猪VHL基因克隆及敲低克隆胚胎的构建

Porcine VHL gene cloning and construction of VHL knockdown cloned embryos

摘要:

通过在细胞和胚胎水平对猪Von Hippel-Lindau (VHL) 基因进行了基因敲低研究，以便为建立VHL疾病模型猪奠定基础。研究首先通过3￠RACE和5￠RACE克隆得到猪VHL基因cDNA 全长序列 (2 725 bp)，Real-time PCR结果表明VHL基因广泛表达于猪的各种组织器官，其中在肾上腺、肝脏、胰腺、心脏和睾丸等组织器官高量表达。进一步在猪iPS细胞中对5条干扰片段进行筛选，获得2条高效的干扰片段，干扰效率分别达到72％ (P=0.0012) 和64％ (P＜0.01)。以稳定干扰VHL基因的猪胎儿成纤维细胞为核供体，构建克隆胚胎，结果表明，克隆胚胎的发育能力与对照组相比没有明显差异，而且在克隆囊胚中VHL基因的干扰效率达到71％ (P＜0.01)。综上所述，文中获得了猪VHL基因全长序列并获得该基因稳定敲低的猪细胞和胚胎，从而为VHL疾病模型猪的构建奠定了良好的基础。

Abstract:

Von Hippel-Lindau(VHL) disease is an autosomal dominant disorder and its clinical manifestation including haemangioblastomas of the central nervous system, renal cell carcinoma, haeochromocytomas, and pancreatic cyst. The deletion, mutation and promoter methylation of VHL gene can cause VHL disease. Swine is considered as an ideal model for human disease because of its physiological and anatomical similarity to human. We cloned pig VHL gene that is 2 725 bp in length. VHL highly expressed in adrenal gland, liver, pancreas, heart and testis. We designed 5 shRNAs and screened the most effective interference RNA fragment with a knockdown efficiency of 72%. Porcine embryonic fibroblasts stably transfected with pGenesil-shRNA vector were used as donor cells for nuclear transfer and there was no significant difference of embryo development compared with the control group. Moreover, VHL was efficiently knocked-down with efficiency of 71% in porcine cloned blastocyst, these results lay a solid foundation for constructing the VHL knock-down model of pig.