Basic Information
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Description
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- TRPM4 is a calcium-activated non selective cation channel which mediated cell membrane depolarization.
- Homozygous mice expressed chimeric TRPM4 and developed normally.
- This model is useful in studying the role of TRPM4 in cadiomyocytes, neurons and cochlea hair cells.
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Targeting strategy
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Gene targeting strategy for B-hTRPM4 mice. The exons 17-20 of mouse Trpm4 gene that encode transmembrane domain, extracellular domain and part of cytoplasmic region is replaced by human counterparts in B-hTRPM4 mice. The genomic region of mouse Trpm4 gene that encodes N- and C- terminal cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained. The chimeric TRPM4 expression is driven by endogenous mouse Trpm4 promoter, while mouse Trpm4 gene transcription and translation will be disrupted.
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mRNA expression analysis
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Analysis of TRPM4 mRNA expression in wild-type C57BL/6N mice and B-hTRPM4 mice by RT-PCR. Brain RNA were isolated from wild-type C57BL/6N mice (+/+) and homozygous B-hTRPM4 mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse Trpm4 and human TRPM4 primers. Mouse Trpm4 mRNA was only detectable in wild-type mice, human TRPM4 mRNA was exclusively detectable in homozygous B-hTRPM4 mice but not in wild-type mice.
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Protein expression analysis
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Western blot analysis of TRPM4 protein expression in homozygous B-hTRPM4 mice. Various tissue lysates were collected from wild-type C57BL/6N mice (+/+) and homozygous B-hTRPM4 mice (H/H), and then analyzed by western blot with anti-TRPM4 antibody (Abcam, ab106200). 40 μg total proteins were loaded for western blotting analysis. TRPM4 was detectable in brain, heart (at a low level), lung, liver, large intestine and stomach.
