Deletion of Gpr128 results in weight loss and increased intestinal contraction frequency

Figure 1 Targeted deletion of Gpr128 in mice. A: Gene targeting strategy. Numbered boxes represent Gpr128 coding exons. The start codon and stop codon are indicated as a star and pound sign, respectively. The targeting vector contains a 7.1-kb 5’ arm and a 5.3-kb 3’ arm. Exons 10, 11 and 12 of the Gpr128 gene were replaced by a PGK-Neo cassette through homologous recombination. The primer pairs for polymerase chain reaction (PCR) genotyping are indicated by arrows (5’ arm: P1, P2; 3’ arm: P3, P4); B: PCR screening for targeted embryonic stem (ES) cell clones. Correctly recombined clones show 7.7 and 5.7 kb bands, respectively. Three recombined ES cell clones show the expected bands as detected with primers P1-P4; C: PCR analysis of genomic tail DNA derived from Gpr128^+/- mouse intercrossing. A 5.4-kb fragment amplified with primers P5 and P6 represents the wild-type (WT) allele. A 5.7-kb band was amplified from the targeted allele with P3 and P4; D: Gpr128 expression in gastrointestinal tissue with two different genotypes by semiquantitive reverse transcription-polymerase chain reaction. A specific Gpr128 fragment, which exists in WT mice, was deleted in Gpr128^-/- mice. The transcript for β-actin was examined as a control for RNA loading and integrity; E: Expression pattern of Gpr128 protein in WT and Gpr128^-/- adult mouse colon revealed by immunofluorescence (original magnification, × 200). M: Marker lane; (-): Negative control without template; S. intestine: Small intestine; P. colon: Proximal colon; D. colon: Distal colon.