Basic Information
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Targeting strategy
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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 will no longer express. -
Protein expression analysis
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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 undetectable.
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mRNA expression analysis
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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.
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Summary
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mRNA expression analysis:
Mouse Gfra1 mRNA was detectable in brain of wild-type C57BL/6 mice. Human GFRA1 mRNA was detectable in homozygous B-hGFRA1 mice.
Protein expression analysis:
GFRA1 protein was detected in the heart, lung, brain and liver of wild-type C57BL/6 mice and homozygous B-hGFRA1 mice, as the antibody is cross-recognize both human and mouse GFRA1.
