DiscussionGlucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are gut peptides secreted in response to food intake that decrease blood glucose levels by stimulating insulin secretion from pancreatic β–cells. G-Protein Coupled Receptors (GPCRs) for GLP-1 and GIP are natively expressed in the rat β–cell line INS1, making it a useful cell line to screen agonists against the receptors at physiological expression levels. GLP-1 and GIP receptors share downstream signalling pathways and there are reports of agonists with dual activity at both receptors (Finan et al., 2013). It is therefore important to distinguish between the contributions of the two receptors when evaluating new GLP1/GIP dual agonist molecules as a part of Structure/Activity Relationship (SAR) determination. Selective antagonists were used to inhibit each receptor to define in vitro pharmacology however at the high concentrations required for complete blockade, we found the antagonists to exhibit non-specific effects. For this reason we created a GLP-1 and a GIP receptor knock out INS1 cell line in which protein for the individual receptors is completely depleted, which is beneficial over RNA interference. Monoclonal cell lines harbouring DNA strand breaks were isolated and assayed for receptor absence at the DNA and protein levels. The downstream production of cAMP in response to GLP-1 or GIP receptor agonism was fully abolished in the respective knock out cell line. Functional assays clearly demonstrate the utility of such receptor knock-out cell lines for assessing the specificity and selectivity of dual agonist molecules in an in vitro screening programme.