Targeting strategy
Gene targeting strategy for B-hSHH mice. The exon 3 of mouse Shh gene that encodes the C domain was replaced by human SHH exon 3 in B-hSHH mice.
Protein expression analysis
Western blot analysis of SHH protein expression in homozygous B-hSHH mice. Various tissue lysates were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hSHH mice (H/H) , and then analyzed by western blot with species-specific anti-SHH antibody. 40 μg total proteins were loaded for western blotting analysis. SHH was detected in stomach, liver and kidney in both wild-type and homozygous B-hSHH mice, as anti-SHH antibody was cross-reactive between human and mouse. SHH was low expression in lung.
mRNA expression analysis
Strain specific analysis of SHH mRNA expression in wild-type C57BL/6 mice and B-hSHH mice by RT-PCR. Stomach RNA were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hSHH mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human SHH primers. Mouse Shh mRNA was detectable only in wild-type C57BL/6 mice. Human SHH mRNA was detectable only in homozygous B-hSHH mice but not in wild-type mice.
