Leahy P, Carmichael G G, Rossomando E F
Department of BioStructure and Function, University of Connecticut Health Center, Farmington, CT 06030, USA.
Nucleic Acids Res. 1997 Jan 15;25(2):449-50. doi: 10.1093/nar/25.2.449.
A protocol for increasing transcription from plasmid expression vectors is presented. A vector containing chloramphenicol acetyltransferase (CAT) gene was digested leaving the transcription cassette intact. Heat inactivation of restriction enzymes followed by ligation of the digestion products yielded concatemers which migrated as a single band in agarose gel electrophoresis. Mouse fibroblasts transfected with the concatemers gave a CAT activity that was 14-fold greater than that of cells transfected with a similar mass (equimolar gene number) of the native plasmid. The effect was independent of promoter type, restriction enzyme, number of restriction sites and with a noted exception, cell line.
本文介绍了一种提高质粒表达载体转录的方案。一个含有氯霉素乙酰转移酶（CAT）基因的载体被消化，使转录盒保持完整。对限制酶进行热失活，然后将消化产物连接，得到串联体，其在琼脂糖凝胶电泳中迁移为单一条带。用串联体转染的小鼠成纤维细胞产生的CAT活性比用相同质量（等摩尔基因数）的天然质粒转染的细胞高14倍。该效应与启动子类型、限制酶、限制位点数量无关，且除了一个显著的例外，与细胞系也无关。
