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Wei J.,Jilin University | Wei J.,Jilin Provincial Key Laboratory of Animal Embryo Engineering | Wei J.,Liaoning Province Academy of Analytic Science | Wei J.,Center for Animal Embryo Engineering of Jilin Province | And 30 more authors.
FEBS Journal | Year: 2012

Hypertriglyceridemia has recently been considered to be an independent risk factor for coronary heart disease, in which apolipoprotein (Apo)CIII is one of the major contributory factors, as it is strongly correlated with plasma triglyceride levels. Although ApoCIII transgenic mice have been generated as an animal model for the study of hypertriglyceridemia, the features of lipoprotein metabolism in mice differ greatly from those in humans. Because of the great similarity between pigs and humans with respect to lipid metabolism and cardiovascular physiology, we generated transgenic miniature pigs expressing human ApoCIII by the transfection of somatic cells combined with nuclear transfer. The expression of human ApoCIII was detected in the liver and intestine of the transgenic pigs. As compared with nontransgenic controls, transgenic pigs showed significantly increased plasma triglyceride levels (83 ± 36 versus 38 ± 4 mg·dL -1, P < 0.01) when fed a chow diet. Plasma lipoprotein profiling by FPLC in transgenic animals showed a higher peak in large-particle fractions corresponding to very low-density lipoprotein/chylomicrons when triglyceride content in the fractions was assayed. There was not much difference in cholesterol content in FPLC fractions, although a large low-density lipoprotein peak was identified in both nontransgenic and transgenic animals, resembling that found in humans. Further analysis revealed markedly delayed clearance of plasma triglyceride, accompanied by significantly reduced lipoprotein lipase activity in post-heparin plasma, in transgenic pigs as compared with nontransgenic controls. In summary, we have successfully generated a novel hypertriglyceridemic ApoCIII transgenic miniature pig model that could be of great value for studies on hyperlipidemia in relation to atherosclerotic disorders. © 2011 FEBS.

Lan Y.,Jilin University | Lu H.,Jilin University | Zhao K.,Jilin University | He W.,Jilin University | And 6 more authors.
Intervirology | Year: 2012

Objective: The specific effect of RNA interference on the replication of porcine hemagglutinating encephalomyelitis virus (PHE-CoV) was explored. Methods: Four species of small interfering RNA (siRNA), targeting different regions of the PHE-CoV spike glycoprotein and replicase polyprotein genes, were prepared by in vitro transcription. After transfection of PK-15 cells with each of the siRNAs followed by infection with PHE-CoV, the cytopathic effect (CPE) was examined by phase-contrast microscope, and viral proliferation within cells was examined by indirect immunofluorescence microscopy, hemagglutination (HA) test, TCID 50 assay and real-time RT-PCR. Results: Examination of CPE demonstrated that the four siRNAs were capable of protecting cells against PHE-CoV invasion with very high specificity and efficiency. At 48 h post-infection, only a few siRNA-treated cells were positive for viral antigen staining, whereas most untreated virus-infected cells were positive. Transfection with siRNAs also suppressed the production of infectious virus by up to 18-to 32-fold as assessed by a HA test and 93-to 494-fold as assessed by TCID 50 assay. Furthermore, treatment with siRNAs caused a 53-91% reduction in the viral genome copy number as assessed by real-time RT-PCR. Conclusion: These results suggested that the four species of siRNAs can efficiently inhibit PHE-CoV genome replication and infectious virus production. Copyright © 2011 S. Karger AG, Basel.

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