Description

• Sodio-taurocholic acid cotransport polypeptide (NTCP) is a 9-step transmembrane protein. NTCP is mainly expressed in the basement membrane side of hepatocytes (blood sinus side), which promotes the absorption of bile acid in portal vein blood into the liver, and is associated with cholestasis, liver fibrosis, and nonalcoholic fatty liver disease. In addition, it is also a functional receptor for human hepatitis B virus (HBV) and hepatitis D virus (HDV). The two marketed drugs targeted are synthetic polypeptides of HBV Pre-S1 structure, which can competitively bind to NTCP to block the entry of hepatitis virus and bile acids into liver cells. There are also oligonucleotide drugs that target NTCP, and ADAR-based RNA editing introduces NTCP nonsense mutations to relieve cholestasis.
• Targeting strategy: A chimeric CDS that encodes P2A and human NTCP signal peptide, extracellular domain, transmembrane, cytoplasmic domain and 3’ UTR is inserted in the exon 3 of mouse Ntcp gene. The chimeric NTCP protein expression will be driven by endogenous mouse Ntcp promoter, while mouse Ntcp gene transcription and translation will be disrupted.
• mRNA expression analysis: Mouse Ntcp mRNA was only detectable in wild-type mice. Human NTCP mRNA was exclusively detectable in homozygous B-NDG hNTCP mice but not in wild-type mice.
• Protein expression analysis: NTCP was detected in liver of both homozygous mice and wild-type mice, and weakly detected in kidney, as anti-NTCP antibody is cross reactive between human and mouse.

Targeting strategy

Gene targeting strategy for B-NDG hNTCP mice. A chimeric CDS that encodes P2A and human NTCP signal peptide, extracellular domain, transmembrane, cytoplasmic domain and 3’ UTR is inserted in the exon 3 of mouse Ntcp gene. The chimeric NTCP protein expression will be driven by endogenous mouse Ntcp promoter, while mouse Ntcp gene transcription and translation will be disrupted.

Protein expression analysis

Western blot analysis of NTCP protein expression in homozygous B-NDG hNTCP mice . Various tissue lysates were collected from wild-type B-NDG mice mice (+/+) and homozygous B-NDG hNTCP mice (H/H), and then analyzed by western blot with cross reactive anti-NTCP antibody (Abcam, ab131084). 40 μg total proteins were loaded for western blotting analysis. NTCP was detected in liver, and weakly detected in kidney of both homozygous mice and wild-type mice.

mRNA expression analysis

Strain specific analysis of NTCP mRNA expression in wild-type B-NDG mice and B-NDG hNTCP mice by RT-PCR. Liver RNA were isolated from wild-type B-NDG mice (+/+) and homozygous B-NDG hNTCP mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human NTCP primers. Mouse Ntcp mRNA was only detectable in wild-type mice. Human NTCP mRNA was exclusively detectable in homozygous B-NDG hNTCP mice but not in wild-type mice.