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Gene targeting strategy for B-hTGR5 mice.The exons 1-2 of mouseTgr5gene that encode the 5’UTR, promoter, signal peptide, extracellular domain, transmembrane domain, cytoplasmic region and 3’UTR are replaced by human counterparts in B-hTGR5 mice. TheTGR5expression is driven by endogenous humanTGR5promoter, while mouseTgr5gene transcription and translation will be disrupted.

Western blot analysis of TGR5 protein expression in homozygous B-hTGR5 mice. Various tissue lysates were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTGR5 mice (H/H), and then analyzed by western blot with anti-TGR5 antibody (abcam, ab72608). 40 μg total proteins were loaded for western blotting analysis. TGR5 was detected in Lung, spleen, bladder, brain.

Strain specific analysis ofTGR5mRNA expression in wild-type C57BL/6JNifdc mice and B-hTGR5 mice by RT-PCR. Lung RNA were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTGR5 mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or humanTGR5primers. MouseTgr5mRNA was only detectable in wild-type mice. HumanTGR5mRNA was exclusively detectable in homozygous B-hTGR5 mice but not in wild-type mice.