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Cre recombinase RLPs

Cre RNA lentiviral particles

Obtain a rapid burst of Cre expression in mammalian cells with footprint-free delivery of Cre mRNA in nonintegrating, virus-like particles. Cre RNA lentiviral particles (RLPs) contain no viral genome and are generated using a novel packaging technology that loads multiple biologically active mRNAs per particle. RLPs have been shown to mediate transient protein expression at high efficiency for in vitro and in vivo applications involving cell lines, primary cells, stem cells, tissues, and organisms.

Obtain a rapid burst of Cre expression in mammalian cells with footprint-free delivery of Cre mRNA in nonintegrating, virus-like particles. Cre RNA lentiviral particles (RLPs) contain no viral genome and are generated using a novel packaging technology that loads multiple biologically active mRNAs per particle. RLPs have been shown to mediate transient protein expression at high efficiency for in vitro and in vivo applications involving cell lines, primary cells, stem cells, tissues, and organisms.

Popular methods for Cre recombinase-based genome editing present tradeoffs. Transient transfection of a plasmid or mRNA limits the scope of Cre activity, minimizing the likelihood of unwanted effects associated with persistent Cre expression, but is associated with cellular toxicity, is unsuitable for in vivo applications, and is only feasible for cells that can be readily transfected. Transduction of the Cre gene sequence on a viral vector enables Cre expression in a broader range of cell types and can be applied to in vivo studies, but is associated with random genomic integration, may require activation and/or selection of target cells, and may result in undesirable persistence of Cre expression.

Cre recombinase RLPs enable researchers to overcome the above limitations and express Cre more rapidly and efficiently in mammalian cells by delivering nonviral Cre mRNA in lentivirus-derived packaging in the absence of a viral genome. In vitro studies involving RLP-mediated delivery of a luciferase reporter demonstrated that reporter expression was maximal at 4–24 hours post application and had declined rapidly at 30 hours. By contrast, expression of luciferase delivered via transfection was undetectable until 8 hours and peaked at 24 hours, while expression via lentiviral integration was undetectable until 24 hours. In an in vivo study involving a loxP mouse line (ROSA26-YFP), local injection of Cre RLPs into muscle yielded a zone of reporter gene expression that was comparable in size to a zone generated via injection of purified Cre lentivirus (Prel et al. 2015).

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Cat. # Product Size Price License Quantity Details
0059VCT RLP-CRE-11 2 x 20 uL USD $1802.00

License Statement

ID Number  
353 LentiFlash Technology: This product is covered by the French pending patent application FR1554381 and the PCT application PCT/FR/051152. The purchase price of this product includes limited, nontransferable rights to use this product solely for internal research purposes. Please contact Vectalys for information on purchasing a license to use these products for commercial purposes: Parc Technologique du Canal, Bâtiment Canal Biotech II, 3, rue des satellites, 31400 Toulouse (France), TEL: +33(0)5 61 28 70 75, EMAIL: sandy.darrigan@vectalys.com.

Cre RLPs (RNA lentiviral particles) consist of biologically active Cre recombinase mRNAs packaged into nonintegrating, virus-like particles, and are used to mediate a transient burst of Cre expression for a wide array of in vitro and in vivo applications involving Cre-Lox recombination. Footprint-free Cre delivery via RLPs avoids the risks of random genomic integration and persistent Cre expression associated with viral transduction, and the cellular toxicity and target-cell limitations attributed to RNA or plasmid-based transfection. Cre RLPs are generated using a novel packaging technology that employs a phage RNA-binding protein to load multiple mature mRNA molecules per particle. RLPs have been demonstrated to yield robust Cre expression with high efficiency in cell lines, primary cells, stem cells, tissues, and organisms.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Image Data

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Dose-dependent, RLP-mediated expression of ZsGreen1 observed over time

Dose-dependent, RLP-mediated expression of ZsGreen1 observed over time
Dose-dependent, RLP-mediated expression of ZsGreen1 observed over time.The indicated volumes of CRE-ZsGreen1 RLPs were applied to TE671 LoxP-LacZ cells in a 12-well format, and ZsGreen1 expression at the indicated time points was monitored by fluorescence microscopy. At 6 hours post application, ZsGreen1 expression was detectable in each cell population and varied according to the amount of RLPs applied. For each RLP dosage tested, ZsGreen1 expression was highest at 24 hours and greatly reduced at 48 hours post application.

Back

Kinetics of Cre recombinase activity compared for RLP-, lentivirus-, and gesicle-mediated delivery

Kinetics of Cre recombinase activity compared for RLP-, lentivirus-, and gesicle-mediated delivery
Kinetics of Cre recombinase activity compared for RLP-, lentivirus-, and gesicle-mediated delivery.�To compare the kinetics of Cre recombinase activity using various delivery methods, Cre was expressed in TE671 LoxP-LacZ cells via application of RLPs, lentivirus, or gesicles (cell-derived nanovesicles carrying Cre protein), respectively, in 2-fold serial dilutions. Cells from each treatment were harvested and lysed at the indicated time points (8, 24, and 48 hours, respectively), and the expression of the LacZ reporter was assayed over a 1-hour period using the Luminescent Beta-galactosidase Detection Kit II (Cat. # 631712). For each delivery method, the MOI corresponding to the highest treatment concentration is indicated in the graphs corresponding to the 8-hour timepoint. In contrast with viral delivery, application of Cre RLPs (Cre RNA) and Cre gesicles (Cre protein) yielded detectable Cre recombinase activity after 8 hours, with Cre gesicles providing the fastest response. For viral delivery, Cre activity was detectable after 24 hours, but at a low level relative to the other methods. At 48 hours, cumulative Cre activity was comparable for the three delivery methods.

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0059VCT: RLP-CRE-11

0059VCT: RLP-CRE-11
0057VCT RLP-CRE-ZsGreen1-11 2 x 20 uL USD $1802.00

License Statement

ID Number  
352 LentiFlash Technology: This product is covered by the French pending patent application FR1554381 and the PCT application PCT/FR/051152. The purchase price of this product includes limited, nontransferable rights to use this product solely for internal research purposes. Please contact Vectalys for information on purchasing a license to use these products for commercial purposes: Parc Technologique du Canal, Bâtiment Canal Biotech II, 3, rue des satellites, 31400 Toulouse (France), TEL: +33(0)5 61 28 70 75, EMAIL: sandy.darrigan@vectalys.com.

Cre-ZsGreen1 RLPs (RNA lentiviral particles) consist of biologically active Cre recombinase and ZsGreen1 mRNAs packaged into nonintegrating, virus-like particles, and are used to mediate a transient burst of Cre and fluorescent protein expression for a wide array of in vitro and in vivo applications involving Cre-Lox recombination. Footprint-free Cre delivery via RLPs avoids the risks of random genomic integration and persistent Cre expression associated with viral transduction, and the cellular toxicity and target-cell limitations attributed to RNA or plasmid-based transfection. Cre-ZsGreen1 RLPs are generated using a novel packaging technology that employs a phage RNA-binding protein to load multiple mature mRNA molecules per particle. RLPs have been demonstrated to yield robust Cre and ZsGreen1 expression with high efficiency in cell lines, primary cells, stem cells, tissues, and organisms.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Image Data

Back

0057VCT: RLP-CRE-ZsGreen1-11

0057VCT: RLP-CRE-ZsGreen1-11

Back

Dose-dependent, RLP-mediated expression of ZsGreen1 observed over time

Dose-dependent, RLP-mediated expression of ZsGreen1 observed over time
Dose-dependent, RLP-mediated expression of ZsGreen1 observed over time.The indicated volumes of CRE-ZsGreen1 RLPs were applied to TE671 LoxP-LacZ cells in a 12-well format, and ZsGreen1 expression at the indicated time points was monitored by fluorescence microscopy. At 6 hours post application, ZsGreen1 expression was detectable in each cell population and varied according to the amount of RLPs applied. For each RLP dosage tested, ZsGreen1 expression was highest at 24 hours and greatly reduced at 48 hours post application.

Back

Kinetics of Cre recombinase activity compared for RLP-, lentivirus-, and gesicle-mediated delivery

Kinetics of Cre recombinase activity compared for RLP-, lentivirus-, and gesicle-mediated delivery
Kinetics of Cre recombinase activity compared for RLP-, lentivirus-, and gesicle-mediated delivery.�To compare the kinetics of Cre recombinase activity using various delivery methods, Cre was expressed in TE671 LoxP-LacZ cells via application of RLPs, lentivirus, or gesicles (cell-derived nanovesicles carrying Cre protein), respectively, in 2-fold serial dilutions. Cells from each treatment were harvested and lysed at the indicated time points (8, 24, and 48 hours, respectively), and the expression of the LacZ reporter was assayed over a 1-hour period using the Luminescent Beta-galactosidase Detection Kit II (Cat. # 631712). For each delivery method, the MOI corresponding to the highest treatment concentration is indicated in the graphs corresponding to the 8-hour timepoint. In contrast with viral delivery, application of Cre RLPs (Cre RNA) and Cre gesicles (Cre protein) yielded detectable Cre recombinase activity after 8 hours, with Cre gesicles providing the fastest response. For viral delivery, Cre activity was detectable after 24 hours, but at a low level relative to the other methods. At 48 hours, cumulative Cre activity was comparable for the three delivery methods.

Overview

  • Efficient delivery of active Cre mRNA via nonintegrating, virus-like particles (RLPs) enables rapid, transient Cre expression in mammalian cells
  • Footprint-free RNA delivery avoids the use of a viral genome and risks associated with random genomic integration or persistent Cre expression and does not require activation or selection of target cells
  • Suitable for a wide array of in vitro and in vivo applications, including use in immortalized cell lines, dividing and nondividing primary cells, stem cells, tissues, and various model organisms
  • Available RLP formulations include Cre mRNA only (RLP-CRE-11, 0059VCT), or a combination of Cre and ZsGreen1 mRNAs (RLP-CRE-ZsGreen1-11, 0057VCT)

More Information

Applications

  • Gene knockout or knockin studies involving loxP-based site-specific recombination
  • Transient Cre expression in a wide array of in vitro and in vivo systems, including cell lines, primary cells, stem cells, mouse models, etc.

References

Prel, A. et al. Highly efficient in vitro and in vivo delivery of functional RNAs using new versatile MS2-chimeric retrovirus-like particles. Mol Ther Methods Clin Dev. 2:15039 (2015).

Additional product information

Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.


Tools for delivering Cre recombinase

Rapid, efficient Cre recombinase delivery for genome modification

See how gesicle technology outperforms plasmid transfection for efficient delivery of Cre recombinase in most cell types.

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