Basic Information
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Description
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- GPR65, also called TDAG8 (T-cell death associated gene 8 protein), is a pH-sensing G-protein coupled receptor (GPCR) activated by acidic extracellular pH through the protonation of several histidine residues in the receptor’s sequence. GPR65 is involved in cancer cell metastasis and proliferation, immune cell function, inflammation, and blood vessel formation.
- GPR65 is primarily expressed in immune cells and leukocyte-rich tissues such as circulating peripheral leukocytes, spleen, thymus, and tonsils. Expression is also detected pain relevant loci such as the dorsal root ganglia neurons and particularly small diameter neurons responsible for nociception.
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Targeting strategy
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Gene targeting strategy for B-hGPR65 mice(C). The exon 2 of mouse Gpr65 gene that encode signal peptide, extracellular domain, transmembrane domain, cytoplasmic region and 3’UTR are replaced by human counterparts in B-hGPR65 mice(C). The promoter and 5’UTR region of the mouse gene are retained. The human GPR65 expression is driven by endogenous mouse Gpr65 promoter, while mouse Gpr65 gene transcription and translation will be disrupted.
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mRNA expression analysis in humanized B-hFPR1 mice
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Strain specific analysis of GPR65 mRNA expression in wild-type BALB/c mice and B-hGPR65 mice(C) by RT-PCR. Lung RNA were isolated from wild-type BALB/c mice (+/+) and homozygous B-hGPR65 mice(C) (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human GPR65 primers. Mouse Gpr65 mRNA was detectable only in wild-type BALB/c mice. Human GPR65 mRNA was detectable only in homozygous B-hGPR65 mice(C) but not in wild-type mice.
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Protein expression analysis
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Western blot analysis of GPR65 protein expression in homozygous B-hGPR65 mice(C). Various tissue lysates were collected from wild-type BALB/c mice (+/+) and homozygous B-hGPR65 mice(C) (H/H), and then analyzed by western blot with anti-GPR65 antibody (Thermo, AGR-043-50UL). 40 μg total proteins were loaded for western blotting analysis. GPR65 was detected in lung and kidney in wild-type mice and homozygous B-hGPR65 mice(C), as the antibody is cross-recognize both human and mouse GPR65.
