Stable Cell Line Service for Assay Development

Stable Cell Line Services for Assay Development (SC1394)

In vitro assays based on gene engineered cells play crucial roles in modern drug discovery processes. Especially for GPCRs and ion channels, multiple trans-membrane proteins, the cell-based assays are dominant in chemical library screening. There are many stable cell lines and assay techniques are developed to model human bodies. With expertise on cell line generation and assay development, GenScript offers your Gene-to-Assay solution for your drug discovery project.

GenScript has developed a Ready-to-Assay GPCR & Ion Channel Cell Line Collection which is available as product for your order. If you can't find the stable cell lines of your interests, please choose the service to customize your stable cell lines with our available assays or other assays upon your specifications.

GenScript also offers a lentiviral based service, CellPower™, to generate a stable cell line with any cell and any gene

Flexible Solutions for Any Target

Transfection
mRNA
Protein
Structure (Binding)
Function
Any cell line (Amaxa® Nucleofector® technology from Lonza)
qPCR
WB or FACS
Radioisotope-based Fluorescence-based, luminescence-based, or electrophysiology-based

Service Package:

GenScript utilizes 2-round selection strategy for GPCR and ion channel stable cell line generation upon different applications. To reduce the cost and timeline, GenScript chooses HTS-compatible fluorescence- or luminescence-based assays to screen stable clones in the 1st round selection. In 2nd round selection, radioisotope- and electrophysiology-based assays are available upon your applications. For nuclear receptors, transporters, and other targets which have no ready HTS-compatible assays, GenScript offers mRNA or protein level assays as alternative solutions.

In addition, GenScript can develop a new assay upon your specifications. Please feel free to contact us for technical support.

Target Class
Type
1st Round Selection
2nd Round Selection
GPCRs Gq-coupled Calcium assay Radioligand binding assay
Gi/Gs-coupled Calcium assay (Gqi5/Gα15), cAMP assay, or luciferase reporter gene assay
Ion Channels Calcium channels Calcium assay Radioligand binding assay or manual patch clamp assay
Potassium channels Thallium flux assay or membrane potential assay
Sodium channels Membrane potential assay
Chloride channels YFP reporter gene assay
Nuclear Receptors, Transporters, etc.
  Upon client's specifications Upon client's specifications
Note: CHO-K1, CHO-K1/Gα15, CHO-K1/Gqi5, HEK293, 293T, 293EC18, 293EC18/CRE-Luc, 1321N1, RH7777, and U-2OS are available as starting cell lines. For special requirements, please feel free to contact us.

  • Case Studies
  • 1st Round Selection
  • 2nd Round Selection

Adenosine Receptor A1 Stable Cell Line Generation

A1 receptor is a Gαi-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 Gαi-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.

  Selection of clones   Stability test of clones  
 
▲ 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 nM1,2, 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

Quotations and Ordering

To request a quotation, please download and complete the Stable Cell Line Service for Assay Development and send it back by email or fax.

To order, please send the completed Quotation Form to us by email or fax with a formal PO (Purchase Order) or credit card information. You can also submit PO/credit card information by phone or via our Secure Online Messaging System.

Our customer service representatives are available 24 hours a day, Monday through Friday to assist you.