... in recombinase-dependent sequences. Recombinase-dependent sequences may be subject to recombinase-independent...pre-recombined population that does not require the recombinase for transgene expression. Once packaged in AAV...pre-recombined DNA could lead to transgene expression in recombinase-negative animals, producing misleading results... hundreds of viral vectors and estimate that recombinase-independent recombination occurs in 0.1-0.6% ...transgene expression but limits the likelihood of recombinase-independent expression. Next-generation sequencing...parallel because the risk of accidental errors increases. In addition to human error, fragments of DNA ...to the reference sequence of the plasmid used to create the viral prep. In a clean viral prep, like AAV...

...transcription or Cre recombinase activity only in the presence of GFP. These systems, known as T-DDOG and Cre-DOG,...Although Cre isn’t a modular protein, Tang et. al were able to create a split version of Cre that is only...Podcast Interview with Connie Cepko Combining Cre and GFP with Cre-DOGs The T-DDOG results showed that GFP ...started with another biological workhorse, Cre recombinase, which induces recombination at LoxP sites.... of interest blocks transcription when Cre is absent. When Cre is present, it removes the STOP cassette...when dimerized by the GBPs/GFP. The new system, CRE-DOG (Cre Dependent On GFP), is activated by GFP and derivatives... cord. Cre-DOG is also suitable for optogenetic studies, and since cells infected with AAV-Cre-DOG retain...

...site-specific serine recombinase. First, the prime editor installs the recombinase “landing sites,” then...then the recombinase handles the installation of the donor sequence. The latest work evolves and engineers...engineers Bxb1 recombinase variants to optimize PASSIGE efficiency (Pandey et al., 2024). The best-performing...editing installs two sequences containing attB/P recombinase landing sites on opposite DNA strands. When the...edited sequence with the attB/P landing site. A recombinase integrates a sequence from a donor plasmid containing... in mammalian cells via continuously evolved recombinases and prime editing. Nature Biomedical Engineering...A., Tao, Y. A., Duby, J. E., Steinbeck, B. J., McCreary, J., Pierce, S. E., … & Liu, D. R. (2024). Efficient...

...limiting amount of AAV are injected into the brain of Cre-expressing mice to trigger recombination and expression... 2019. Sparse Predictive Activity through Recombinase Competition (SPARC), a genetic tool that allows...expressed and the cells are not labeled. PhiC31, a recombinase. It specifically integrates an attP site into...variety of fly cell types. Because the PhiC31 recombinase can work in other model organisms including mouse...site upstream of the stop cassette results in decreased efficiency of recombination and preferentially...

...encoding Cre recombinase under the control of the synapsin promoter. It can be used to drive Cre expression...be to inject your Cre-dependent mCherry mouse with the pmSyn-EBFP-Cre AAV. Because Cre expression is controlled... could breed your Cre-dependent mCherry line with a separate line that expresses Cre in neurons, but both...expression of any Cre-dependent gene of interest in neurons. This ability to be paired with any Cre-controlled...interest makes pmSyn-BFP-Cre highly modular. Further uses of pmSyn1-EBFP-Cre Of course there are also ... cre-recombinase...AAV that became available in 2017 is pmSyn1-EBFP-Cre from Hongkui Zeng’s lab. This AAV has had over 150...

...next experiment. Nucleic acid based machines Recombinase-based State Machines Enable Order-dependent Logic...inputs are received by a cell. These so-called recombinase-based state machines could potentially be used...development. Read this post to learn more about recombinase-based state machines and how they function. Adapting...reminds us that, although fluorescent proteins are incredibly useful, chemical fluorophores can still be used...Dangerous Viruses Contributed by guest blogger Tessa Cressy from the labs of Dr. Elke Mühlberger and Dr. Rachel...CombiGEM and CRISPR for Combinatorial Genetic Screening CombiGEM is a technology that can be used to quickly...barcode small libraries of CRISPR gRNAs to use for screening experiments. Get the full details on how to make...

...can remove the gene through the addition of Cre recombinase (Figure 2). Figure 2: A knockout targeting... can remove a floxed resistance gene using Cre recombinase. If GFP is under control of an endogenous promoter...you'll first remove the resistance marker using Cre recombinase. Since there are 3 loxP sites, recombination...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...so that it can be removed post-selection through Cre-mediated recombination.   Knockout/Knock-in...hence knock out the gene) through a second round of Cre recombination.  The future of knockout plasmids Although...

...pAAV-AiE0452h-minBG-iCre(R297T)-BGHpA (Alias: CN3781) Enhancer AAV for Cre recombinase expression in striatal...pAAV-AiE0779m_3xC2-minBG-iCre(R297T)-BGHpA (Alias: CN3779) Enhancer AAV for Cre recombinase expression in striatal...pAAV-AiE0450h-minBG-iCre(R297T)-BGHpA (Alias: CN3778) Enhancer AAV for Cre recombinase expression in striatal...in combination with Cre-dependent transgenic mouse lines or co-infection with Cre dependent viral vectors...PHPeB CN1851-rAAV-hI56i-minBglobin-iCre-4X2C-WPRE3-BGHpA AAV vector for Cre expression in forebrain GABAergic...pAAV-AiE0441h_3xC2-minBG-iCre(R297T)-BGHpA (Alias: CN4825) Expression of iCre(R297T) in striatal medium...network function. Browse plasmids or viral preps created with support from the BRAIN Armamentarium Project...

... or DIO. Lox sites are recognized by the recombinase Cre. Cre first flips the sequence between the first...Lox sites Lox sites, in combination with the Cre recombinase, are used to spatially and temporally control...flipped transgene. By restricting the availability of Cre, lox sites lets researchers control the spatial and... activation of a transgene. Figure 2: FLEX Cre-Lox system.  WPRE The Woodchuck Hepatitis Virus...which aids in nuclear export of mRNA and helps increase the expression of your transgene. The WPRE is ...

...105550 pAAV.GFAP.Cre.WPRE.hGH GFAP Cre-expression Cre Wilson 107738 pAAV-hSyn-Cre-P2A-dTomato Syn Cre-expression... Fishell Recombinases 105540 pENN.AAV.hSyn.HI.eGFP-Cre.WPRE.SV40 Syn Cre-EGFP expression Cre Wilson 105550...Cre-expression Cre Larsen 140135 pAAV-EF1a-iCreV EF1a light-inducible recombinase iCreV Zeng 140136 pAAV-EF1a-iDreV...Activator, Cre-dependent DREADD Roth 44362 pAAV-hSyn-DIO-hM4D(Gi)-mCherry Syn Inhibitor, Cre-dependent ...sensor, Cre-dependent GCaMP Kim , GENIE 104492 pGP-AAV-syn-FLEX-jGCaMP7f-WPRE Syn Calcium sensor, Cre-dependent...sensor, Cre-dependent GCaMP Kim , GENIE 104496 pGP-AAV-CAG-FLEX-jGCaMP7f-WPRE CAG Calcium sensor, Cre-dependent...Activator, Cre-dependent ChR2 Deisseroth 127090 pAAV-CAG-DIO-ChR2(H134R)-eYFP CAG Activator, Cre-dependent...

...infectious virus, plasmids expressing both the Cre and Flp recombinases to remove genes flanked by either loxP... a variety of reporters as well as the Cre and Flp recombinases. Genes can be cloned into these transfer...tdTomato under EF-1 promoter when Cre is expressed by another vector pCDH-CB-iCre-P2A-tdTomato-T2A-Puro 72255... 72253 Expresses copGFP under EF-1 promoter when Cre is expressed by another vector pCDH-EF1-DIO-tdTomato...72255 NOT AVAILABLE YET Cre is coexpressed with tdTomato and Pac (puromycin N-acethyl-transferase) pCDH-CB-copGFP-T2A-iCre...pCDH-CB-copGFP-T2A-iCre 72256 Expresses copGFP and iCre under from the CB promoter pCDH-CB-iCre 72257 Express... pAdx-CMV-iCre-p2A-copGFP 73350 Expresses iCre and EGFP from the CMV promoter pAdx-CMV-iCre-P2A-tdTomato...

...vector from the Gage lab encodes GFP and Cre-recombinase. The Cre-lox system is a technology that can be... Mammalian shRNA Tools for RNAi! Cre-Lox technology CAG-GFP-IRES-CRE (Plasmid #48201) This retroviral ...vector deposited by Dr. Tyler Jacks. It carries CRE recombinase and a puromycin resistance cassette. No time...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...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...

...instead of using piggyBac™, you could use recombinase technology (Cre/loxP) to remove the donor cassette. While...mutants [7], it is generally not as efficient as Cre recombinase. However, the use of negative selection largely... to DNA toxicity. More importantly, unlike Cre recombinase, the PBx is highly specific, and has not been...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...

...This is achieved through site-specific recombinases such as Cre or Flp that induce DNA recombination at...cells with an inactive opsin gene; in Cre expressing cells, however, Cre induces the recombination of the ...al. "Adeno-associated virus 2/9 delivery of Cre recombinase in mouse primary afferents." Scientific reports...recombination. Purification and properties of the Cre recombinase protein." Journal of Biological Chemistry 259.3...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...

... PMID: 30678704. Sparse cell labeling using recombinase competition Article contributed by Aliyah Weinstein...Weinstein Sparse Predictive Activity through Recombinase Competition (SPARC) is a fully genetic tool for... 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...

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

...Channelrhodopsins, Halorhodopsins, Archaerhodopsins Recombinases Cre, Flp, Dre Biosensors Calcium Sensors, Glutamate...

... 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...

...It’s an alternative to the conventional Cre or Flp recombinases used to excise markers. It has the advantage...doxycycline, but it can also be regulated spatially with Cre-recombination. Read the paper in Frontiers in Molecular...Cas12a inhibitors Using a bioinformatics and a screening approach, the Jennifer Doudna lab identified three...

...protein genes, Cre recombination is required before fluorescence can occur. Cre recombinase can be used ...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...melanogaster and is under the control of Flp recombinase. The XFPs in Flybow are membrane-tethered, making...