Targeting strategy
Gene targeting strategy for B-hGFRA3 mice. The exons 1~8 of mouse Gfra3 gene that encode the full-length protein were replaced by human GFRA3 exons 1~8 in B-hGFRA3 mice.
Protein expression analysis
Western blot analysis of GFRA3 protein expression in homozygous B-hGFRA3 mice. Various tissue lysates were collected from wild-type C57BL/6(+/+) mice and homozygous B-hGFRA3 mice (H/H), and then analyzed by western blot with species-specific anti-GFRA3 antibody. 40 μg total proteins were loaded for western blotting analysis. GFRA3 was detected in heart, liver and kidney.
mRNA expression analysis
Strain specific analysis of GFRA3 mRNA expression in wild-type C57BL/6 mice and B-hGFRA3 mice by RT-PCR. Heart RNA were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hGFRA3 mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human GFRA3 primers. Mouse Gfra3 mRNA was detectable only in wild-type C57BL/6 mice. Human GFRA3 mRNA was detectable only in homozygous B-hGFRA3 mice but not in wild-type mice. And the PCR products were confirmed with Sanger sequencing.
