• RSPO3 is an ~31 kDa secreted protein, which is positive modulators of Wnt/ beta-catenin signaling.
• LGR5 also known as GPR49, is a seven‑transmembrane glycoprotein receptor in the Lgr family of cell surface receptors, which modulates Wnt signaling in the presence of the ligand R-spondin.
• The exons 2~5 of mouse Rspo3 gene were replaced by human RSPO3 exons 2~5 in B-hRSPO3/hLGR5 mice.
• The full-length protein of human LGR5 gene were constructed into a chimeric CDS vector and inserted into the mouse exon 1 in B-hRSPO3/hLGR5 mice. The targeted mice will express the chimeric LGR5 protein, while mouse LGR5 will no longer express.
• RSPO3 protein was detected in stomach of homozygous B-hRSPO3/hLGR5 mice.
• LGR5 protein was detected in stomach, colon, frontal lobe, kidney, liver and lung of homozygous B-hRSPO3/hLGR5 mice.

Strain specific analysis of LGR5 mRNA expression in wild-type C57BL/6 mice and B-hRSPO3/hLGR5 mice by RT-PCR. Stomach RNA was isolated from wildtype C57BL/6 mice (+/+) and homozygous B-hRSPO3/hLGR5 mice (H/H; H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human LGR5 primers. Mouse Lgr5 mRNA was detectable only in wild-type C57BL/6 mice. Human LGR5 mRNA was detectable only in homozygous B-hRSPO3/hLGR5 mice but not in wild-type mice.

Strain specific analysis of RSPO3 mRNA expression in wild-type C57BL/6 mice and B-hRSPO3/hLGR5 mice by RT-PCR. Stomach RNA was isolated from wildtype C57BL/6 mice (+/+) and homozygous B-hRSPO3/hLGR5 mice (H/H; H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human RSPO3 primers. Mouse Rspo3 mRNA was detectable only in wild-type C57BL/6 mice. Human RSPO3 mRNA was detectable only in homozygous B-hRSPO3/hLGR5 mice but not in wild-type mice.

Western blot analysis of LGR5 protein expression in homozygous B-hRSPO3/hLGR5 mice. Various tissue lysates were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hRSPO3/hLGR5 mice (H/H; H/H), and then analyzed by western blot with anti-LGR5 antibody. 40 μg total proteins were loaded for western blotting analysis. LGR5 was detected in stomach, colon, frontal lobe, kidney, liver and lung.

Western blot analysis of RSPO3 protein expression in homozygous B-hRSPO3/hLGR5 mice. Various tissue lysates were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hRSPO3/hLGR5 mice (H/H; H/H), and then analyzed by western blot with anti-RSPO3 antibody. 40 μg total proteins were loaded for western blotting analysis. RSPO3 was detected in stomach.