Targeting strategy
Gene targeting strategy for B-hHBEGF mice. The exons 2~4 of mouse Hbegf gene that encode the extracellular domain were replaced by human HBEGF exons 2~4 in B-hHBEGF mice.
Protein expression analysis
Western blot analysis of HBEGF protein expression in homozygous B-hHBEGF mice. Various tissue lysates were collected from wild-type C57BL/6N (+/+) mice and homozygous B-hHBEGF mice (H/H), and then analyzed by western blot with anti-HBEGF antibody. 40μg total proteins were loaded for western blotting analysis. HBEGF were detected in kidney, liver and spleen. The antibodies showed cross-recognition.
mRNA expression analysis
Strain specific analysis of HBEGF mRNA expression in wild-type C57BL/6N mice and B-hHBEGF mice by RT-PCR. Heart and lung RNA were isolated from wild-type C57BL/6N mice (+/+) and homozygous B-hHBEGF mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse and human HBEGF primers. As the coding region is only about 630bp, the same primers were used to amplify both mouse Hbegf and human HBEGF gene. PCR products in heart and lung for human HBEGF were confirmed via Sanger Sequencing.
