关键词: D374Y突变体, 体细胞核移植, 五指山小型猪近交系, LDLR

Abstract: 【Objective】 Proprotein convertase subtilisin/kexin type 9 (PCSK9) is the third major gene of autosomal dominant hypercholesterolemia (ADH). Gain-of-function mutations of PCSK9 are directly associated with human familial hypercholesterolemia, such as the D374Y mutant. In vitro detection experimental results on the degradation of LDLR by the PCSK9 D374Y mutant showed that its LDLR degradation ability is ten times stronger than that of the wild-type protein. This ability increases the risk of hypercholesterolemia, and promotes atherosclerotic lesions. The miniature pig is a useful animal model for biomedical research. However the utility of pigs for diseases model preparation is relatively infrequent because of their individual differences. Inbred lines increased the consistency of the genetic background of the experimental animals, thus consequently, would simplify disease progression of the animal model. This study used Wuzhishan minipig intending to make a transgenic pig with gain-of-function D374Y mutation of human PCSK9, and to contribute to the model of atherosclerosis. 【Method】 In this study, D374Y-PCSK9 over-expressing plasmids were transferred into Wuzhishan porcine embryonic fibroblasts by electroporation, and somatic cell nuclear transplantation and embryo transfer were used to make D374Y-PCSK9 over-expressing pig. Using transgenic pig liver, the human PCSK9 expression was assessed by PCR, Southern blot and Western blot in DNA, RNA and protein levels. At the same time, istochemical staining and hematoxylin-eosin (HE) staining were used to identify the pathological changes in liver between D374Y-PCSK9 transgenic pigs and wild-type Wuzhishan pigs. 【Result】 The positive cloned cells appeared for 3 days, and colony point was formed for 7 days. The PCR results showed that the D374Y-PCSK9 fragments could be spliced into a complete fragment. That means the D374Y-PCSK9 over-expressing plasmids were successfully integrated into fibroblasts of Wuzhishan pig. The screened positive cloned cells were used as donor cells for somatic cell nuclear transfer (SCNT) and transplanted into recipient sows, and finally, the transgenic pig was got. PCR and Southern blot detection results of the DNA samples demonstrated that the entire D374Y-PCSK9 gene was integrated into the genomes. RT-PCR and QPCR were performed using primers specifically recognizing human or pig PCSK9 mRNA. The results showed that the human PCSK9 gene could be normally transcribed in the pig liver and did not influence the transcription of the endogenous pig PCSK9. Surprisingly, the expression of the human PCSK9 gene could be detected in other organs including the heart, spleen, lung, and kidney, whereas the expression levels of the endogenous pig PCSK9 in these tissues were very low. The western blot results of the proteins were similar to the RNA detection levels. Therefore, the human D374Y-PCSK9 gene integrated into pig genomes was normally transcribed and translated. Besides, transgenic pig’s liver had pathological changes were detected by HE staining and histochemical staining and the results showed that the LDLR level in the transgenic pigs’ liver was significantly lower than wild-type controls’.【Conclusion】 In conclusion, human PCSK9 protein was successfully over-expressed in Wuzhishan inbred miniature pigs. Compared with wild type pigs, transgenic pigs had a low LDLR level in liver. At the same time, there were obvious pathological changes in transgenic pigs’ liver.

Key words: D374Y, SCNT, Wuzhishan Minipig Inbred, LDLR