Stable Cell Line Services for Assay Development

Adenosine Receptor A1 Stable Cell Line Generation

A1 receptor is a G_αi-coupled GPCR which theoretically cannot be assayed by calcium mobilization assay. We co-expressed A1 receptor with Gα15, a promiscuous Gα protein, in CHO-K1 cells and selected a best clone as the primary clone and developed calcium mobilization assay.

1. Biology of Adenosine Receptor A1

Activation of A1 receptor, a ubiquitous G_αi-coupled GPCR, elicits an inhibition of adenylate cyclase and causes decrease in cAMP concentration. Adenosine antagonists are widely used in neonatal medicine.

2. Stable Clone Screening

We transfected CHO-K1 cells with the ADORA1 gene which encodes A1 receptor and got 96 primary clones by antibiotic selection. These 96 clones were further screened for the positive clones through calcium mobilization response (Fig. 1). Based on the long-lasting signal intensity profile over several months in culture, Clone 32 and Clone 7 were selected as the primary and backup clones for CHO-K1/ADORA1/Gα15 cell line, respectively (Fig. 2). All the further studies were performed on the primary clone, Clone 32.

▲ Figure 1

▲ Figure 2

3. 29-Passage Stability Test

We compared EC₅₀ values of NECA, an ADORA1 agonist, at various passages of Clone 32. The variation of EC₅₀ values of Clone 32 from passage 1 to 29 passages was within a small window (see below). Based on the pharmacology study, our results showed our Clone 32 has a high level of passage stability. The EC₅₀ values that we have obtained matched very well with those reported in the literature (EC₅₀ value: 20 ~ 173 nM^1,2, Tab. 1).

Passage Number	P1	P5	P10	P15	P20	P23	P26	P29
EC₅₀ (n=2) nM	32±6	68±5	41±7	43±2	72±6	65±4	56±9	45±7

▲ Table 1

1. Yolande Cordeaux et al. (2004) Coupling of the human A1 adenosine receptor to different heterotrimeric G proteins: evidence for agonist-specific G protein activation. Brit. J. Pharmacol., 143: 705-714.
2. Cordeaux Y et al. (2000) Influence of Receptor Number on Functional Responses Elicited by Agonists Acting at the Human Adenosine A1 Receptor: Evidence for Signaling Pathway-Dependent Changes in Agonist Potency and Relative Intrinsic Activity. Mol Pharmacol. 58(5):1075-84

Premium Package

No.	Milestone	Description	Timeline	Deliverable	Comment
1	Gene Synthesis	If you need to synthesize the gene in GenScript	2 weeks	Gene in pUC57 and sequencing report	Quote ($0.35/bp)
2	Subcloning and Plasmid Preparation	Supercoiled plasmid	2 weeks
3	Transfection	5 independent transfections	1 week
4	First Assay Screening	Screening 5*96 mixed clones	1 week	Mixed clones	Paused if no good results.
5	Second Assay Screening	Screening positive mixed clones from the previous step	2 weeks	Mixed clones	Paused if no good results.
6	Isolation of Single Clones	From 5 best mixed clones	2 weeks
7	Third Assay Screening	Screening minimal 50 single clones	2 weeks	Single clones	Paused if no good results.
8	16-Passage Stability Test	For 5 best single clones	6 weeks	QC report (EC50 comparison)
9	Expansion and Cryo-preservation		-	5 clones (2 vials per clone)
10	Mycoplasma and Viability Test		-	QC report (mycoplasma negative and viability >90%)

Medium Package

No.	Milestone	Description	Timeline	Deliverable	Comment
1	Gene Synthesis	If you need to synthesize the gene in GenScript	2 weeks	Gene in pUC57 and sequencing report	Quote ($0.35/bp)
2	Subcloning and Plasmid Preparation	Supercoiled plasmid	2 weeks
3	Transfection	2 independent transfections	1 week
4	First Assay Screening	Screening 2*96 mixed clones	1 week	Mixed clones	Paused if no good results.
5	Second Assay Screening	Screening positive mixed clones from the previous step	2 weeks	Mixed clones	Paused if no good results.
6	Isolation of Single Clones	From 2 best mixed clones	2 weeks
7	Third Assay Screening	Screening minimal 20 single clones	2 weeks	Single clones	Paused if no good results.
8	4-Passage Stability Test	For 3 best single clones	2 weeks	QC report (EC50 comparison)
9	Expansion and Cryo-preservation		-	3 clones (2 vials per clone)
10	Mycoplasma and Viability Test		-	QC report (mycoplasma negative and viability >90%)

Basic Package

No.	Milestone	Description	Timeline	Deliverable	Comment
1	Gene Synthesis	If you need to synthesize the gene in GenScript	2 weeks	Gene in pUC57 and sequencing report	Quote ($0.35/bp)
2	Subcloning and Plasmid Preparation	Supercoiled plasmid	2 weeks
3	Transfection		1 week
4	First Assay Screening	Screening 96 mixed clones	1 week	Mixed clones	Paused if no good results.
5	Second Assay Screening	Screening positive mixed clones from the previous step	2 weeks	Mixed clones	Paused if no good results.
6	Isolation of Single Clones	From 1 best mixed clones	2 weeks
7	Third Assay Screening	Screening minimal 10 single clones	2 weeks	Single clones	Paused if no good results.
8	4-Passage Stability Test	For 2 best single clones	2 weeks	QC report (EC50 comparison)
9	Expansion and Cryo-preservation		-	2 clones (2 vials per clone)
10	Mycoplasma and Viability Test		-	QC report (mycoplasma negative and viability >90%)

Manual Patch Clamp Package

No.	Milestone	Description	Timeline	Deliverable	Comment
1	Patch Clamping	For 4 clones (12 cells per clone)	2 weeks		Paused if no good results.
2	15-Passage Current Stability Test	For 1 best clone	5 weeks
3	Properties	I-V relationship, inactivation curve, etc.	1 week
4	Pharmacology Study	One reference compound	1 week	1 clone (2 vials) and data report

Radioligand Binding Package

No.	Milestone	Description	Timeline	Deliverable
1	Reagent preparation	Radio-labeled ligand	3 weeks
2	Membrane Preparation	4 clones (10 mg per clone)	2 weeks
3	Assay Development	Radioligand binding assay	2 weeks	1 clone (2 vials) and data report

Stable Cell Line Services for Assay Development (SC1394)

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