23-02-2013, 04:18 PM
High-throughput stable cell line generation
HIGH.pdf (Size: 545.75 KB / Downloads: 23)
Introduction
Stably transfected cell lines are used extensively in drug discovery. Cell lines expressing a target of interest, such as a G-protein coupled receptor (GPCR) or a reporter gene, form the basis for most cell-based compound screening campaigns. In establishing new assays for high-throughput screening, creation of the appropriate cell line is a bottleneck. Typically, a stable cell line is created by transfection with a plasmid encoding the target of interest or reporter gene construct, and an additional gene which allows for chemical selection of successfully transfected cells (usually an antibiotic resistance gene). Through a lengthy selection process and subsequent limiting dilution to obtain clones, the desired stable cell line is generated. This process is time consuming and takes approximately 2-3 months, usually yielding 5-10 usable clones and allowing little control over the end result throughout the process.
The Laser-Enabled Analysis and Processing (LEAP™) system has been developed for high-throughput laser-mediated cell elimination for cell purification (Koller et al. 2004). LEAP images all cells within a well, selects a specific population of cells by gating, and eliminates selected cells at >103 per second.
LEAP can select cells of interest based on fluorescent properties, morphological properties, or a combination of both. By replacing the antibiotic resistance gene used for chemical selection with a gene encoding a fluorescent protein, transfected cells can be selected based on fluorescence. These cells can then be purified using LEAP by specifically eliminating non-fluorescent cells using laser elimination. By selecting cells that remain fluorescent and proliferate over a period of time, stable cell lines are isolated.
Validation & Results
GPCRs are a major target class in drug discovery. Cell lines engineered to express target GPCRs are commonly used in a high-throughput setting. Due to the high throughput achievable, a preferred assay for GCPR screens is Ca2+ flux, for example using the FLIPR® instrument.
Conclusion
Based on fluorescent selection and highly precise elimination of non-transfected cells, LEAP was used to generate stable cell lines in 3 weeks vs. the normal 2-3 months by classical methods. LEAP generation of cell lines include: (1) shorter time to functional cell lines; (2) reduced labor requirements, (3) no requirement for antibiotics, allowing (4) greater numbers of cell lines to be generated.