... how Cre-lox recombination works. Advances in Cre-lox recombination have included inducible Cre, such...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...remaining cassette can invert as long as Cre remains expressed; once Cre is removed from the system, the fluorescent...

.... 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...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...mice, conditional knockout/knockin mice, use the Cre-lox recombination system to express the transgene...

...efficiency of Cre recombination varies for different constructs and cell types. Second, Cre may induce recombination...expression of Cre from the standard UAS/GAL4 system is toxic to proliferating cells. A Cre-estrogen receptor...Cre-lox recombination is an incredibly useful molecular biology tool, but like any biological system,... developmental aberrations. In multiple systems, Cre itself, without the presence of a floxed construct...contrast, Dre recombinase, the close relative of Cre, displayed an excision efficiency of only 70%. To...nontoxic. In contrast, 0/79 offspring carried both Cre and GAL4, further emphasizing the toxicity of this...more information about limitations and caveats of Cre usage: Vagaries of conditional gene targeting. Schmidt-Supprian...

...heterozygous for each mutated allele.  Cre-lox expressing strains Cre-lox strains are a common example of... expressing both Cre and LoxP occurs. But how are these mice made to begin with? Cre-lox mice are made...strain that expresses Cre with a strain that expresses a floxed gene of interest. Cre may be expressed globally...using Cre-lox Read our genome engineering blog posts Resources on Addgene.org Find more about Cre-lox ...cover why and how to cross mice to create double knockout lines and Cre-lox lines, and how to properly control...carriers at those alleles. Some alleles, such as Cre, function when hemizygous, meaning that only one ... be either hemizygous or null at one locus (e.g. Cre) and heterozygous at the other. 3. Breed your F1 ...

...combines the Cre/lox system with optogenetics. Cre activity is controlled by red light by splitting Cre and fusing... Figure 4: The C-terminus of Cre (CreC) and the N-terminus of Cre (CreN) are fused to the truncated Phytochrome... previous Cre/lox or drug-induced Cre systems (ex: tamoxifen). As the name suggests, CreLite combines ... dimerize, bringing each terminus of Cre together to restore Cre activity. As soon as red light or PCB...proteins separate, inactivating Cre. This is critical for reducing Cre toxicity in long-term experiments... and PIF6APB binding, bringing the two halves of Cre together. Image from Eisenhoffer et al., 2019. ...express various fluorescent proteins controlled by Cre recombination. Each cell can have multiple copies...

...Mary Dunlop’s lab generated a photosensitive Cre, Opto-Cre-Vvd, by splitting and joining the protein regions...C-terminal and N-terminal Cre fragments together and permitting recombinase activity. Opto-Cre-Vvd is precise, ...light. Unlike other split-Cre systems which can take up to 24 hours to cut, Opto-Cre-Vvd is fast and can cut...pAAV-hSyn-Cre-P2A-dTomato is now available in AAV1 and AAV8, as well as AAV5 and AAVrg. Find the Cre-expressing... you'll find in this post: Blue-light inducible Cre for use in bacteria CRISPR-Casɸ mGold, a new yellow...the viral service A fast blue-light inducible Cre for precise control of bacterial gene expression ...recombinase activity were exposed to blue light. Cre-Vvd showed substantially improved activity as compared...

...PI 60226 AAV:ITR-U6-sgRNA(backbone)-pEFS-Rluc-2A-Cre-WPRE-hGHpA-ITR Renilla EF1α AAV9-CRISPR knockout ...promoter/enhancer regions. Blue/white screening can be used for screening of transformants in cloning process...transcriptional reporters. However, they are not secreted, so cells must be lysed or the substrate must ...to measure luminescence. Gaussia luciferase is secreted and more stable, making time course experiments...size. NanoLuc® Luciferase is small, bright, and a secreted version of the protein is available – these attributes...luciferase mutants : A set of three sensors that are increasingly structurally destabilized versions of Firefly.../green light and red light, respectively, for increased usefulness in in vivo applications. LuxSit-i :...

...dependent Cre. The two segments come together in the presence of GFP to form a functional Cre enzyme that...ChR2 for targeted expression DREADDs GFP dependent Cre and FLPo And more!  New lentiviral preps SARS-...control plasmids in new serotypes, as well as a new Cre dependent GFP control plasmid for expression in forebrain...future therapies. Our popular plasmid encoding a Cre dependent version of the DREADD hM3D is now also ...Roth   Recombinases Recombinase systems such as Cre-lox allow for more accurate cell and tissue targeting...recombinases from the Cepko Lab are now available as AAV1. CRE-DOG constructs 69570 and 69571 together encode the...restrict gene activation to cells expressing both Cre and GFP. The GFP dependent FLPo, Flp-DOG (75469),...

...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...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...when crossed with mice expressing the appropriate Cre allele. The Confetti mouse contains the Brainbow ...

...cells with an inactive opsin gene; in Cre expressing cells, however, Cre induces the recombination of the ...achieved through site-specific recombinases such as Cre or Flp that induce DNA recombination at defined recognition...loxP and lox511) in opposite orientations. Since Cre does not cause recombination between mismatched recognition...specific neuronal cells? This can be achieved using Cre-dependent viruses, such as the optogenetic FLEx vectors...combination with transgenic animals or rAAVs expressing Cre under a specific cell-type promoter. Once injected...behind the mechanisms of sleep and anesthesia. In Cre transgenic mice, they targeted the dopamine (DA) ...of FLEx vectors combined with the use of several Cre transgenic mice targeting different brain areas enabled...

...Flp-dependent Cre AAV in AAV9 and AAVrg. See our new additions here: Cre vectors pAAV-EF1a-fDIO-Cre (121675...expression. This is a Cre-dependent switch that expresses nuclear mCherry in the absence of Cre, and expresses...expresses nuclear EGFP in the presence of Cre. Biosensor AAV (calcium sensors and GABA sensors) Calcium sensors...sensor (see below), in addition to our inventory of Cre-dependent GABA sensors. See all our GABA sensor AAV...reduced. This variant has an approximately 12-fold increase in brightness after ~100 action potentials. This...121675-AAV9, 121675-AAVrg)  pENN.AAV.hSyn.Cre.WPRE.hGH (105553-AAV5) Dre vector AAV phSyn1(S)-DreO-bGHpA...

... Mammalian shRNA Tools for RNAi! Cre-Lox technology CAG-GFP-IRES-CRE (Plasmid #48201) This retroviral ...vector from the Gage lab encodes GFP and Cre-recombinase. The Cre-lox system is a technology that can be...the pLKO.1 vector can be found here. Cre-Lox technology Puro.Cre empty vector (Plasmid #17408)   This ...of mouse transgenics. Experimental uses include: Cre-dependent gene expression, gene knockout, and selection... vector deposited by Dr. Tyler Jacks. It carries CRE recombinase and a puromycin resistance cassette. ...shRNA), mutation/editing (CRISPR-Cas9) or deletion (Cre), together with expression of fluorescent or enzymatic...CRISPR pooled libraries that allow functional screening of thousands of genes in genome-wide experiments...

... of Cre-dependent mouse lines for overexpressing genes within discrete cell populations makes Cre-dependent...Constitutive or inducible Cre recombinases can be introduced by AAV or by mating with Cre-expressing mice. ...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...

...by injecting a Cre-dependent AAV into Broca’s area of a mouse specifically expressing Cre in the hippocampus... However, the Cre-dependent AAV cargo would only become activated in the presence of Cre recombinase, ...genetically controlled function. By using AAVrg in Cre transgenic mouse lines, you can access and manipulate...hippocampus (Fig. 3). The Cre-dependent AAV would access the neurons (Fig. 3A) that project to Broca’s area...a retrograde recombinase-expressing vector (e.g. Cre or FLP) to transduce the projection pathway neurons..., 2022) and substantia nigra (Tervo, 2016). Discrepancies in transduction efficiency can have a major ...

...want to create a conditional knockout. To make a conditional knockout, researchers often use the Cre/Lox ...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...example, you can remove a floxed resistance gene using Cre recombinase. If GFP is under control of an endogenous... you'll first remove the resistance marker using Cre recombinase. Since there are 3 loxP sites, recombination...hence knock out the gene) through a second round of Cre recombination.  The future of knockout plasmids Although...

...transferable skill: creativity. I’ve heard quite a few scientists state that they’re not creative, usually saying... artistic, and therefore not creative. However, if we think of creativity as a form of innovation, it’...clear that creativity is absolutely a transferable skill developed during PhD training. Creativity is required...innovation. If you identify as a non-creative or if you find yourself in a creative rut, read on!   Fig ...had to think creatively to solve a problem. But how do you intentionally engage in a creative problem-solving...Grant, a creativity expert at Stanford conducted an experiment in which people were asked to create a device... 2: Blugene likes to spark creativity in out on the open sea Creativity comes in many forms. Whether...

...provide informative content to help researchers in creating and sharing knowledge, related to the types of...Identify needed content Let’s take a look at how we create this content, using the example of our Antibody...Tweet, followed by very detailed answers from our incredible scientists. Expert reviews on blog posts are ...need to reach out to an external expert to help us create accurate content. Regardless of the process, everything...

...right for you. Screen variations Forward and reverse screens Forward and reverse screens involve manipulating... suppressor screens These types of screens are very similar to forward and reverse screens, except that...approaches. Screens and selections The common goals The confusion in differentiating between a screen and a ...reverse screen – all known genes are assayed for their role in a particular capacity. Drug screens, on the... time screening hits that aren’t “real” or novel for their purposes… or worse yet have a screen that doesn...phenotype. These screens employ the same downstream manipulations as a forward or reverse screen (CRISPR, drug...component. Alternative screening techniques While CRISPR and drugs dominate the screening scene, there are ...

...variety of screens, including interference, activation, and knockout. Most CRISPR screens rely on lentiviral..., AAV vector screens may be AAVantageous!       In vivo, in vitro, ex vivo CRISPR screens can be carried... Figure 1: Overview of in vitro AAV CRISPR screens. Created with BioRender.com. The in vitro option... For in vivo screens, mice are directly infected with the CRISPR library. In vivo screens can become costly...engineered in labs. This vastly increases the possibilities when designing screens. Each serotype has its own...CRISPR Delivery Genome-wide Screening Using CRISPR Performing In Vivo CRISPR Screens Using the FITS Approach...Forward genetics screens are a valuable part of the molecular biology toolbox to identify new target ...

...genome-wide screens? The goal of a genome-wide screening experiment is to generate and screen a population...transfer vectors to create the CRISPR library. CRISPR libraries have been created to knock out, activate...the steps involved in a CRISPR screen? Performing a forward genetic screen using CRISPR libraries is a multi-step...sorting and screened for a particular phenotype. For example, mutant cells can be used in drug screens to identify...means to carry out so-called “forward genetic screening experiments”, it is certainly the most robust....libraries are being used to perform genome-wide screens and highlight some of the reagents that have been...CRISPR can be readily scaled up for genome-wide screening due to the broad range of potential target sequences...