Gassmann M, Donoho G, Berg P
Department of Biochemistry, Beckman Center, Stanford University Medical School, CA 94305-5425.
Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1292-6. doi: 10.1073/pnas.92.5.1292.
We have constructed and characterized a polyoma virus-based plasmid that is maintained as an autonomously replicating extrachromosomal element (episome) in mouse embryonic stem (ES) cells. Plasmid pMGD20neo contains the polyoma origin of replication harboring a mutated enhancer (PyF101), a modified polyoma early region that encodes the large tumor (T) antigen only, and a gene that confers resistance to G418 (neo). After transfection, the plasmid replicates in ES cells and is maintained as an extrachromosomal element in 15% of G418-resistant clones. Integration of the plasmid DNA is undetectable for at least 28 cell generations. In one clone, the transfected DNA persists unaltered as an episome at 10-30 copies per cell for at least 74 cell generations in the presence of G418. Cells that maintain the autonomously replicating plasmid can efficiently replicate and maintain a second plasmid that carries the polyoma origin of replication. Independent vector-containing ES cell lines showed no significant alteration of the karyotype, and two cell lines yielded several chimeric animals when introduced into blastocysts, suggesting that the presence of an episomal element and expression of polyoma large T do not eliminate the ES cells' ability to populate an embryo. This system offers an efficient means for manipulating and analyzing various aspects of gene expression in ES cells.
我们构建并鉴定了一种基于多瘤病毒的质粒，该质粒在小鼠胚胎干细胞（ES细胞）中作为自主复制的染色体外元件（附加体）维持。质粒pMGD20neo包含携带突变增强子（PyF101）的多瘤病毒复制起点、仅编码大T抗原的修饰多瘤病毒早期区域以及赋予对G418（新霉素）抗性的基因（neo）。转染后，该质粒在ES细胞中复制，并在15%的G418抗性克隆中作为染色体外元件维持。在至少28个细胞世代中未检测到质粒DNA的整合。在一个克隆中，转染的DNA在G418存在的情况下，以每个细胞10 - 30个拷贝的形式作为附加体持续不变地存在至少74个细胞世代。维持自主复制质粒的细胞能够高效复制并维持携带多瘤病毒复制起点的第二个质粒。含有独立载体的ES细胞系的核型未显示出明显改变，并且当将两个细胞系导入囊胚时产生了几只嵌合体动物，这表明附加体元件的存在和多瘤病毒大T抗原的表达并未消除ES细胞在胚胎中定殖的能力。该系统为操纵和分析ES细胞中基因表达的各个方面提供了一种有效的手段。
