Biamonti G, Della Valle G, Talarico D, Cobianchi F, Riva S, Falaschi A
Nucleic Acids Res. 1985 Aug 12;13(15):5545-61. doi: 10.1093/nar/13.15.5545.
We have constructed a number of plasmids selectable in both E. coli and mouse or human cells. Human DNA sequences were inserted and the recombinant plasmids were used to transfect either mouse or human cells by the Ca-phosphate precipitation technique. We have observed that: (i) competent cells uptake large amounts of plasmid DNA; (ii) input plasmids persist in transformed mammalian cells as free unreplicating circular molecules for up to 20 generations; such persistence does not depend on the presence of selective markers; (iii) plasmids incorporated into mouse L-cells undergo widespread rearrangements (in the absence of replication) entailing mostly deletions of both human and bacterial sequences which yield smaller products; the latter appear to be more stable in a subsequent transformation cycle. Surprisingly such rearrangements are almost totally absent in transformed human KB-cells. This property of human KB-cells may prove useful for the development of a vector apt at cloning and expressing human DNA sequences. Unlike what has been observed in yeast, no "autonomously replicating sequence" can be detected in mammalian cells by randomly cloning human DNA sequences into a selectable plasmid and screening for an increased transformation efficiency.
我们构建了一些在大肠杆菌以及小鼠或人类细胞中均可选择的质粒。将人类DNA序列插入其中，然后通过磷酸钙沉淀技术，利用重组质粒转染小鼠或人类细胞。我们观察到：（i）感受态细胞摄取大量质粒DNA；（ii）导入的质粒作为游离的、未复制的环状分子，在转化的哺乳动物细胞中持续存在长达20代；这种持续性不依赖于选择标记的存在；（iii）整合到小鼠L细胞中的质粒会发生广泛的重排（在无复制的情况下），主要导致人类和细菌序列的缺失，从而产生较小的产物；后者在随后的转化周期中似乎更稳定。令人惊讶的是，在转化的人类KB细胞中几乎完全不存在这种重排。人类KB细胞的这一特性可能对开发适合克隆和表达人类DNA序列的载体有用。与在酵母中观察到的情况不同，通过将人类DNA序列随机克隆到可选择的质粒中并筛选提高的转化效率，在哺乳动物细胞中无法检测到“自主复制序列”。
