.... In tamoxifen-inducible Cre (Cre-ER) systems, Cre is fused to the ligand binding domain of estrogen receptor...the Esr1 domain that blocks Cre expression. Then, Cre is expressed and Cre-mediated recombination of floxed...in specific cells or under certain stimuli. Cre recombinase catalyzes the recombination of DNA sequences...that regulates Cre expression. The most common drug used to induce the expression of Cre is tamoxifen. ...doxycycline mediated activation of Cre to either induce or repress Cre expression using TetON or TetOFF ...using Cre-lox Read our genome engineering blog posts Resources on Addgene.org Find more about Cre-lox ... vector, creating the floxed gene segment. Crossing of mice containing a floxed gene to a Cre-expressing...

... functional portion of the Nfix gene using Cre recombinase and to generate Nfix-negative neural stem cells...burden of immune symptoms on CDG patients and to increase their quality of life by reducing their risks ...ligand binding sites in T4 lysozyme by deficiency-creating substitutions." Journal of molecular biology 277.2...

...levels of certain genes. A popular method uses Cre recombinase on transgenic flox animals to introduce modifications...light or noise should be avoided as this will also increase stress levels and affect behavior. A fact to consider... also to minimize the damage of tissue due to increased pressure of the added liquid. The volume should...editing/deletion using CRISPR. In some cases, a decrease of the expression efficiency can be observed after...

...floxed dCas9 so that it may later be excised by Cre recombinase. Addgene has several great options: pLV hUbC-dCas9...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 ...

...Constitutive or inducible Cre recombinases can be introduced by AAV or by mating with Cre-expressing mice. ... of Cre-dependent mouse lines for overexpressing genes within discrete cell populations makes Cre-dependent...the Cre-dependent SPH construct for generating the SPH mouse. (B) AAV plasmids used to express CRE under...importantly, the expression of this construct is Cre-dependent, enabling spatiotemporal control over its..., how many sgRNA expression cassettes will fit). Cre-dependent expression of the SPH activator paired ...populations.  The Yang Lab’s AAV plasmids used to express Cre under neuron-specific promoters, including calmodulin-dependent...and hSyn). Created with BioRender.com. Before using gRNAs in vivo, they should be screened for activity...

...CNS and PNS! What is the CREATE method?  CREATE stands for Cre Recombinase-based AAV Targeted Evolution...cell-type-specific Cre, the polyA sequence is inverted only in the Cre-expressing tissues. This inversion creates a template...using this library to infect an animal expressing Cre in a cell type of interest, a scientist can isolate...28671695. 2. Deverman BE, Pravdo PL, Simpson BP, et al. Cre-dependent selection yields AAV variants for widespread...rounds of infection and screening by PCR enrich for capsid variants with increased ability to infect the...to reach the brain. Delivering the CREATE AAV library via IV screens for capsids that are able to cross...that determines tropism) have been developed that increase transduction efficiency, none allow for simple...

...strategies that combine prime editing with a recombinase to install large sequences (up to 10 kb) into... in mammalian cells via continuously evolved recombinases and prime editing. Nature Biomedical Engineering..., the Liu lab created and evaluated 19 PE1 variants with RT mutations known to increase activity, enhance... initial nick site (to avoid creating a double strand break), increasing editing efficiencies 2-3-fold... Figure 1: An overview of prime editing. Created with BioRender.com. How does it work? First... between the template and primer binding site, increase processivity, or improve thermostability. What...fold. However, the process of double nicking increases indel formation slightly. Designing the sgRNA with...

...researchers injected Cre-expressing AAV-5 into the DRN of a transgenic mouse harbouring a Cre-dependent IL-1...injected AAV2 particles into a Drd1-Cre transgenic mouse to deliver Cre-dependent expression of  Channelrhodopsin...-1 beta overexpression cassette. Expression of Cre in these neurons turns on overexpression of IL-1 beta... (Howerton at al. 2014). Addgene has several recombinase-expressing ready-made AAV vectors which can be...2014). Scientists from the University of Geneva (Creed et al., 2015) used optogenetics to support these...transmission from the mPFC projections to the NAc and decreased the locomotion of the animal. This confirms the...behavior in rats in the forced swim test as seen by increasing the ratio of time spent actively swimming versus...

... at Addgene New serotypes for the Cre-dependent Flpo recombinase pEF1a-DIO-FLPo-WPRE-hGHpA, which is...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...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...

... The old standby of CRE/flp didn’t work – it turns out these tyrosine recombinases are as bad at integrating...likely do it using Prime editing alongside some creative (and significant!) protein engineering. They combined...insert being “dropped” in the wrong place, while increasing the number of potential applications. But they...

...AAV serotypes, and the ever-growing variety of recombinase systems. Picking Ligands Picking the right ligand...further modified to create ultrapotent PSEMS (uPSEMs) that have drastically increased potency. Download ...signaling pathways and can lead to such responses as decreased heart rate (Redfern et al., 1999) or altered neuron...firing. Other DREADDs, such as the KORD receptor, increase this threshold and silence neurons. DREADD-activated...actuator modules, were developed with the goal of creating receptors with less endogenous activation and ... Higher potency means lower drug doses, which decreases the risk of off target effects. These synthetic...

...dynamics in fine structures. We have expanded our recombinase-dependent virus selection to now include Flp-... aaRSs in these processes. To create the antibody toolbox, they screened antibody candidates using a synthetic...fourteen aminoacyl tRNA synthetases (aaRS) have been created, characterized, and validated by the Gräslund lab...cross-reactivity, antibody candidates underwent affinity screening to select for slow off-rate binders. The best ...pathways By Gabrielle Clouse Claudia Vickers’ lab has created a novel nanocompartment for transporting proteins...

...sequence of interest (red) is amplified using Recombinase Polymerase Amplification (RPA) or Reverse-transcription...dLwaCas13a as a programmable RNA-binding protein, creating the fluorescent fusion protein dLwaCas13a-NF for...

...resistance markers, and other enzymes such as Cre-recombinase drastically expanding the base-editing reporter...Base editors create specific point mutations in the genome, but they’re inefficient compared to CRISPR...experiments. In the past few years, scientists have created an array of base editing reporters that can help...Cas9-mediated editing in real time The Harris lab created ACE, a reporter that monitors APOBEC (a family ...and a downstream constitutively active eGFP. To create the inactive mCherry, the lab introduced a 43 base-pair...DSB in their previous reporter, the Harris Lab created a panel of eGFP reporters to quantify the on-target...complexes in real-time (St. Martin et al., 2019). To create the eGFP reporters the Harris lab individually ...

...PMCID: PMC4165888. 6.Witten, Ilana B., et al. "Recombinase-driver rat lines: tools, techniques, and optogenetic...using Chr2 stimulation should result in robust increases in c-fos expression in the targeted brain region...

.... Flipbow employs the Flp recombinase/FRT system in place of Brainbow’s Cre/loxP, allowing for simultaneous...all-in-one versions which express self-excising Cre recombinase from the same transgene as the Brainbow cassette... 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 ...SUMO tags in the FP sequence for separation from Cre-based Brainbow-expressing cells. Autobow plasmids...

... from a sample is first amplified using RPA (recombinase polymerase amplification) or reverse transcriptase...the presence of your targeted nucleic acid. To increase the sensitivity of the assay, targeted DNA or ...non-specific (trans) ssDNA and harnessed this ability to create a DNA detection platform called DETECTR. How does...

... 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...

...and/or off-target effects (e.g. recombinase-dependent vectors) increases with the amount of virus you add...from Aschauer et al., 2013, Figure 1. Used under Creative Commons Attribution License. Within the nervous...can become methylated over time, resulting in decreased expression (Damdindorj et al., 2014; Nieuwenhuis...from Griffin et al., 2019, Figure 4. Used under Creative Commons Attribution 4.0 International License....vice versa. The total volume needed, however, increases with the size of the region you want to cover ...

...lentiviral vector, but transient expression of Cre recombinase allows for deletion of the inserted transgenes...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...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 ...