Targeting strategy
Gene targeting strategy for B-hGFRA1 mice. The exons 5-10 of of human GFRA1 gene that encodes the extracellular region was constructed into a chimeric CDS vector and inserted into the mouse exon 6. The targeted mice will express the chimeric GFRA1 protein, while mouse GFRA1 protein will no longer express.
Protein expression analysis
Western blot analysis of GFRA1 protein expression in homozygous B-hGFRA1 mice. Various tissue lysates were collected from wild-type C57BL/6 mice(+/+) and homozygous B-hGFRA1 mice (H/H), and then analyzed by western blot with anti-GFRA1 antibody. 40 μg total proteins were loaded for western blotting analysis. GFRA1 protein was detected in the heart, lung and brain of wild-type C57BL/6 mice and homozygous B-hGFRA1 mice, as the antibody is cross-recognize both human and mouse GFRA1. β-Actin is expressed at very low levels in the heart and undetectableand.
mRNA expression analysis
Strain specific analysis of GFRA1 mRNA expression in wild-type C57BL/6 mice and B-hGFRA1 mice by RT-PCR. Brain RNA was isolated from wild-type C57BL/6 mice(+/+) and homozygous B-hGFRA1 mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human GFRA1 primers. Mouse Gfra1 mRNA was detectable only in wild-type C57BL/6 mice. Human GFRA1 mRNA was detectable only in homozygous B-hGFRA1 mice but not in wild-type mice. And the PCR products were confirmed with Sanger sequencing.
