(A) A schematic of the gene that is expressed in the hGLP-1R mice. (B) The protein that is produced from this gene is a fusion of the mouse signal peptide (beige residues) and the human GLP-1R protein (white residues). The blue residues are those that differ between mouse and human GLP-1R. The signaling peptide is cleaved, leaving behind a human GLP-1R protein containing the C-terminal FLAG epitope (green residues). (C) cDNA was generated from total RNA isolated from islet and lung of hGLP-1R, mGlp-1r, and Glp1r^−/− mice for RT-PCR. The 5′ P915 primer annealed in human exon 8, while the 3′ P913 primer annealed to the FLAG region, a unique site within the hGLP-1R gene. This PCR product is a 588 bp fragment only observed in the hGLP-1R mice. (D) A schematic of the gene that is expressed in the Glp-1r^−/− mice. (E) Once the splice event occurs between human exon 2 and mouse exon 2, a frame-shift mutation nullifies downstream protein expression. (F) The final protein product in Glp-1r^−/− mice is a 98-aa truncation mutant. The first 36 aa’s of the mature protein encode a fraction of the GLP-1R extracellular domain, and the remaining 40 aa’s constitute missense sequence that shows no similarity to known proteins. The RT-PCR and DNA sequence analyses of the Glp-1r^−/− gene product demonstrate the Glp-1r^−/− mouse does not code for a functional GLP-1R. (G) A schematic of the wild-type (mGlp-1r) gene. (H) Using the same primer pair, PCR products from Glp-1r^−/− (372 bp) and mGlp-1r (275 bp) mice differ in size by 97 bp. (I) The wild-type GLP-1R protein is 463 aa’s including the signaling peptide.

https://doi.org/10.1371/journal.pone.0132875.g001