Weidle U H, Buckel P, Grummt F
Department of Genetics, Boehringer Mannheim GmbH, Penzberg, F.R.G.
Gene. 1988 Dec 20;73(2):427-37. doi: 10.1016/0378-1119(88)90507-0.
We have constructed a new expression vector for mammalian cells. The vector contains a truncated tk gene for amplification under selective conditions, a sequence putatively supporting the replication of plasmid DNA in eukaryotic cells (murine autonomously replicating sequence) and an expression cassette for the cDNA to be studied. As a model cDNA we have used that of human tissue-type plasminogen activator (t-PA). Analysis of Hirt supernatants and chromosomal DNA from L cells, prepared six weeks after isolation of the clones indicated a 50- to 500-fold amplification of the expression construct in the cells. Concomitantly, the expression of t-PA was dramatically increased. Our data are consistent with episomal persistence of the expression construct, with a head-to-tail mode of integration into the mouse genome and with coexistence of both episomal plasmids and head-to-tail integrates. In tk-deficient cell lines other then L-cells, such as mouse mastocytoma or rat hepatoma cells, a strong selection against the persistence of the expression construct was noted. After long-term propagation of the L-cells under selective conditions the expression of the indicator gene continually decreases, but finally a constant plateau level of expression is established. Expression could be restored to the original level by blocking more efficiently the de novo synthesis of nucleosides.
我们构建了一种用于哺乳动物细胞的新型表达载体。该载体包含一个截短的tk基因，用于在选择性条件下进行扩增；一个推测可支持质粒DNA在真核细胞中复制的序列（小鼠自主复制序列）；以及一个用于待研究cDNA的表达盒。作为模型cDNA，我们使用了人组织型纤溶酶原激活剂（t-PA）的cDNA。对克隆分离六周后制备的L细胞的Hirt上清液和染色体DNA进行分析，结果表明细胞中表达构建体有50至500倍的扩增。与此同时，t-PA的表达显著增加。我们的数据与表达构建体的游离型持久性、以头对头方式整合到小鼠基因组以及游离型质粒和头对头整合体共存相一致。在除L细胞之外的tk缺陷细胞系中，如小鼠肥大细胞瘤或大鼠肝癌细胞，观察到对表达构建体的持久性有强烈的选择作用。在选择性条件下对L细胞进行长期传代培养后，指示基因的表达持续下降，但最终会建立一个恒定的平台期表达水平。通过更有效地阻断核苷的从头合成，表达可以恢复到原始水平。
