牛Nanog基因克隆及其在皮肤成纤维细胞中的表达

Cloning of Bovine Nanog Gene and Its Expression in Skin Fibroblast Cells

本研究克隆了牛Nanog基因,构建其真核表达载体,并转染皮肤成纤维细胞,获得能够用作核移植供核细胞的稳定转染细胞株。从6周龄的胎牛原始生殖嵴中提取总RNA,通过RT-PCR扩增Nanog基因,将其克隆到pMD-18T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pCDNA3-FLAG表达载体上,挑选序列正确的真核表达质粒pCDNA3-Nanog转染牛皮肤成纤维细胞,获得了稳定转染的细胞株。用RT-PCR和Western Blotting分别检测NanogmRNA和FLAG-Nanog融合蛋白的表达,用免疫染色法验证该细胞株是否具有干细胞特征。结果表明:(1)从胎牛原始生殖嵴中克隆了序列正确的Nanog全长编码序列;(2)所构建的pFLAG-Nanog重组质粒能够在皮肤成纤维细胞中高效表达;(3)所获稳定转染的细胞株能表达ES细胞表面抗原SSEA-4和多能性维持因子Nanog、Oct-4,表明其具有一定的多能性。为进一步研究Nanog基因功能,尤其是探讨它在家畜早期胚胎发育、生产转基因动物,以及胚胎干细胞建系中的作用奠定了基础。

The present study is to construct a eukaryotic expression vector of bovine Nanog, transfecting it into skin fibroblast, and further to obtain stable Nanogtransfected cell line, which can be used in nuclear transfer. By means of reverse transcription polymerase chain reaction （RT-PCR）, cDNA of Nanog was cloned from tissues of fetal bovine primodial genital ridges at the age of about six weeks. It was inserted into pMD18-T vector, then into vector pCDNA3FLAG by gene recombination technique. After being confirmed by restriction enzyme digestion and sequencing, the recombinant plasmid FLAG-Nanog was transfected into skin fibroblast cells. Selecting for two months with neomycine （G418）, we obtained a stably transfected cell line. RTPCR and Western Blotting were respectively used to detect the expression of Nanog mRNA and that of FLAG-Nanog fusion protein. In addition, immunostaining assay was performed to investigate whether the cell line is characteristic with stem cells. The results showed that （1） complete cording sequences （CDS） of Nanog was cloned from fetal bovine primodial genital ridges; （2） its eukaryotic expression vector was constructed, which expressed efficiently in the skin fibroblast cellsl （3）Stage Specific Embryonic Antigen 4 （SSEA-4）, factor Oct-4 as well as Nanog, required to maintain pluripotency of ES cells, was readily detectable in these Nanog-expressing fibroblast cells, indicating that the cell line is to some extent pluripotent. Our present study will lay a good foundation for further studying functions of Nanog, especially its role on early embryo development, production of transgenic animals, and derivation of embryonic stem cell lines from farm animals.