牛c-myc基因的克隆及其在皮肤成纤维细胞中的表达

Cloning of bovine c-myc gene and its expression in skin fibroblast cells

摘要:

为了构建包含牛c-myc基因编码序列的重组载体，以胎牛原始生殖嵴为材料，用RT-PCR方法克隆出牛c-myc 基因的编码序列，将其亚克隆至pMD19-T载体，再从酶切鉴定和测序正确的质粒上切下目的片段，定向克隆到pIRES2-AcGFP1-Nuc表达载体上，挑选序列正确的重组真核表达质粒转染牛皮肤成纤维细胞，用RT-PCR和Western blotting分别检测c-myc mRNA和蛋白的表达。结果表明，从胎牛原始生殖嵴中正确克隆了c-myc基因的全长编码序列，所构建的重组质粒能够在皮肤成纤维细胞中有效

Abstract:

In order to construct a eukaryotic expression vector of bovine c-myc gene, the coding sequence (CDS) of c-myc gene was amplified from bovine primordial genital ridges by RT-PCR. The CDS was subcloned into pMD19-T vector, and then inserted into vector pIRES2-AcGFP1-Nuc. After confirmed by restriction enzyme digestion and sequencing, the recombined plasmid was transfected into skin fibroblast cells. RT-PCR and Western Blotting were used to detect the expression of c-myc mRNA and protein, respectively. The results show that the complete CDS of c-myc gene was cloned from fetal bovine primordial genital ridges. The eukaryotic expression vector of bovine c-myc gene was constructed and efficiently expressed in the skin fibroblast cells. The present study will lay a good foundation for further study of c-myc gene function and bovine induced pluripotent stem cells from somatic cells by defined factors.