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The AAVpro Helper Free System (AAV2-CRE Recombinase) is a kit for the preparation of serotype 2 adeno-associated virus (AAV2) particles to deliver a Cre recombinase gene cassette to mammalian cells. This kit enables the preparation of high-titer AAV2 particles without a helper virus. Simply transfect the three included plasmids into HEK 293T cells to prepare the virus. This kit also includes an AAV Extraction Solution for efficient virus isolation.

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Analysis of ZsGreen1 expression in HEK 293 cells infected with AAV2-Cre particles

Analysis of ZsGreen1 expression in HEK 293 cells infected with AAV2-Cre particles. AAV2-Cre viral particles were prepared using the AAVpro Helper Free System (AAV2-CRE Recombinase) and purified using the AAVpro Purification Kit (AAV2). HEK293 cells engineered to express ZsGreen1 after Cre-mediated recombination were infected with either 100 vg/cell or 12,500 vg/cell of AAV2-Cre viral particles and analyzed by flow cytometry. The proportion of ZsGreen1+ cells increased with the amount of AAV2-Cre particles used for infection.

Cre Recombinase Gesicles are cell-derived nanovesicles used to deliver active Cre recombinase protein directly into target cells. When they are applied to your target cells, the gesicles fuse with the plasma membrane and deliver a temporary dose of Cre recombinase, which facilitates recombination at loxP sites contained within the cell. As an added benefit, Cre Recombinase Gesicles contain CherryPicker protein, a membrane-targeted version of the mCherry fluorescent protein that helps to confirm the delivery of the Cre recombinase to your target cells.

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293T cells are used to produce Cre Recombinase Gesicles, which allow rapid and efficient genome modification in the target cells of your choice

293T cells are used to produce Cre Recombinase Gesicles, which allow rapid and efficient genome modification in the target cells of your choice. Nanovesicle-inducing glycoproteins stimulate gesicle formation from the 293T cell line. iDimerize technology incorporates Cre recombinase into the gesicles and associates it with CherryPicker red fluorescent protein. After the gesicles pinch off from the producer cells, they fuse with the plasma membrane of the target cells and release Cre recombinase into the cell. The CherryPicker protein provides convenient visualization of delivery to your cells.

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ZsGreen1 reporter assay for efficiency of Cre Recombinase Gesicles with various cell lines

ZsGreen1 reporter assay for efficiency of Cre Recombinase Gesicles with various cell lines. pLVX-LoxP-ZsGreen1 contains ZsGreen1 cDNA separated from the EF1a promoter by a floxed stop cassette. In the presence of Cre recombinase, the stop cassette is removed, permitting high-level expression of ZsGreen1. All cell lines were transduced with this vector, followed by selection in puromycin to create stable lines. These lines were then exposed to a dilution series of recombinase-containing gesicles, centrifuged at 1,000 x g for 30 min at 32°C, and incubated for an additional 3 hr at 37°C. After 3 hr, media was exchanged and cells were permitted to grow for an additional 48 hr, at which time they were analyzed by FACS for ZsGreen1 expression.

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LacZ Reporter Assay for Cre Recombinase Gesicles

LacZ Reporter Assay for Cre Recombinase Gesicles. Panel A. Gesicle effectiveness was tested using a reporter cell line in which the lacZ gene is only expressed following removal of a stop codon by Cre recombinase activity. Cells were either transfected for 6 hr with a plasmid expressing Cre recombinase, or treated with 20 µl Cre Recombinase Gesicles for 3 hr. 24 hr after treatment, cells were stained for lacZ expression using the Beta Galactosidase Staining Kit (Cat. No. 31780). Panel B. The high concentration of blue cells in the center image indicate high recombinase activity. Red fluorescence in the right-hand image provides easy visualization of gesicle delivery to target cells.

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Visualization of ZsGreen1 reporter assay for Cre Recombinase Gesicles in various cell lines

Visualization of ZsGreen1 reporter assay for Cre Recombinase Gesicles in various cell lines. pLVX-LoxP-ZsGreen1 contains ZsGreen1 cDNA separated from the EF1a promoter by a floxed stop cassette. In the presence of Cre recombinase, the stop cassette is removed, permitting high-level expression of ZsGreen1. All cell lines were transduced with this vector, followed by selection in puromycin to create stable lines. These lines were then exposed to a dilution series of recombinase-containing gesicles, centrifuged at 1,000 x g for 30 min at 32°C, and incubated for an additional 3 hr at 37°C. After 3 hr, media was exchanged and cells were permitted to grow for an additional 48 hr, at which time they were imaged by fluorescence microscopy for ZsGreen1 expression.

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631449: Cre Recombinase Gesicles

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.

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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.

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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

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.

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0057VCT: RLP-CRE-ZsGreen1-11

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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.

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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.