GPR120 in control of pancreatic polypeptide secretion from male mouse islets via phospholipase C-mediated calcium mobilization

GPR120-IRES-EGFP mice were generated on C57BL/6J background. The sequence of IRES-EGFP was inserted into the genome behind the gene termination codon TAA of GPR120 gene by targeted integration with CRISPR/Cas9 technology to generate the knockin (KI) mice that express EGFP under the control of GPR120 expression. In brief, the recombinant donor vector containing IRES-EGFP element was constructed and identified by double enzyme digestion. The Cas9 mRNA, guide RNA and the donor vector were transferred into the fertilized eggs of C57BL/6J mice by microinjection and the founder mice were generated. The positive founder mice with right homologous recombination were mated with wild C57BL/6J mice to obtain F1 generation. The F1 mice were back-crossed with wild type C57BL/6J mice for 6 generations and then the homologous KI mice were mass-produced for experiments.

The GPR120 expression in mouse islets was knockdown by GPR120 siRNA transfection, and the reduction of GPR120 expression was observed by western blot after 3 days culture. The specificity of PP ELISA kits was measured by pre-absorption with PP antibodies. Samples with PP antibody pre-absorption had undetectable PP levels while the samples without antibody treatment were measured positively for PP levels.