Construction Of The Liver-specific Expression Of HNPC1L1in Transgenic Bama Miniature Pig

As a member of the Niemann-Pick protein family, Niemann-Pick type C1Like1(NPC1L1) plays an extremely important role in the regulation of cholesterolabsorption and metabolism process and closely relates to lipid metabolism disorders.But the molecular mechanism of NPC1L1in metabolic diseases especially in lipidmetabolism disorders is still not clearly explained up to now. Therefore, the depthstudy of the molecular mechanism of NPC1L1is extremely important significancefor the prevention and treatment of such diseases.The distribution of NPC1L1is species-specific. It mainly expresses in the smallintestine in mice and hardly expresses in the liver whereas it high expresses in theliver in humans. So it may not be suitable to study the role and mechanism ofNPC1L1in mouse models and need to find or build a more appropriate model.Pigs, especially miniature pigs, are very similar to human in anatomy,metabolism and pharmacokinetic. Especially in terms of disease characteristics, theirgenetic and phenotypic characteristics are more closer to human and make thembecome animal models widely used in the study of disease.With the improvement of transgenic and somatic cell nuclear transfertechnology, transgenic pigs as models for the study of human diseases receive moreand more peoples attention.This study aims to establish transgenic Bama miniature pigs in order to revealthe molecular mechanism of NPC1L1in hepatic lipid metabolism. Firstly, weconstructed a eukaryotic expression vector pLiv-11-Neor/Kanr-hNPC1L1usinghuman NPC1L1gene, pLiv-11and pEGFP-C1plasmid. Human NPC1L1canspecifically express in livers driven by the apoE promoter/human apoE hepaticcontrol region on reconstructed vector. After linearized by SpeI, the vector wastransfected into the minipig fbroblast cell. By G418selection, we harvest positivecell clones with liver-specific expression of human NPC1L1. They were used as donor cells to generate eleven transgenic Bama miniature piglets and seven of themare positive transgenic piglets by PCR identification. RT-PCR and Western blotingtest proved that the human NPC1L1gene only specifically expressed in the livers.Immunohistochemistry analysis showed that NPC1L1localized on the hepaticcanalicular. In addition, we also investigated the superoxide dismutase (SOD)activities and the levels of maleic dialdehyde(MDA) as a biomarker of lipidperoxidation. These data reveal that liver-specific expression of NPC1L1resulted inthe severe lipid peroxidation. Real-time quantitative PCR detection showed thatNPC1L1plays an important role in lipid metabolism disorders and liver disease byaffecting the transcription of certain genes involved in lipid synthesis.In our study, we successfully generated transgenic Bama miniature pigs withliver-specific expression of human NPC1L1. we confirmed that NPC1L1localizeson the hepatic canalicular and accelerated the liver lipid peroxidation. All these showthat NPC1L1plays an important role in lipid metabolism and related diseases. Ourfinding further reveals the molecular mechanism of NPC1L1in hepatic lipidmetabolism and clarifies pathogenesis of diseases related to lipid metabolism. It hasgreat significance for finding new therapeutic target and developing new treatment.