Widely used for genome modification (e.g., knockout and knockin studies), Cre recombinase has conventionally been delivered to cells via a plasmid or viral gene delivery. While widely adopted, these conventional methods have some drawbacks, stemming from the use of nucleic acids for the introduction of Cre recombinase to target cells. These disadvantages include inefficient excision, unintended recombination events due to persistent expression, and lengthy protocols.
Cre Recombinase Gesicles are cell-derived nanovesicles that deliver active Cre recombinase protein directly to your target cells. Simpler to use than either plasmid or viral gene delivery, these gesicles allow you to quickly and efficiently flox a broad range of cell types on demand. This one tool simultaneously saves time and opens up new experimental possibilities. Please visit our overview page for a summary of the advantages of this system and general information on Cre recombinase.
Results
Concentrated Cre recombinase introduced directly into target cells
Cre Recombinase Gesicles overcome the shortcomings of viral and plasmid Cre recombinase delivery by enabling the introduction of Cre protein directly to target cells. This is accomplished without the co-delivery of its coding gene, thus minimizing the chance of unintended recombination events.
How we make them (Figure 1):
- 293T cells express both Cre recombinase and a particular nanovesicle-inducing glycoprotein responsible for elevating gesicle production from the cell membrane.
- iDimerize technology is used to enrich Cre recombinase in the gesicles. During gesicle formation, a dimerizer ligand forces interaction between Cre recombinase and the membrane-localized CherryPicker protein.
- Loaded Cre Recombinase Gesicles are harvested from the supernatant, thus providing a concentrated stock for use on target cells.
How you use them (Figure 2):
- Simply apply a few microliters of Cre Recombinase Gesicles to your target cells in the presence of Polybrene. We recommend a brief plate centrifugation step at 1,000g to enhance gesicle-to-cell contact.
- The gesicles fuse with the target cell membrane. Your cells will be transiently labeled by the CherryPicker red fluorescent protein, allowing visual confirmation of delivery.
- At the same time, active Cre recombinase is released into your target cells; the lack of dimerizer ligand in your target cell culture medium allows the Cre protein to dissociate from the CherryPicker protein.
Figure 1. Mechanism of nucleic-acid-free genome modification with gesicle technology. Gesicle production is stimulated by over-expression of a nanovesicle-inducing glycoprotein. Cre recombinase is enriched in the gesicles using iDimerize technology. Loaded gesicles merge with target cells to deliver active Cre recombinase.
Figure 2. LacZ reporter assay for Cre Recombinase Gesicle activity. Top Panel. 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. Bottom Panel. 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.
Gesicles outperform plasmid transfection
Cre Recombinase Gesicles make it possible to flox your cells on demand. Cre recombinase is only present when you need it, and acts quickly and reliably within your target cells. In contrast, recombinase delivery by plasmid transfection is followed by a delay in expression due to transcription and translation of the protein. Gesicles avoid this delay because the protein is present and active as soon as the gesicles are applied to the cells. This high level of efficiency is easily visible in the reporter assay described above, where expression of lacZ is only possible when an upstream loxP-flanked stop codon is excised by Cre recombinase. Following staining for LacZ expression, cells display markedly faster Cre recombinase activity following the gesicle treatment compared to plasmid transfection (Figure 3).
Figure 3. Comparison of Cre recombinase activity from gesicle delivery and plasmid transfection. Cre recombinase activity was tested using the LacZ reporter cell line, wherein the lacZ gene is only expressed following Cre recombinase activity. Delivery via Cre Recombinase Gesicles resulted in faster activity than via plasmid transfection.
Efficient delivery and high expression in most cell types
This protocol requires no preparation or pretreatment of your cells and is gentle enough to use with any cell type, including dividing and nondividing cells, primary cells, and cell lines. Cre Recombinase Gesicles maintain efficient delivery and high levels of activity regardless of cell type (Figure 4).
Figure 4. ZSGreen1 reporter assay. Gesicles were tested using a reporter cell line where ZsGreen1 is only expressed following Cre recombinase activity. Figure. Fluorescence microscopy images of HepG2, Jurkat, and Raji cell lines exposed to Cre Recombinase Gesicles. Table. FACS analysis of each cell line after exposure to Cre Recombinase Gesicles.
Conclusions
Cre Recombinase Gesicles provide a fast, easy method for efficient genome modification without the use of nucleic acids. Recombinase expression is kept under strict control—unintended recombination events are less likely due to the lack of persistent expression of Cre. Since no transcription or translation steps are needed, target cells are floxed much faster than with plasmid or viral delivery methods. Gesicles show excellent performance in most cell types, thus expanding research possibilities.
Methods
For ZsGreen1 reporter assays, cell lines were transduced with the pLVX-LoxP-ZsGreen1 vector, with subsequent selection in puromycin to create stable lines. The vector 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 expression of ZsGreen1. Stable lines were exposed to 20 µl Cre Recombinase Gesicles, centrifuged at 1,000g for 30 min at 32°C, and incubated for an additional 3 hr at 37°C. Media was then exchanged and cells were permitted to grow for an additional 48 hr, at which time they were imaged by fluorescence microscopy and analyzed by FACS for ZsGreen1 expression.
The same vector was used for LacZ reporter assays, substituting lacZ for ZsGreen1 cDNA. Cells were either transfected (using Xfect Transfection Reagent) for 6 hr with a plasmid expressing Cre recombinase, or treated with 20 µl of Cre Recombinase Gesicles for 3 hr. 24 hr after treatment, cells were stained for LacZ expression using the Beta-Galactosidase Staining Kit.
