Enhanced reporter gene expression in cells transfected in the presence of DMI-2, an acid nuclease inhibitor
Enhanced reporter gene expression in cells transfected in the presence of DMI-2, an acid nuclease inhibitor
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DOI：
10.1038/sj.gt.3300721
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发表时间：
1998-09-01
期刊：
影响因子：
5.1
通讯作者：
Korfhagen, TR
中科院分区：
文献类型：
--
作者：
Ross, GF;Bruno, MD;Korfhagen, TR
细胞核酸酶活性是成功将外源基因递送至哺乳动物细胞的潜在障碍。我们测试了这样的假设：在特定 DNase 抑制剂存在下的转染可以增强外源基因产物的表达。我们使用了 DMI-2，一种链霉菌的聚酮化合物代谢物。菌株 560 增强人肺腺癌细胞系 H441 中细菌氯霉素乙酰转移酶 (CAT) 的表达。先前已证明 DMI-2 可以抑制猪 DNase II，这是一种内体/溶酶体区室中含有的酸性 pH 核酸酶。在0.1-1μg/ml DMI-2存在下转染H441细胞引起：（1）当细菌质粒与表面活性蛋白A-聚赖氨酸或转铁蛋白-聚赖氨酸复合时，CAT活性增强10倍； (2) 接触 lipofectin-DNA 复合物的细胞中 CAT 活性增强 1.5 至 2 倍； (3)磷酸钙共沉淀对转染无影响。单独的 DMI-2 没有表现出固有的转染活性。在使用 SP-A-聚赖氨酸和含有 β-半乳糖苷酶报告基因的质粒的实验中，DMI-2 增加了转染细胞的数量。 DMI-2的甲醇分解产物不抑制DNase II并且不提高转染效率。综上所述，数据支持以下假设：核酸酶作用是外源基因表达的显着障碍，并且特定核酸酶的抑制可能促进转染。
Cellular nuclease activity is a potential barrier to the successful delivery of foreign genes to mammalian cells We tested the hypothesis that transfection in the presence of a specific DNase inhibitor can enhance the expression of foreign gene products. We have used DMI-2, a polyketide metabolite of Streptomyces sp. strain 560 to enhance the expression of bacterial chloramphenicol acetyltransferase (CAT) in the human lung adenocarcinoma cell line H441. DMI-2 has been shown previously to inhibit porcine DNase II, an acid pH nuclease contained in the endosomal/lysosomal compartment. Transfection of H441 cells in the presence of 0.1-1 mu g/ml DMI-2 caused: (1) 10-fold enhancement of CAT activity when the bacterial plasmid was complexed with either surfactant protein A-poly-lysine or transferrin-poly-lysine; (2) 1.5- to two-fold enhancement of CAT activity in cells exposed to lipofectin-DNA complexes; (3) no effect on transfection via calcium phosphate co-precipitation. DMI-2 alone showed no inherent transfection activity. In experiments using SP-A-poly-lysine and plasmid containing the beta-galactosidase reporter gene, DMI-2 increased the number of transfected cells. Methanolysis products of DMI-2 did not inhibit DNase II and did not enhance transfection efficiency, Taken together, the data support the hypothesis that nuclease action is a significant barrier to expression of-foreign genes and inhibition of specific nucleases may facilitate transfection.