Cheng S M, Lee S G, Kalyan N K, McCloud S, Levner M, Hung P P
Microbiology Division, Wyeth Laboratories Inc., Radnor, PA 19087.
Gene. 1987;58(2-3):299-303. doi: 10.1016/0378-1119(87)90385-4.
Using the gene coding for tissue plasminogen activator (tPA) as a reporter gene, a transient gene expression system has been established. Vectors containing the full-length cDNA of tPA with its signal sequences were introduced into mammalian recipient cells by a modified gene transfer procedure. Thirty hours after transfection, the secreted tPA was found in serum-free medium and measured by a fibrin-agarose plate assay (FAPA). In this assay, tPA converts plasminogen into plasmin which then degrades high-Mr fibrin to produce cleared zones. The sizes of these zones correspond to quantities of tPA. The combination of transient tPA expression system and the FAPA provides a quick, sensitive, quantitative and non-destructive method to examine the strength of eukaryotic regulatory elements in tissue-culture cells.
利用编码组织型纤溶酶原激活剂（tPA）的基因作为报告基因，建立了一个瞬时基因表达系统。通过改良的基因转移程序，将含有tPA全长cDNA及其信号序列的载体导入哺乳动物受体细胞。转染30小时后，在无血清培养基中发现分泌的tPA，并通过纤维蛋白-琼脂糖平板测定法（FAPA）进行测量。在该测定法中，tPA将纤溶酶原转化为纤溶酶，然后纤溶酶降解高分子量纤维蛋白以产生清除区。这些区域的大小与tPA的量相对应。瞬时tPA表达系统和FAPA的结合提供了一种快速、灵敏、定量且非破坏性的方法，用于检测组织培养细胞中真核调节元件的强度。
