描述：
Premo™ FUCCI Cell Cycle Sensor is a fluorescent, two-color sensor of cell cycle progression and division in live cells. It allows accurate and sensitive cell cycle analysis of individual cells or a population of cells by fluorescence microscopy, flow cytometry, or high-content imaging.
The Premo™ FUCCI Cell Cycle Sensor is delivered by highly efficient BacMam 2.0 technology, enabling cell cycle studies in essentially any cell type. Provided in a ready-to-use format—simply add, incubate, and image—Premo™ FUCCI affords highly efficient transient expression in cell lines, primary cells, and stem cells.
Premo™ FUCCI Cell Cycle Sensor is:
• Accurate: Cell-cycle controlled expression of bright GFP and RFP indicators for live cell analysis of individual cells or populations
• Highly Efficient: >90% transduction of a wide range of mammalian cell lines, including primary cells, stem cells, and neurons
• Fast and Convenient: Simply add Premo™ FUCCI Cell Cycle Sensor to your cells in complete medium, incubate overnight, and analyze
• Safe: BacMam reagents are non-integrating and non-replicating in mammalian cells, lack observable cytopathic effects, and are suitable for biosafety level (BSL) 1 handling
Study Cell Cycle in the Relevant Cellular Context
If you are studying cell cycle regulators, cell differentiation, or are developing anti-cancer compounds against cell cycle-related targets, Premo™ FUCCI is a superbly sensitive and accurate sensor tool. The efficient transduction and transient expression allows you to conduct these experiments in the biologically relevant cell types.
Simple, Quick Cell Cycle Visualization
Simply add the ready-to-use Premo™ FUCCI cell cycle sensor to your cells in complete medium, incubate overnight, then visualize cell cycle progression in populations of cells using fluorescence microscopy, flow cytometry, or high content imaging instruments. Cells change from red in the G1 to yellow in the G1/S interphase and green in S, G2, and M phases, as fusions of emGFP and TagRFP coupled to two cell cycle-regulated proteins are expressed and degraded.
How It Works
Premo™ FUCCI is derived from the fluorescence ubiquitination cell cycle indicator (FUCCI), developed by Miyawaki and colleagues (Ref 1). FUCCI is based on two cell cycle-regulated proteins, geminin and Cdt1, fused to one green (emGFP) and red (TagRFP) fluorescent protein, respectively. As Cdt1 and geminin are present only during specific phases of the cell cycle, the fluorescent protein partner is similarly cell-cycle dependent. Ubiquitination by specific ubiquitin E3 ligases target the fusion constructs in the proteasome for degradation. E3 ligases display temporal regulation of activity, resulting in biphasic cycling of geminin and Cdt1 levels during the cell cycle. Geminin-GFP is degraded in the G1 phase; the presence of Cdt1-TagRFP is indicated by red fluorescence within nuclei. During the S, G2, and M phases, however, Cdt1 is degraded and only geminin-GFP remains, giving cells green-fluorescent nuclei. During the G1/S transition, when Cdt1 levels are decreasing and geminin levels are increasing, both proteins are present, giving a yellow-fluorescent nuclear signal. This dynamic color change (red to yellow to green) clearly displays progression through the cell cycle and division.
Powered by BacMam 2.0 Technology
BacMam technology is based on an insect virus (baculovirus) for efficient transduction and transient expression in mammalian cells. The Premo™ FUCCI cell cycle sensor uses BacMam 2.0 technology to enable cell cycle studies in essentially any cell type, from cell lines to stem cells by incorporation of a mammalian expression cassette for the FUCCI construct. Compared with BacMam 1.0 technology, BacMam 2.0 does not require the use of an enhancer and can be used in complete medium. There is no need to purify plasmid or worry about vector integrity and quality. No lipids, dye-loading chemicals, or other potentially harmful treatments are required. BacMam 2.0 incorporates elements that greatly enhance transduction efficiency and expression levels: a pseudotyped capsid protein for more efficient cell entry, an enhanced CMV promoter, and the Woodchuck hepatitis post-transcriptional regulatory element (WPRE).
Titratable Expression that Lasts Up to Two Weeks
The transient transgene expression lasts from about 5 days in transformed cell lines to up to two weeks in slowly dividing cells, such as some primary cell types; we have imaged terminally differentiated neurons more than three weeks after transduction. In addition, BacMam technology permits defined optimization because it gives you the ability to precisely titrate expression levels. Baculoviruses do not replicate in mammalian cells, nor are viral genes expressed, giving them an excellent safety profile and lack of cytopathic effects on mammalian cells.
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