...effective gene activators when fused with dCas9. Recently, synthetic CRISPR-Cas gene activators have been ...zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALENs) required scientists...fusing dCas9 to transcriptional repressors or activators and targeting promoter regions. You might sometimes...mice and human cells. The simplest dCas9-based activators and repressors consist of dCas9 fused directly...directly to a single transcriptional activator (e.g. VP64) or repressor (e.g. KRAB; Figure 9A). Other examples...co-expression of epitope-tagged dCas9 and antibody-activator effector proteins; long-term imaging of proteins...instead of a two-component system (Figure 9C) SAM activators - co-expression of dCas9-VP64 with a modified...

...mutations of these regulatory elements on gene expression. Empty backbones for inserting regulatory elements before...are commonly used to investigate the effect of regulatory elements, such as promoters, enhancers and untranslated...Optimized STARR-seq ( S elf- T ranscribing A ctive R egulatory R egion) plasmids : Genome-wide screening of enhancer...cloning to test for the presence of transcriptional regulatory region in specific cell types. Used to identify...backbone plasmids into which you can clone your regulatory element or gene of interest into to create a ...Lentiviral expression of firefly luciferase and dTomato. A gene of interest can also be inserted into the...Reporter Constructs Already know what gene or regulatory element that you need a luciferase reporter for...

...Fibroblasts Hepatocytes Retroviral Mouse Direct conversion of mouse fibroblasts to hepatocyte-like cells.... 2016 Mar 7;7:10869. Hu CRISPRa Human CRISPR activator system for reprogramming human cells to pluripotency...genes Human pluripotent reprogramming with CRISPR activators. Nat Commun. 2018 Jul 6;9(1):2643. Otonkoski ...generation OCT4 and SOX2 Work as Transcriptional Activators in Reprogramming Human Fibroblasts. Cell Rep....2013 Apr 13;60C:97-106. Gearhart Fibroblasts Hematopoietic Progenitor Cells Lentiviral Mouse Direct reprogramming...reprogramming of murine fibroblasts to hematopoietic progenitor cells. Cell Rep. 2014 Dec 11;9(5):1871-...1871-84. Lacaud Fibroblasts Hematopoietic Progenitor Cells Lentiviral Human Cooperative Transcription Factor...

...-nls-dTomato EF1a GCaMP6f dTomato Cre dependent rg* Ting 51085 AAV-hSyn1-GCaMP6f-P2A-nls-dTomato Syn GCaMP6f...Rose 51082 AAV-EF1a-DIO-GCaMP6s-P2A-nls-dTomato EF1a GCaMP6s dTomato Cre dependent 1 Ting 105714 pAAV-Ef1a-fDIO-GCaMP6s...Larsen 51084 AAV-hSyn1-GCaMP6s-P2A-nls-dTomato Syn GCaMP6s dTomato Constitutive 1, rg* Ting 68717 pAAV-CAG-Flex-mRuby2...CAG CaMKIIa Dlx EF1a GFAP/GfaABC1D Synapsin E2 regulatory element Activity Cre-dependent Flp-dependent ... GCaMP6f dTomato Constitutive 1, rg* Ting 52924 pZac2.1 gfaABC1D-lck-GCaMP6f GfaABC1D GCaMP6f none Constitutive...

...Goldstein Nematode: gRNA design and cloning Cas9-sgRNA construct PDF 355.3 KB Goldstein Nematode: Injection... dCas9 activators sgRNA dual expression: pAC2 , pAC152 , pAC153 , pAC154 ; pmax dCas9 activator expression...cow, dog, pig, Thale cress, rice (Oryza sativa), tomato, corn, monkey (macaca mulatta). Cas-Designer searches...rice fish, mouse, silk worm, stickleback, tobacco, tomato, frog ( X. laevis and X. tropicalis ), and zebrafish...pAC91 , pAC92 , pAC93 , pAC94 , pAC95 ; dCas9 activator gateway donors: pAC84 , pAC1 , pAC147 , pAC148... ; gateway destination: pAC90 PDF 1.0 MB Katic Nematode: Cas9 and gRNA use Cas9 (pIK86) ; gRNA empty backbone...

...Suntag” Cas9 activator that recruits up to twenty copies of the p65-HSF1 transcriptional activator to each ...and engineered combinations of transcriptional activators to induce gene transcription at genomic sites... vitro settings due to the large size of Cas9 activators, which largely precludes delivery by adeno-associated...will fit). Cre-dependent expression of the SPH activator paired with AAV sgRNA delivery make the SPH mouse...calmodulin-dependent kinase II alpha (aCaMKII; excitatory neurons) and Synapsin-1 (pan-neuronal) promoters...Neuron-optimized viral vectors Alternatively, Cas9 activators can be delivered to the CNS by lentivirus, which...expression of dCas9-VPR (Figure 2), a popular Cas9 activator developed by the Church Lab. Neuron-specific expression...

...NLucTom Knock-in of NLuc-tdTomato at endogenous MECP2 locus Castaneus MECP2-NLuc-tdTomato mouse reporter cell... working with the RSRT along with individual laboratories to assemble a Rett Syndrome plasmid resource...The MECP2 protein is a global transcriptional regulator of thousands of genes and studies have suggested...contains a Nuclear receptor Co-Repressor 1/Silencing Mediator of Retinoic acid and Thyroid hormone receptor ...contact information by following the link to their laboratory website. Mouse Line Mutation (DNA) Background...contact information by following the link to their laboratory website. Cell Line Mutation (DNA) Mutation (protein...prenatal LMD microarray data, ISH image data, and anatomic reference atlases of prenatal and adult human ...

...editing assemblies are sensor-actuators, either transcriptional regulators or chromatin-modifying enzymes...s chromatin status into account (Fig 1a). The laboratory of Karmella Haynes at Emory recently contributed...with examples of potential synthetic chromatin mediators and probes. Image from Priode et al., 2021. ...flexibility of the single-pot system will allow investigators to rapidly create multiple synthetic chromatin-modifying...interactions By Jennifer Tsang GFP-based Touching Nexus (G-baToN) is a new tool developed by Rui Tang in Monte Winslow...label cells after cell-cell interactions. Using G-baToN, “sender” cells express GFP on the surface and “...receiver cell. Image from Tang et al., 2020.   G-baToN can also be applied to other fluorophore/antibody...

...dCas9) can be fused to a transactivator and used as a transcriptional activator . ID Name Insert Antibiotic...dCAS9 (D10A, H840A) none Expresses dCAS9-VP64 activator with 2A GFP Zhang 61425 lenti dCAS-VP64_Blast ...Hygromycin lenti vector encoding the MS2-P65-HSF1 activator helper complex with a 2A Hygro resistance marker...Celltag library contains 19973 barcodes to combinatorially index cells for single-cell analysis of clonal... Celltag library contains 4934 barcodes to combinatorially index cells for single-cell analysis of clonal... Celltag library contains 5737 barcodes to combinatorially index cells for single-cell analysis of clonal...

...single-molecule biophysics Fast and responsive voltage indicator ONE-GO biosensors kit Antibodies for neurodegeneration...lab recently "broke the PE efficiency barrier in tomatoes" by combining several approaches (Vu et al., 2024...chaperone, RT: reverse transcriptase. Right: T1 tomato plants that inherited an edited gene to confer ...the team was able to drive prime editing in both tomatoes and Arabidopsis, suggesting the improvements may... species. In addition, they generated offspring tomato plants that inherited the genomic edit but not ...prime editing enables heritable desired edits in tomato and Arabidopsis. Nature Plants, 10(10), 1502–1513...2024.12.20.629623.  A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic...

...finally decide the LEGO laboratory was good enough. There are no pre-made LEGO laboratory sets so we had to ...to show people the very human struggles in the laboratory routine. We hope that sharing and laughing at...the LEGOs were mirrored by the nearly infinite regulatory capabilities of gene pathways just waiting to...was creating a LEGO stage that resembles a real laboratory. It only took one night to build the first model...use our imaginations to mold it all into a full laboratory. It is priceless to see our “toys” gaining life...working as scientists in a custom made, colorful laboratory. When we started, the hardest part of the process...building each scenario, I go “WOW, now I have my own laboratory” (the main character is much more than a piece...

...Cancer Institute! Sonic Hedgehog, an important regulator of embryonic development The largest conference...the hedgehog signalling pathway, which is a key regulator of all bilateral animal development. SHH plays...publication, Ivics et al 1997. Spaghetti Squash, a regulator of cellular contraction You may think that a gene...vessels). Spaghetti squash (Sqh) is the Drosophila Regulatory Light Chain (RMLC) of nonmuscle myosin II (Karess... Jordan and Karess, 1997), which contains the regulatory regions that are necessary for myosin activity...monster, a meiotic arrest gene required for spermatogenesis One of our smaller conference rooms is named... meiotic arrest gene that is necessary for spermatogenesis. Flies that lack cookie monster are sterile...

...doi: 10.1038/s41592-022-01597-x. New glutamate indicator iGluSnFR3 Improving on the glutamate sensor iGluSnFR...PDGFR.codonopt    Aggarwal A, et al. Glutamate indicators with improved activation kinetics and localization...have developed a new genetically-encoded voltage indicator (GEVI) called JEDI-2P (Liu et al. 2022) optimized...Sustained deep-tissue voltage recording using a fast indicator evolved for two-photon microscopy. Cell. 2022 ...has generated a new genetically encoded calcium indicator using small monomeric NIR fluorescent proteins... of a Small Near-Infrared Fluorescent Calcium Indicator. Front Cell Dev Biol. 2022 Jun 29;10:880107. doi...they developed a family of fluorescent potassium indicators with a range of binding affinities (Torres Cabán...

...genes. We’ve created enough regulatory elements, CRISPRs, repressors, activators. We’ve been putting together...CRISPR/Cas genetic circuits are linked to the regulatory networks within E. coli host cells. ...in building genetic circuits is getting enough regulatory proteins that don’t interfere with each other...programmable that you can make a very large number of regulators. That basically means larger genetic circuits...that could conceivably be as large as natural regulatory networks. Addgene: What kinds of things could... pattern, shape. All of that is controlled by regulatory networks in the plant. If we want to access that...CRISPR is how much easier it is to work with than regulatory proteins. There are still things we haven’t solved...

...In-Fusion cloning molar ratio calculators Lab math calculators These calculators can help you with common...lab. Molarity calculator Promega's Biomath Calculators Primer resuspension calculator (sign-in required...Golden Gate Reactions Cloning ligation reaction calculators These links can help you calculate how much insert...assembly, or In-Fusion cloning.  NEB Ligation Calculator: This tool works for any cloning method. You ...required.)  DailyCalcs Science Calculator app for iPhone Reagent and Data Repositories These links can help...

...transcriptional regulator to modulate expression at that locus. The use of regulators allows a greater...shifted in recent years toward tinkering with regulatory networks and multiplex gene control. However,...approaches are often met with the challenges of regulatory bottlenecks and their scope is limited by the...scaffolds that can recruit various transcriptional regulators (Zalatan et al. 2015).  These scaffolds are stretches...levels dependent upon the type of transcriptional regulator or the number of designed scaffold binding sites...with distinct programs controlled by each dCas9 regulatory switch. By no means limited to the field of metabolic...scaffolds can recruit various transcriptional regulators to alter gene expression at the targeted site...

.... enhanced signaling of mTOR, a key metabolic regulator, correlates with poor prognosis in many cancers...arget o f r apamycin (mTOR) is a key metabolic regulator controlling cell growth and proliferation... caused by inactivating mutations in negative regulators of mTORC1. The hyperactivation of mTORC1 in cancer...Ras-related GTP binding Raptor Also known as RPTOR; Regulatory associated protein of MTOR, complex 1 Rheb Ras...Catalytic Subunits PIK3CA PIK3CB PIK3CD PIK3CG Regulatory Subunits PIK3R1 PIK3R2 PIK3R3 PIK3R4 PIK3R5 PIK3R6...Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic and regulatory subunits - Class I PKC PKC alpha PKC beta PKC...

...Constructs Kits and Collections Other Resources The nematode Caenorhabditis elegans ( C. elegans or “the worm... approach has also been developed by the Fire laboratory and described in Efficient Marker-Free Recovery...contains 29 plasmids used to generate transgenic nematodes, including C. elegans and C. briggsae. Other related...A database featuring behavioral and structural anatomoy of C. elegans. WormBase - A consortium of biologists...genomics and biology of C. elegans and related nematodes. WormBook - A comprehensive, open-access collection...related to the biology of C. elegans and other nematodes. Wormbuilder - Describes methods to engineer the...

...to their laboratory. A single mouse click on a gene of interest will direct the investigator to a list... cancer are yielding promising results in the laboratory, and Addgene is accelerating this research by...proto-oncogenes and tumor suppressors to the accelerator and brake pedal of a vehicle, respectively. In... (now an oncogene), this is analogous to the accelerator of a vehicle being stuck down. Similarly, a mutation...cells still need to successfully infiltrate the circulatory or lymph systems to spread, or metastasize. Many...Many cancerous cells will die trying, as the circulatory and lymph systems are not ideal environments ...involved in the Ras pathway alone and thousands of investigators studying them, how do we ensure that the research...

...target genes using dCas9-based activators The simplest of the dCas9 activators consists of dCas9 fused to ... dCas9 and antibody-activator fusion proteins. The Synergistic Activation Mediator complex, generally ...exploited to localize factors such as transcriptional activators and repressors to target genes. Repressing target...activation domain - VP64.  Later generations of activators include the SunTag system which facilitates recruitment...generally the strongest activator, consists of a dCas9-VP64 fusion and a modified gRNA that is capable of interacting...with an additional RNA-binding transcriptional activator. Plasmids for activating target genes in a variety...fusion, both targeted to the promoter region. Cas9 activators include Cas9 fused to VP64, VP64 along with other...