Basic Information
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Targeting strategy
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Gene targeting strategy for B-hNTRK1/hNGF mice.
The exons 1-10 of mouse Ntrk1 gene that encode the the extracellular domain were replaced by human NTRK1 exons 1-10 in B-hNTRK1/hNGF mice. The exon 3 of mouse Ngf gene that encodes the the full-length protein was replaced by human NGF exon 3 in B-hNTRK1/hNGF mice.
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Protein expression analysis
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Western blot analysis of NTRK1 protein expression in homozygous B-hNTRK1/hNGF mice. Various tissue lysates were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hNTRK1/hNGF mice (H/H;H/H), and then analyzed by western blot anti-m/hNTRK1 antibody. 80 μg total proteins were loaded for western blotting analysis. NTRK1 was detected in hippocampus,cerebellum and cerebrum.
NGF expression analysis in homozygous B-hNTRK1/hNGF mice by ELISA. Plasma were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hNTRK1/hNGF mice (H/H;H/H)(n=3, famale, 6-week-old), and analyzed by ELISA with NGF ELISA kit. NGF was detected in plasma of wild-type mice and homozygous B-hNTRK1/hNGF mice, as the ELISA kit is cross-recognize both human and mouse NGF.
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mRNA expression analysis
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Strain specific analysis of NTRK1 and NGF mRNA expression in wild-type C57BL/6 mice and B-hNTRK1/hNGF mice by RT-PCR. Brain RNA were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hNTRK1/hNGF mice (H/H; H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human NTRK1 and NGF primers. Mouse Ntrk1 and Ngf mRNA were detectable only in wild-type C57BL/6 mice. Human NTRK1 and NGF mRNA were detectable only in homozygous B-hNTRK1/hNGF mice but not in wild-type mice. And the PCR products were confirmed with Sanger sequencing.
