High-throughput stable cell line generation

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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.

In addition, fluorescence level may be used to identify cell lines with a specific desired expression level of the transfected construct. The fluorescent reporter gene may also be replaced by a variety of fluorescent cell physiology read outs, enabling the selection of cells based on functional responses.

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  Fig. 1. Classical cell line generation vs. cell line generation on LEAP.

In addition, fluorescence level may be used to identify cell lines with a specific desired expression level of the transfected construct. The fluorescent reporter gene may also be replaced by a variety of fluorescent cell physiology read outs, enabling the selection of cells based on functional responses.

Purifying transfected cells based on fluorescence using LEAP confers several advantages:

Speed. Fluorescence selection of stably transfected cells eliminates the need for chemical selection and limiting dilution, reducing the time needed to generate a stable cell line from 2-3 months to ~3 weeks.

No antibiotic selection. This factor eliminates the risk of artifacts, such as up-regulation of drug transporters, due to the chemical selection process.

Greater control. Cell line generation progress is monitored throughout the process. Failure to generate cell lines is detectable within 10 days.

Validation and 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.

Compound libraries are screened against the appropriate cell line to identify molecules that modulate the response or activity of the receptor. To validate the generation of stable cell lines using LEAP, we chose to express GPR91 along with GFP in CHO-K1, one of the most frequently used cell lines for high-throughput screening. GPR91 was recently de-orphanized and shown to be a receptor for succinic acid (He at al. 2004) and has been implicated in diabetes-induced hypertension (Toma et al. 2008). CHO-K1 cells were transfected with a plasmid encoding GPR91 and GFP and seeded into 96-well plates at a density of 1000 cells/well. After six days, cells were processed using LEAP. Cells that did not express GFP were eliminated by photothermal elimination. Plates were then returned to the incubator for further growth. The purification process was repeated on days 8 and 12, after which pure GFP expressing colonies were obtained (Fig. 2). To assess functionality of the generated cell lines, a dose-response curve against succinic acid, the cognate ligand of GPR91, was generated. Cells were seeded in a 384-well C-lect™ plate, labeled with Fluo4NW® (Invitrogen) and assayed in a FLIPR® instrument (MDS) upon addition of ligand. Resulting peak responses were plotted against ligand concentration to generate a dose response curve (Fig. 3). The EC50 for succinic acid was 47.6 uM matching reported values in the literature (He et al. 2004).

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  Fig. 2. Purification of a single stably transfected colony using LEAP. “Before” is prior to the first purification round, “After” is after purification of positive cells and growth. Red = all viable cells, green = GFP positive cells. Histograms on right show the distribution of GFP-positive cells before and after processing on the LEAP and subsequent growth. LEAP was instructed to select cells within the gating window highlighted in yellow

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.

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  Fig. 3. Functionality of a generated GPR91 cell line. Ca2+ elevation in response to a dose titration of succinic acid was measured on a FLIPR®. The measured EC50 was 47.6 uM.

References

1. Koller et al, Cytometry, Part A 61A:153–161, 2004
2. He W, Miao FJ, Lin DC, Schwandner RT, Wang Z, Gao J, Chen JL, Tian H, Ling L. Nature. 2004 May 13;429(6988):188-93.
3. Toma I, Kang JJ, Sipos A, Vargas S, Bansal E, Hanner F, Meer E, Peti-Peterdi J. J Clin Invest. 2008 Jun 5.