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We narrowed to 13 results for: EGFP-Cre cassette

Showing: 1 - 13 of 13 results

  1. Tips for CRISPR Gene Editing in Mice

    Type
    Blog Post
    Published
    June 28, 2016, 10:59 a.m.
    ...protein (EGFP) reconstitution. (a) Scheme of validation for DSB mediated EGFP expression cassette reconstitution...annealing) took place and reconstituted the EGFP expression cassette. (b) pCAG-EGxxFP target plasmid and pX330...MCS) between EGFP fragments. The pX330 plasmid contains humanized Cas9 expression cassette and sgRNA expression...halves of EGFP to recombine by homology directed repair, and resulting in the expression of EGFP. Using ...pCAG-EGxxFP target plasmid contains overlapping 5′ and 3′ EGFP fragments under the ubiquitous CAG promoter. The...expression cassette. The target sgRNA can be cloned directionally into the BbsI site. (c) The pCAG-EGxxFP...indicates where your target is inserted, disrupting the EGFP fluorescent signal, for details on this plasmid,...

  2. 15 Hot Plasmids from 2017

    Type
    Blog Post
    Published
    Jan. 9, 2018, 3:02 p.m.
    ...along with EGFP while retaining an emission spectrum that is easily separable from that of EGFP. This makes...kanamycin cassettes and were carefully designed to allow for easy removal of the resistance cassette via Cre...variants of the polycistronic cassettes and discovered reprogramming discrepancies, which they traced to a ...spectra (N- and C-terminal tags for mTagBFP, TagRFPt, EGFP, mVenus, mCerulean3, mKOFP2) and empty vectors that...the transcription factors from a polycistronic cassette. However, it was unknown whether variations in...in gene expression from these polycistronic cassettes could lead to comparable experimental results. Using... Klf4 isoform (KLF4s or KLF4L). Polycistronic cassettes that contained KLF4s had overall diminished KLF4...

  3. Choosing Your Perfect Empty Backbone

    Type
    Blog Post
    Published
    Aug. 19, 2014, 3:39 p.m.
    ...selection cassette for growth and cloning in bacteria and a mammalian selection cassette to screen for positive... allow you to visualize YGOI in vivo (try pcDNA3-EGFP). If you can get away with generating transiently...cells, you will not need a mammalian selection cassette (e.g. 1435 pSG5L Flag HA). You can also upgrade...with an integrating vector expressing a selection cassette with 5′ and 3′ flanking sequences matching the...fusion proteins. Pick your promoter, selection cassette and visualization vehicle to conduct your custom...your applications and experimental design. You can create a new strain with a disrupted gene by transforming...

  4. Evolution of Brainbow: Using Cre-lox for Multicolor Labeling of Neurons

    Type
    Blog Post
    Published
    April 24, 2015, 2:39 p.m.
    ... how Cre-lox recombination works. Advances in Cre-lox recombination have included inducible Cre, such...possibilities. The remaining cassette can invert as long as Cre remains expressed; once Cre is removed from the...Cai et al. also created an Autobow construct containing both Cre and XFPs. Cre production drives recombination...continued utility of Cre-lox. Check out our previous blog post, Plasmids 101: Cre-lox, if you need a quick...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 recombinations...system employs Cre-mediated deletion, the design of Brainbow-2.1 cleverly combines Cre-mediated deletion...

  5. Hot Plasmids - June 2020 - Barcoded CRISPR Library, Sparse Cell Labeling, Calcineurin Reporter, and DNA Staining Dye Alternative

    Type
    Blog Post
    Published
    June 2, 2020, 1:15 p.m.
    ...remove a stop cassette upstream of an effector/reporter gene. In cells where the stop cassette is removed...fluorescent protein to the DNA. They created plasmids for purification of tTALE-eGFP and tTALE-mCherry. In comparison... Figure 1: Initial steps in the CRISPR-pooled screening using sgRNAiBAR. Image from Zhu et al., 2019.  ... Peking University has developed a new CRISPR screening method that makes use of guide RNAs with internal...false-positive rate discovery rate, conventional CRISPR screens require a low multiplicity of infection (MOI) during... annotated human gene, it enables genome-wide screening with a high MOI, which is beneficial for a low... calcineurin dynamics and can serve as a drug-screening platform to identify new antifungals that target...

  6. Plasmids 101: Knockout/Knock-In Plasmids

    Type
    Blog Post
    Published
    Dec. 1, 2016, 3:30 p.m.
    ...want to create a conditional knockout. To make a conditional knockout, researchers often use the Cre/Lox ...resistance cassette has been placed into an intron.    Figure 4: A targeting vector to create a floxed...researchers usually remove it after selection using the Cre/Lox recombination system. After “floxing” the resistance... you can remove the gene through the addition of Cre recombinase (Figure 2). Figure 2: A knockout...so that it can be removed post-selection through Cre-mediated recombination.   Knockout/Knock-in...targeted gene. Upon recombination, the GFP/NeoR cassette is inserted in place of exon 2. Thus, the targeted...example, you can remove a floxed resistance gene using Cre recombinase. If GFP is under control of an endogenous...

  7. Plasmids 101: Multicistronic Vectors

    Type
    Blog Post
    Published
    Sept. 9, 2014, 8:20 p.m.
    ...individual expression cassettes, having the genes expressed from the same cassette is sometimes advantageous...Multicistronic Element Expression Type MSCV-IRES-EGFP IRES Retroviral pMSCV-pBabeMCS-IRES-RFP IRES Retroviral...and you can then insert the whole multicistronic cassette into a backbone as a single unit. Although it ... or the creation of bicistronic or multicistronic vectors. Unlike promoters which will create unique mRNA...resistance gene. These reporters allow you to easily screen or select for cells that are expressing your gene...interest at high levels. Unlike vectors that express screenable or selectable markers from a unique promoter,...now download Addgene's new eBook! Our goal was to create a one-stop reference guide for plasmids. We've ...

  8. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    Published
    Jan. 6, 2015, 5:21 p.m.
    ...sequence in between two fragments of EGFP in pCAG-EGxxFP. The EGFP fragments contain 482bp of overlapping...express self-excising Cre recombinase from the same transgene as the Brainbow cassette, simplifying experiments... short hairpin) and CRUSH (Conditional RUSH) use Cre-mediated recombination to turn on or off the expression...a dual-color RNAi “sensor” mouse strain in which Cre expression causes the induction of both DsRed fluorescence...Mice carrying the R26DsRedR; CRUSH-GFP; and Nestin-Cre alleles showed efficient GFP knockdown in eyes and...sequence to be integrated and a removable integrase cassette. They validated this methodology in two differents... recombination events at the genetic level. Upon Cre/loxP recombination, each transgene expresses one ...

  9. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    Published
    Dec. 23, 2015, 3:30 p.m.
    ...containing Cas9, EGFP, mCherry, iRFP, tdTomato, luciferase, LacZ, puromycin or CreERT2 can be combined ...separate reporter gene (EGFP, iRFP, IFP1.4, puromycin, neomycin or luciferase) to create a final lentiviral...result in a translational fusion of the antibiotic cassette to the gene of interest. The gene to be expressed... missing codons from the antibiotic resistance cassette, a ribosome binding site, SacI restriction site...heterologous promoter outside of the Multisite Gateway® cassette, you can specifically express your new chimeric...scientists to target RNA-based protein-binding cassettes, artificial aptamers, pools of random RNA sequences...utilize a U6 promoter and contain the RNA cargo cassette inserted within the sgRNA core (ie an INT construct...

  10. Easi-CRISPR: Generating Knock-In and Conditional Mouse Models

    Type
    Blog Post
    Published
    April 5, 2018, 12:42 p.m.
    ...commonly used cassettes, such as fluorescent proteins (EGFP, mCherry etc.), recombinases (Cre, Flp etc.),...But CRISPRing typical mouse models, including creating Cre-dependent conditional alleles, has remained ..., we’ll use the example of inserting a 2A-FlpO cassette into the Fgf8 gene. Miura et al. (2018) suggest...plasmid pmScarlet_C1! After creating the T7-DNA template, you'd use ivTRT to create the ssDNA construct as ...CRISPR genome editing has made it easier to create knockout alleles in a variety of species, including...repair template is used. The original design for creating a conditional allele, or floxed mouse, from Yang... and inject the ES cells into a mouse embryo to create chimeric mice. This laborious process takes ~8-...

  11. Multicolor Animals: Using Fluorescent Proteins to Understand Single Cell Behavior

    Type
    Blog Post
    Published
    March 5, 2019, 1:08 p.m.
    ...fly. As a stop cassette is located directly upstream of the fluorescent protein genes, Cre recombination...developing fish via Cre protein microinjection or crossing of Zebrabow animals to an inducible Cre line. The Schier...OFP (orange) (Livet et al., 2007). Brainbow uses a Cre/lox system to label neurons. Genes encoding the fluorescent...gene directly following the promoter is expressed. Cre-mediated deletion of one or several of these genes...system. One such system is Brainbow 2.0, which uses Cre to trigger the inversion of two genes aligned head-to-head...head-to-head. Another variation, Brainbow 2.1, uses Cre to trigger the inversion of two tandem sets of genes...incorporates the highly photostable fluorophores mOrange2, EGFP, and mKate2 (red) to solve the problematically low...

  12. Hot Plasmids - February 2022

    Type
    Blog Post
    Published
    Feb. 22, 2022, 2:15 p.m.
    ...within a single cassette provides increased simplicity for combinatorial genetic screens, and this optimized...perform multiplex editing within a single expression cassette is advantageous to Cas9’s larger expression requirements...2: comparison of Cas 9 vs. Cas 12a expression cassettes when encoding multiple guides. While two guides...expanded the genetic screening toolkit by optimizing a Cas12a-based method for screens. Cas12a’s ability ...underutilized in genetic screens because of its lowered editing rate. This research aimed to increase the potential...parallel. Lastly, this Cas12a-based screening method was used to create a genome-wide library capable of ...imaging of cellular targets Optimized genetic screening technique using Cas12a for multiplexed gene perturbation...

  13. 27 Hot Plasmids from 2016

    Type
    Blog Post
    Published
    Dec. 22, 2016, 3:03 p.m.
    ... CAG-driven loxP-stop-loxP cassette into the Rosa26 locus, allowing for Cre-dependent expression of a ...Hess et al. successfully evolved wild type GFP to EGFP using CRISPR-X and subsequent FACS sorting. They...of a good screening experiment can be found on our blog post entitled “Genome-wide Screening Using CRISPR...Receptor-ome Expression and Screening via Transcriptional Output-TANGO (PRESTO-TANGO) to screen the NCC-1 library...and later create stable cell lines expressing it. The Church lab fixed this issue by creating a new dCas9...  CRISPR pooled libraries enable researchers to screen the entire genome for genes that regulate a wide...CRISPR knockout library and used this library to screen for genes involved in West Nile Virus (WNV) induced...
Showing: 1 - 13 of 13 results