... 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... recombination events at the genetic level. Upon Cre/loxP recombination, each transgene expresses one ...Flp recombinase/FRT system in place of Brainbow’s Cre/loxP, allowing for simultaneous use of the two systems...SUMO tags in the FP sequence for separation from Cre-based Brainbow-expressing cells. Autobow plasmids... all-in-one versions which express self-excising Cre recombinase from the same transgene as the Brainbow...

...sensors include syn-jYCaMP1s (slow variant), the Cre-dependent syn-FLEX-jYCaMP1s, and the axon-targeted...syn-FLEX-axon-jYCaMP1s. Find these AAVs at Addgene Cre-dependent EYFP AAV in the new serotype PHP.V1 that...Find this AAV at Addgene New serotypes for the Cre-dependent Flpo recombinase pEF1a-DIO-FLPo-WPRE-hGHpA...biotechnology tool to optimize another. The authors have created a yeast-based directed evolution approach with ...your studies.  Find the plasmids to set up your screening systems at Addgene! Schematic of the evolution...following a DNA miniprep protocol!  First, the authors created the pMAK backbone by modifying pMAL-T-Avi-His/BirA...exported to the periplasm of bacteria where it can be secreted into the supernatant. Four plasmids containing...

...in CRISPR genome editing.   2. Plasmids 101: Cre-lox Cre recombinase can be used to generate inversions...community members. In addition we’ve experienced incredible growth with our readership expanding from ~10,000...next revolution in biological research. We’re incredibly happy to see that one of our career posts has...protein of interest. 4. Plasmids 101: Blue-White Screening Getting a lot of empty vector contaminants in ...Addgenie Jessica Welch explains how Blue-White screening could be a potential solution. 5. Cpf1: A New ...

... the expression of a Cre recombinase contained within the knock-in. Including Cre-mediated marker excision...kilobases of additional sequence being introduced. Cre-mediated excision of the selective cassette minimizes...experimental manipulation. In some cases, injection of Cre is required, however the development of the self-...variable amounts of experimental manipulation and screening.  A co-selection strategy for CRISPR editing in... with inefficient sgRNAs in my initial direct screening efforts, which were similar to the approach detailed...low efficiency (0.13%) and laborious handling (screening 768 F1 animals). Recovering rare editing events...conditional-lethal mutation to identify edits with minimal screening effort. Selecting for repair of a lethal mutation...

...recombinase that can be used in combination with Cre for intersectional control of specific neuronal populations... We now offer a Flp-expressing AAV, as well as a Cre-dependent Flp-expressing AAV. See our recombinase...

...CAG-FLEX: CAG promoter, FLEXed for expression in Cre-expressing cells hSynap: Human Synapsin-1 promoter...hSynap-FLEX: Synapsin-1 promoter, FLEXed for expression in Cre-expressing cells. hSynap-FLP: Synapsin-1 promoter...they created three affinity variants – A184S (increased affinity for glutamate), S72A (decreased affinity...as: iGluSnFR3 v82: increased affinity and slower kinetics iGluSnFR3 v857: decreased affinity and faster... forms of neuronal signaling and regulation. To create iGluSnFR, the Looger lab wedged GFP between two...to glutamate, conformational changes lead to an increase in fluorescence intensity from the inserted GFP...iGluSnFR, the Looger lab swapped eGFP for sfGFP to create SF-iGluSnFR. This improved variant has higher expression...

...piggyBac™, you could use recombinase technology (Cre/loxP) to remove the donor cassette. While this method...mutants [7], it is generally not as efficient as Cre recombinase. However, the use of negative selection...likely due to DNA toxicity. More importantly, unlike Cre recombinase, the PBx is highly specific, and has ...applications, including transgenesis, gene-trap screens, and gene editing. How does the piggyBac™ system...of the major uses of the piggyBac™ system is to create stable transgenic cell lines. The traditional method...site-specific nuclease (such as CRISPR/Cas9) to create a double-stranded DNA break. After DNA cleavage... editing? There are primarily two other ways to create specific point mutations by gene editing. First...

... be achieved using specialized promoters, Flp or Cre dependent gene switches, and serotype selection. ...cell-specific gene expression and viral infection. Cre or Flp dependent switches control gene expression...

... floxed dCas9 so that it may later be excised by Cre recombinase. Addgene has several great options: pLV...T2A-GFP for removable dCas9 expression and pLM-CMV-R-Cre. Selection for cells stably and highly expressing...are dioxygenases that oxidize 5-methylcytosine to create rare DNA bases that are recognized by distinct ...

...targeting inhibitory or excitatory synapses or that are Cre-inducible, researchers can explore synaptic integration...preps at Addgene hSyn1-SIO-stGtACR1-FusionRed is a Cre-dependent, soma-targeting inhibitory channelrhodopsin...brighter in worms than split-sfCherry3. It was created by Maria Ingaramo, Jerome Goudeau, Cynthia Kenyon...Addgene The Cas Hybrid for Multiplexed Editing and screening Applications (CHyMErA) Knockout Libraries express...

...useful tool for marking and tracking cells is the the Cre/lox recombination system. A researcher can flank ...expression is turned on or off in cells expressing Cre. The result of a system like this is a mosaic of ...respectively. The permanent labelling of cells using cre-lox is advantageous when compared to photoactivable... In the past 15 years efforts have been made to create transgenic mice expressing fluorescent protein ...wavelength as it has been shown that even a small incremental change of the excitation wavelength can significantly...called Brainbow, the Ubow mouse strain has been created to fate map Langerhans cells and follicular dendritic...microscopy and new constructs have facilitated the creation of mouse lines that can be used in fate mapping...

...Cell type-specific manipulation with GFP-dependent Cre recombinase.” Nat. Neurosci. 18(9) (2015): 1334-1341...

...More About Designing gRNAs Against Your Favorite CREs Here Additional Resources on Addgene.org Find ... cre... to make out the details. To increase the resolution, the Xu lab created a dCas9-based CAPTURE (CRISPR... smaller pieces. Sticky ends created are then ligated together to create loops of DNA. This is key to ...that, when bound by transcription factors, can increase a gene’s transcription. Sometimes enhancers are...method The original CAPTURE method requires the creation of a stable cell line that co-expresses three ...DNA into smaller pieces, and proximity ligation creates chimeric circles of DNA that are distant linearly...

...bacteriophage integrase phiC31. The popular UAS/Gal4 and Cre/Lox gene regulatory systems have also been successfully...zebrafish transgenesis with Tol2 and application for Cre/lox recombination experiments." Methods in cell biology...two revolutionary large-scale random mutagenesis screens produced ~1,500 zebrafish mutants that are still...deactivation tools for zebrafish Random mutagenesis screens provided a major toehold into understanding how...et al., 2019). Generating zebrafish transgenics Creating transgenic organisms by introducing foreign DNA...one-eyed pinhead mutant from the random mutagenesis screens. Microscopy-based lineage tracing can help illuminate...

...But CRISPRing typical mouse models, including creating Cre-dependent conditional alleles, has remained ...fluorescent proteins (EGFP, mCherry etc.), recombinases (Cre, Flp etc.), and tetracycline drug induction systems...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...product as a template for reverse transcription to create your ssDNA. Note that the T7 promoter will not ...guanine (i.e. GG); this design has been shown to increase T7 promoter activity. To avoid re-cutting by Cas9...

...(Guy et al., 2007). They accomplished this using Cre-Lox mouse models in which the endogenous MECP2 gene...excision of the Stop cassette using tamoxifen-inducible Cre not only halted the neurological damage but completely...two MECP2 labs (Gail Mandel and Stuart Cobb) to create the Gene Therapy Consortium. Leveraging their combined...

... Ace2N-mNeon-A122D and Voltron2 are available as Cre-dependent AAV1 vectors and are suitable for extended...found that administering these AAVs with a dilute Cre-expressing vector resulted in a sparsely-labeled ...CRISPR screens By Emily Bentley Building on their CROPseq method for optical pooled CRISPR screening, Paul...Multiplexed perturbation and decoding in pooled CRISPR screens Improved prime editor PE7 Antibodies to facilitate...multiplexing approach to Cas9-based pooled CRISPR screens called CROPseq-multi (Walton et al. 2024). This...enrichment, single-cell sequencing, and optical pooled screens in human cells. Addgene offers vectors for CROPseq-multi...multiplexed perturbation and decoding in pooled CRISPR screens. bioRxiv 2024.03.17.585235; doi: https://doi.org...

...formation in iPSC-derived and iPSC-transplanted mice. Cre-deletable or Tet-inducible lentiviruses address some...considered safe for clinical applications. Excisable Cre-lox lentiviral vectors: This approach is the same...standard lentiviral vector, but transient expression of Cre recombinase allows for deletion of the inserted transgenes...insertional mutagenesis. Random integration also creates heterogeneous iPSC cell lines, which can complicate...insertional mutagenesis and random integration creates variation between iPSC clones. Retroviruses are...which is removed by culturing cells at 38℃, but screening cells for the presence of transgenes is still ...

...Systemic capsids packaging Cre-dependent transgenes can also be paired with Cre-expressing transgenic mouse...with appropriate cell type-specific promoters or Cre lines. Spatial transcriptomics suggests that AAV-...Huber, N., Pasca, S. P., & Gradinaru, V. (2016). Cre-dependent selection yields AAV variants for widespread...Deverman, B. E., & Gradinaru, V. (2020). Multiplexed Cre-dependent selection yields systemic AAVs for targeting...have developed techniques like CREATE (Deverman et al., 2016), M-CREATE (Ravindra Kumar et al., 2020),...primates (NHPs). These engineered capsids have increased transduction efficiency in the CNS, with greater...

...Combinations of classical genetic tools, for example Cre/loxP or Flp/FRT, with AAV vectors are common approaches... strategies Combining different approaches can increase the specificity of cell targeting even further...delivery of AAV at the injection site results in increased specificity of the targeted cell population.  ...are also developing ways to improve delivery by increasing transgene cargo capacity, enhancing anterograde...