...Versatile Viral Tool for Gene Expression in Mammals Recombinase-based State Machines Enable Order-dependent Logic...fluorophores other than fluorescent proteins can be incredibly useful in FRET experiments. Professor Fessenden's... also really helped put this technology into a concrete context. I recommend this post to anyone wondering...

...63704 pRetroX GFP T2A Cre CMV/MSV Tetracycline inducible expression of GFP T2A Cre fusion in mammalian ...overexpression plasmid; deletion of dsRed by Cre recombinase results in the rapid loss of dsRed and the ...largeTcDNA MoMLV Expression of Large T antigen for creation of immortalized cells (see Weinberg Lab page for...pBABE-hygro-hTERT MoMLV Expression of hTERT for creation of immortalized cells; additional plasmids include...cloning and gene expression; select with puromycin or screen for GFP Bartel 32702 pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE... post-transcriptional regulatory element that increases export to the cytoplasm Hahn 11375 MDH1-PGK-GFP...

...fluorescent proteins (EGFP, mCherry etc.), recombinases (Cre, Flp etc.), and tetracycline drug induction...But CRISPRing typical mouse models, including creating Cre-dependent conditional alleles, has remained ...marker, likely by breeding to a mouse line with a recombinase. If it turned out your chimeric mice did not ...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-...

...genetic tools: activators, inhibitors, sensors, recombinases, and more. With the help of adeno-associated...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...

...DreO-bGHpA 121675  AAV9  pAAV-EF1a-fDIO-Cre (FLP-dependent CRE) 55634  AAV1  pAAV-EF1a-mCherry-IRES-... constitutive (pAAV-Syn-Archon1-KGC-GFP-ER2) and cre-dependent version (pAAV-Syn-FLEX-rc [Archon1-KGC-...membrane-embedding voltage detector. Find Archon1 and its cre-dependent version at Addgene! Fluorescent protein...Chimeric channels for neuronal manipulation Recombinases Plasmid Serotype Name 50363  AAV5  AAV ...

...extracted from a sample and subject to isothermal recombinase polymerase amplification (RPA) using a commercially...contains SSB, strand displacement DNA polymerase, recombinase, and the ssDNA-FQ reporters that exhibit fluorescence...based detection of SARS-CoV-2 genetic material (CREST) Sensitivity: 10 copies of RNA per microliter Time...

...CRISPR genome editing.   2. Plasmids 101: Cre-lox Cre recombinase can be used to generate inversions, deletions...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 ...

...open-reading frame (DIO). Upon coexpression with cre recombinase (either from a mouse line or viral co-injection...specificity dependent on the promoter of the cre recombinase while still taking advantage of the robust ...an appropriate mouse line expressing a particular cre, viral expression of your opsin is no longer required...tracing with fluorescent proteins in nontransgenic and cre driver mice. Current protocols in neuroscience / ...: PMC4242517.  7. Madisen L, et al. A toolbox of Cre-dependent optogenetic transgenic mice for light-induced... us to show causality in neural circuits with incredible temporal and spatial precision (I’m not going...optogenetics has already been proven. Therefore, a concrete, well defined, and robust behavioral output is...

...pAAV-hSyn Con/Fon hChR2(H134R)-EYFP that is both Cre and Flp dependent. Plasmid Serotype Name 124603...mCherry)-EGFP 50457 AAV2 pAAV-hSyn-DIO-EGFP Recombinases Plasmid Serotype Name 87306 AAVrg, AAV5...

...made with BioRender.   Grow plasmids in recombinase-deficient strains Since plasmid multimerization...plasmid in a bacterial strain that is deficient in recombinase protein RecA can reduce the frequency of multimerization...generated by SnapGene software.  Thanks to increasing affordability and accessibility, more researchers...by growing seed stocks at 30°C compared to 42°C. Screen multiple colonies to isolate the monomer Sometimes... 1984; Summers et al., 1993). In these cases, screening multiple bacterial colonies using one of the detection...DNA-forming sequences and WRN deficiency independently increase the frequency of base substitution in human cells... self-control and a sense of direction are the secrets of multicopy plasmid stability. Mol Microbiol. ...

...RNAi, empty backbones, fluorescent proteins, and Cre expression plasmids. Large collections or kits are.... elegans by combining expression of the FLP recombinase from either a heat-shock promoter or a tissue-specific...

...will only be expressed in cells that express cre recombinase. Typically, this feature is used to attain ...Expression of hM4D(Gi)-mCherry from this virus is cre-dependent, meaning that once injected, the DREADD...cell-specific expression in transgenic animals, where cre expression is restricted to a particular cell type...the first time, the Roth Lab recommends verifying cre expression prior to use. The lab additionally recommends...expressing cell lines, which can then be used both for screens (using pooled CRISPR knockout libraries) and individual...Secondly, you can use a LentiCas9-Blast viral prep to create a single stable Cas9-expressing cell line and then...monoclonal Cas9-expression line prior to performing a screen or even a single gene knockout. Top AAV: pAAV-hSyn-DIO-hM4D...

...excised by intentional introduction of the Cre recombinase. This is a time-tested system but does require...control when you turn off expression for good, the Cre-Lox system offers inducible yet permanent gene excision...some permanent knock out plasmids? Check out our Cre vector and Murine LoxP vector.   Turn it off for ...!  Turn it on Turn it on forever Permanently increasing or turning on expression of a new protein is ... gRNA, to your gene of interest to temporarily increase expression. There are several flavors of activators...

.... 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...