June 1, 2014 (Vol. 34, No. 11)
Alison Glaser Field Application Scientist Molecular Devices
A Rapid Method to Screen and Pick Cells with Optimal Expression Levels
The G-protein-coupled receptor (GPCR) family represents the largest and most versatile group of cell surface receptors, and are targets of approximately 50% of pharmaceutical drugs in the marketplace. Identifying key modulators of GPCRs and ligands against known and orphan GPCRs are at the crux of drug discovery efforts. However, the path to identifying GPCR targets is not straightforward given the complexity of signaling pathways and interactions with other cellular components.
Although the human genome project enables GPCR’s genome sequence to be determined, challenges exist in identifying unique GPCRs and their subsequent ligands. Critical to researchers is the need to improve efficiency of target validation while shortening timelines once the target is selected for expression and cloning.
Traditionally, GPCR discovery efforts rely on various functional assays such as measuring levels of cAMP or monitoring cytoplasmic calcium changes. These typically require a much higher concentration of functional GPCRs on the cell surface than endogenous expression levels. While a need exists for an expression system capable of supplying overexpressed GPCRs, it is important to also establish in vitro assays that mimic the activity of endogenously expressed targets. This challenge incites the need to utilize mammalian expression systems capable of providing a range of requisite GPCR protein expression levels.
Identification and Selection of GPCR Expressing Cell Lines
A solution utilizing an automated high-throughput platform that identifies and isolates heterogeneous expressing pools of cells provides significant value and saves considerable resource. The ClonePix™ 2 System from Molecular Devices represents a proven, one-step method of screening large cell populations rapidly. Utilizing white light and fluorescent images in situ, the ClonePix 2 System has the sensitivity to detect both overexpressed and endogenous levels of cell surface protein expression.
The principle of ClonePix technology is based upon plating single cells in a semi-solid matrix and growing into distinct clonal colonies. A fluorescent antibody against the target cell surface protein is also added; the antibody is small enough to diffuse through the matrix and bind to the cell surface. The physical location and morphology of the colonies are identified in white light; the corresponding fluorescent signal is proportional to the level of expression on the cell surface. The morphological and fluorescence criteria can be refined before acceptable colonies are picked into liquid expansion media. These colonies can be scaled up to generate cell lines or be utilized for further characterization studies.
Detecting Endogenous Muscarinic 1 Receptor
A transfected CHO-M1 cell line expressing muscarinic 1 cholinergic receptor (M1) was chosen to demonstrate the feasibility of using the ClonePix 2 System to detect cell surface proteins. CHO-M1 and parental untransfected CHO-K1 cells (negative control) were plated in Molecular Devices CloneMediaCHO semisolid media at low density and incubated until discrete colonies were formed.
Both a direct-labeled and a dual-label approach were tested. For the direct-labeled approach, a Phycoerythrin (PE)-labeled anti-M1 antibody was used. For the dual-label approach, an unconjugated rabbit anti-M1 antibody and a PE-labeled anti-rabbit polyclonal antibody were used. A dual label approach was done to determine feasibility in cases where a labeled primary antibody is unavailable.
The ClonePix 2 system (Figure 1) utilized brightfield to identify the colonies’ morphology and location, and fluorescence to identify the M1 expression. Positively expressing CHO-M1 cells produced a range of fluorescent signals with both the direct-labeled and dual-antibody approach. The parental line CHO-K1 did not produce a fluorescent signal (Figure 2). Clones were ranked by fluorescent intensity and binned into groups based upon intensity: high, medium, low, and no signal.
Colonies picked by the ClonePix 2 System were deposited into 96-well plates containing liquid expansion media, cultured for one week, and transferred to 384 well plates. Picked colonies were functionally corroborated with the FLIPR Tetra System using the FLIPR Calcium Assay 6 Kit. The kit measures transient intracellular calcium mobilization as a result of activation of the M1 G-protein coupled IP3 sensitive pathway. Carbachol was used as the muscarinic agonist.
In the high fluorescent clones (Figure 3A) picked by the ClonePix 2 System, carbachol produced a fourfold increase in fluorescent read from background. In the mixed medium and low (Figure 3B) fluorescent picked clones, carbachol produced a four- and twofold increase in fluorescent read from background, respectively. Finally in CHO-K1 negative control group (Figure 3C), carbachol failed to elicit any significant change in fluorescent read from background. Changes in baseline fluorescence intensity were normalized to background fluorescent reads of each 384-well cell plate before the addition of 40 nM carbachol.
These results support a positive correlation between membrane-bound G-protein coupled muscarinic receptor expression level and functional activity. The lack of calcium fluorescent signal in the CHO-K1 negative control group further confirms that ClonePix 2 System can accurately distinguish between clones that are positive or negative for cell surface expression. Relative differences in the expression of membrane-bound M1 GPCR resulted in commensurable fluorescence intensities as recorded for high, low, and no (negative control) M1 expression respectively.
To meet the demands of high-throughput screening and selectivity profiling, robust cloning and expression systems are needed. Establishing cell lines expressing GPCRs of interest provide great advantage in characterization studies of receptor-mediated signaling and screening campaigns of novel therapeutic drug candidates. Identification and isolation of GPCR clones provide a source of GPCR proteins for a variety of applications including cell-based functional assays for hard-to-express GPCRs.
The ClonePix 2 System is a platform enabling the identification and isolation of GPCR clones in an accurate and rapid manner. Utilizing white light and fluorescent based imaging in situ, the ClonePix 2 System has both the sensitivity and specificity to quantitatively detect various expression levels of cell surface protein, thus providing a solution to screening both endogenous and overexpressed levels of GPCR protein.
The ability to screen and rapidly pick large pools positive expressing cells improve the throughput required for cell line development. Thus the ClonePix 2 System provides a unique solution to shorten time lines involved in target validation and development of GPCR expressing cell lines.
Alison Glaser (firstname.lastname@example.org) is a field application scientist at Molecular Devices.
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