...those optogenetic controls, gene expression levels reliably increased in vitro by a factor of 10 to 20...functions, Konermann needed tools to manipulate the expression of specific genes in the brain in reversible ...Zhang showed they could package LITEs in viral vectors and genetically target them to specific cell populations...demonstrated use of the LITEs to control gene expression in mouse neurons and in the brains of living ...system could be by applying it to altering gene expression via epigenetics with the addition of histone ...different effectors. I’d encourage people to get creative with them for their own uses.” Konermann said ...found here.) So tell us, readers, how will you get creative with LITE? Reference: Silvana Konermann et al...

...promoter is expressed. Cre-mediated deletion of one or several of these genes permits the expression of genes...copy of the transgene expressing either YFP or CFP, and cells that do not express Cre fluorescing with the...activate the expression of a different fluorescent protein. Therefore, each cell could express a unique combination...fluorescent protein genes, Cre recombination is required before fluorescence can occur. Cre recombinase can be...developing fish via Cre protein microinjection or crossing of Zebrabow animals to an inducible Cre line. The Schier...any cell type when crossed with mice expressing the appropriate Cre allele. The Confetti mouse contains...so that XFP are only expressed following a recombination event. Tissue-specific Cre recombination in this...

...were detected over a 10-week period. Their expression decreased over time but they were not silenced.    ...), now allows scientists to identify the gene expression patterns unique to the few cells that successfully...Unfortunately scRNA-seq only captures the gene expression of cells at a single time point, making it difficult...difficult to investigate how early expression patterns influence reprogramming outcomes. CellTag from ...single-cell level, thus linking ancestral gene expression patterns with reprogramming outcomes (Biddy et...MEFs were reprogrammed into iEPs by retroviral expression of transcription factors Foxa1 and Hnf4α, a process...lineage trees and comparing them with the gene expression changes of more than 100,000 cells undergoing...

...working on developing new surface display vectors to improve expression and trafficking of these biosensors....gray (PAG), with red neurons expressing tdTomato and green neurons expressing cFos. Image from Elizabeth...B) CR-Cre mice demonstrate pain response due to blue light stimulation of dual virus-expressing CR neurons...verification of the dual virus expression in CR neurons of the spinal cord. Created with BioRender.com. Image... Figure 2: Micrographs of the biosensor expressed in the human cell line, Hep3B. From left to right...opioid withdrawal with tdTomato and assessed cFos expression. She will explore the connectivity patterns of...chemogenetics to study the activity of parvalbumin (PV)-expressing neurons, neurons that the lab previously found...

...fundamentally the reporter construct monitors the expression of a nearby gene. The reporter gene serves as...as a proxy for the endogenous gene – its expression levels (high/low) and patterns (tissue/timing) can...that the gene which GFP was monitoring was being expressed in the same pattern. These systems suggest the...function of a gene. For example, they eliminate the expression of the gene (by CRISPR knockout, shRNA depletion...Tissue-specific genes – the genes of interest may not be expressed in the tissue/cell line you are working with. ...the gene as a result but will still allow for expression pattern monitoring. Enhancer traps Like promoter...trapping of endogenous enhancers to stimulate the expression of the reporter. The system is made up of a reporter...

...Genome Engineering Guide CRISPR Mammalian Expression plasmids Cre-Lox plasmids References Prathipati, P.,...be injected, including cells, drugs, DNA, viral vectors, and viruses. All researchers must go through ...choice for administering different drugs and viral vectors. Like IV injections, intraperitoneal injections...for the delivery of various therapies and viral vectors. This injection sounds very disruptive, but surprisingly... This is most often used for delivery of viral vectors, though drug treatments are also possible. One ...representation of common injection routes in mice. Image created with biorender.com.   Needle techniques Intravenous... often used when injecting a cell suspension to create xenograft models. Restraint: back scruffing or ...

... how Cre-lox recombination works. Advances in Cre-lox recombination have included inducible Cre, such...membrane tethered cyan). Without Cre recombination, RFP is expressed. Cre can mediate one of two deletions... construct, OFP (orange), is expressed when Cre is absent; stochastic Cre excision results in one of three...remaining cassette can invert as long as Cre remains expressed; once Cre is removed from the system, the fluorescent...Cai et al. also created an Autobow construct containing both Cre and XFPs. Cre production drives recombination...manipulate gene expression, but other gene manipulation systems remain valuable to biology. Cre-lox recombination...temporally control gene expression, especially in in vivo models, and new Cre-lox based technologies are...

...cells to express Cas9. In these particular experiments, we generated A549 cells stably expressing Cas9 via...wildly variable Cas9 expression (Figure 1). This is a problem because Cas9 expression affects growth rate...variable Cas9-expression was across the different cells (Figure 3).   Figure 2: Cas9 expression in monoclonal...Checking Cas9 expression Overview: By Western blotting (Figure 2, pool), I knew the cells expressed Cas9, but...thought I could see GFP expression with a fluorescence microscope, but the GFP expression was so low (even in...against GFP expression, but I didn’t. 2. In the original publication they see GFP expression in about 20%... MOIs. 2. Create a monoclonal Cas9-expression cell line. Especially if you’re doing a screen. I made a...

...constructs are currently available as Cre-dependent, EF1ɑ-driven viral vectors (AAV1) - and you can also find...B) Cortical slice of HcKCR1-EYFP and tdTomato expressed layer 2/3 neurons in mouse. C) Action potential...voltage traces of action potentials in a HcKCR1-expressing mouse neuron without (top) and with (bottom) ... blot for the myc-tagged protein GFP-smFP-myc expressed from Plasmid 98926. See the Applications section...called JEDI-2P. Using a custom, high-throughput screening platform to apply saturating mutagenesis on 21... 1: Summary of the approach and outcomes of the screen for improved genetically-encoded voltage indicators... in-vivo imaging of voltage fluctuations. Image credit: Liu Z, et al. Cell. 2022.  Find JEDI-2p viral...

... information on viral vectors, including transduction download our Viral Vectors 101 eBook. Bacterial ...can be used to create both transient cell lines, where a gene of interest is expressed but not integrated...bacterial or eukaryotic cells via a virus or viral vector. One example are bacteriophages that attach to ...scientists commonly use phagemids, a DNA cloning vector that contains both bacteriophage and plasmid properties...application of an electrical charge to cells, to increase cell membrane permeability and thus transformation...DNA can be accidentally packaged into the newly created phages. Transfer of this DNA to another cell is...Electroporation on the other hand uses electrical pulses to create transient pores in the cell membrane that genetic...

... single protein is expressed, with the intent of purification, it will be expressed along with all other...The MBP tag, although large, can actually increase expression and solubility of the tagged protein (Fox...other cellular proteins within the expression system (bacteria, yeast, mammalian, etc.). To individually...about that protein that no other protein in the expression system has. This is where affinity tags come ...peptides which do not naturally exist in most expression systems that can be added to the N or C terminus...on: The protein you are trying to purify The expression system If you want the tag to remain on your ...Ready to start tagging? We are here to help with vectors, protocols, antibodies, and more!   Resources ...

...transgenic protein for the HEK 293T cells to express: CD45. Upon expression, CD45 is incorporated into the plasma...CD45, but is separately expressed. (The reason for having a separately expressed transduction reporter ...manage to express CD45. Thus, if transduction efficiency is much higher than CD45 expression, this could... example, point at issues with the double-expression vector.) In our flow experiment, we'll want to identify...CD45+ cells (CD45 expression) and assess our transduction efficiency (eGFP expression).     The first ...detected through a cell surface label. The viral vector also encodes for eGFP, which serves as a transduction...rest of our panel. Lastly, we want to detect the expression of our protein of interest: CD45. I am using ...

...Bradbury et al., 2018), and hybridomas can lose gene expression of the mAb-encoding genes, die, or fail to revive...research materials, such as plasmids and viral vectors. However, it typically takes a few years for a ...antibodies are not monospecific: Hybridomas frequently express additional functional variable regions. mAbs 10...these issues by creating immortal antibody-producing cell lines (hybridomas) that secrete antibodies in ...Neurotechnologies® (BRAIN) Initiative, will allow Addgene to create a much needed open-access recombinant antibody/...School of Medicine on this grant. Dr. Trimmer has created thousands of high-quality antibodies/nanobodies...reagent batch. To supplement this, Addgene will be creating a crowdsourced Data Hub (built off of the concept...

...2018) HDAd5/35++ Adenovirus Vector Expressing Anti-CRISPR Peptides Decreases CRISPR/Cas9 Toxicity in Human...genome editing, activating gene expression, repressing gene expression, and much more. But there’s one... means they can be delivered in vivo using AAV vectors. Having a way to shut off CRISPR-Cas systems prevents... off-target effects Prolonged Cas activity can increase the chance of off-target editing particularly ...

...fluorescently labeled. Using lentiviral vectors to stably or inducibly express tagged iPAK4 from different promoters...reprogram delivery vehicles like lentiviral vectors, CreVLPs, and eVLPs for in vivo and in vitro applications...the Wu Lab is working to create a three-dimensional atlas of protein expression in the adult mouse brain...activation events as bands of induced GFP-iPAK4 expressed under promoters like the tet-responsive element...enable the creation of CAR-T cells directly within the body. Overall, DIRECTED can increase on-target .... The Ting lab recently made two deposits that increase the utility of this technique.  By engineering... O’Neill The Cohen lab and colleagues recently created a system for recording the transcriptional history...

...genes is from Proteobacteria to mammalian expression vectors. And more broadly, their data revealed that...a distantly related organism, the gene may not express well because of these different codon usage biases...closely resembles that of the host organism, it may express protein more efficiently. To classify gene sequences...rare codons, sometimes intentionally modulating expression in ways we don’t quite understand yet,” Kunjapur...synthetic. They also found out that the most common expression system within the Addgene repository is mammalian...series of simulations and machine learning, they created a classifier that predicts whether a gene was natural...

...instance, if you were cloning a gene into an expression vector, you would want the start of the gene to end...Your Insert and Vector by Gel Purification     Now that you’ve cut your insert and vector, unfortunately...unsuccessful 1), in a vector alone plate with many more colonies than the vector + insert plate (unsuccessful... ligation step. 3. Ligate Your Insert into Your Vector In the ligation step, you mix your purified, cut...Resources on Addgene.org Find the Perfect Empty Vector for Your Cloning Experiments Browse Our Molecular... 2 ligations for each plasmid you are trying to create, with varying ratios of recipient plasmid to insert...

...can be expressed in a single vector, pCRISPRainbow-DONOR1, with dCas9 supplied by a separate vector. ...CRISPRainbow gRNA will permit labeling, but a newly expressed standard length gRNA would “switch” the system...scaffold to enhance complex assembly, thereby decreasing the amount of background fluorescence from unbound...CRISPR to label multiple loci in distinct colors. To create a colorful Cas9 toolbox, Ma et al. turned to SpCas9...was fused to a different fluorescent protein to create three distinct colors. The specificity of these...distinct hairpin sequences produces white light. Created with BioRender.com. With this rainbow of colors...combination, for example, with a far-red FP, would increase the available colors to 15! They also envision...

...interest. In our studies, we used AAV viral vectors to express hM4Di in neurons of rhesus monkeys or mice...successful expression of the protein of interest, an alternative could be bicistronic expression of the fluorescent...the fluorescence expressed in the neuropil conceals identification of opsin-expressing cells). For other...proteins provide a versatile method to identify the expression of transgenes of interest. Furthermore, fluorophore...can be a roadblock, rather than an aid, in the expression of the protein of interest. The green fluorescent...’ tags to identify proteins Given the limited expression of hM4Di‐mCherry at the plasma membrane in monkey... mCherry with the HA tag resulted in abundant expression of hM4Di in plasma membranes in both species ...

...years, he and his colleagues built a Molecular Expressions collection including some 3,300 plasmids along... Baird said. “They are all designed in the same vector backbone with very similar restriction enzymes....shutter its doors. “We wanted to give all of these vectors to the scientific community and contribute in a...proposition, explained Michelle Baird, who has created plasmids as a technician in the Davidson lab for...prompted him to deposit the tools he and his team created with the research community at large, "[Fluorescent...