported through the vesicles to the lysosome where they... of an organelle or involved in intracellular transport pathways, it is possible to map the internal organization...this case the cell needs to be fixed and the cell membrane permeabilized in order for the antibodies to reach...used markers for major mammalian organelles and transport pathways. It is best, though not always possible...

...genome edits. But have you heard of another double-stranded break repair mechanism: MMEJ (microhomology-mediated...primary methods for repairing DNA after a double-stranded break. HDR copies the sequence from a repair template...are chewed back to reveal homology, allowing the strands to anneal. DNA synthesis then fills in the gaps...5-25 bp) regions of homology flanking a double-stranded break yield precise deletion of the sequence between...locus flanking the GFP-Puro cassette. Three double stranded breaks are necessary for knock-in: one on either...an insertion locus-specific gRNA. These double stranded breaks allow for two sets of microhomologies (...PITCh-gRNA binding sites. This PITCh plasmic is cotransfected with a plasmid carrying Cas9, the PITCh-gRNA...

...microscopy coupled with techniques that make tissues transparent are enabling microscopes to visualize the cellular...the cortico spinal tract, CST, spinal cord and cranial nerve afferents labelled with a AAV8 expressing...sectioning (OPFOS) (2). Arne Voie, David Burns and Francis Spelman focused a laser beam into a thin sheet ...of clear specimens and for in vivo imaging of transparent organisms. Advances in tissue clearing Similar...attempt to render fixed anatomical preparations transparent was done by Walter Spalteholtz (10).  He used...green fluorescent protein (GFP) (6).   The Dodt and Frank Bradke laboratories later developed the 3DISCO (...spinal cords by combining Ultramicroscopy with tetrahydrofuran-based tissue clearing (13,14). A few years ...