HIGH-LEVEL TRANSIENT EXPRESSION OF A CHLORAMPHENICOL ACETYL TRANSFERASE GENE BY DEAE-DEXTRAN MEDIATED DNA TRANSFECTION COUPLED WITH A DIMETHYLSULFOXIDE OR GLYCEROL SHOCK-TREATMENT
HIGH-LEVEL TRANSIENT EXPRESSION OF A CHLORAMPHENICOL ACETYL TRANSFERASE GENE BY DEAE-DEXTRAN MEDIATED DNA TRANSFECTION COUPLED WITH A DIMETHYLSULFOXIDE OR GLYCEROL SHOCK-TREATMENT
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DOI：
10.1093/nar/12.14.5707
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发表时间：
1984-01-01
影响因子：
14.9
通讯作者：
SOLLNERWEBB, B
中科院分区：
文献类型：
--
作者：
LOPATA, MA;CLEVELAND, DW;SOLLNERWEBB, B
使用含有细菌氯霉素乙酰转移酶基因的质粒，我们分析了通过多种转染条件导入小鼠 L 细胞中的 DNA 的瞬时表达。通过修改 DEAE 右旋糖酐介导的 McCutchan 和 Pagano 转染程序 (1)，加入二甲亚砜或甘油休克，实现了该外源 DNA 的高效摄取和表达。包含休克步骤可以将转染基因的表达增加令人惊讶的～50倍。利用转染后不复制的质粒构建体，我们可以在过夜放射自显影曝光中，从 60 mm 培养皿的转染细胞中提取不到 0.1% 的提取物，轻松检测 CAT 活性。我们已经确定了DNA的量、DEAE-葡聚糖和二甲亚砜处理的量和时间过程、额外DNA的作用以及产生最大表达的转染后的时间。总体而言，这种使用 DEAE-dextrsn 结合休克处理的转染方案简单、直接，并且输入 DNA 始终保持高水平表达。
Using a plasmid containing the bacterial chloramphenicol acetyl transferase gene, we have assayed for transient expression of DNA introduced into mouse L cells by a variety of transfection conditions. High efficiency uptake and expression of this foreign DNA have been achieved by modifying the DEAE dextran mediated trsnafection procedure of McCutchan and Pagano (1) to include a shock with either dimethyl sulfoxide or glycerol. Inclusion of the shock step can increase expreasion of the transfected gene a surprising ˜50 fold. With plasmid constructs that do not replicate after transfection, we can readily detect CAT activity in an overnight autoradiographic exposure from less than 0.1% of an extract from a 60 mm dish of transfected cells. We have determined the amounts of DNA, the amount and time course of DEAE-dextran and dimethyl aulfoxide treatments, the effects of additional DNA, and the time after transfection which yield maximal expression. Overall, this transfection protocol using DEAE-dextrsn coupled to a shock treatment is simple, straightforward, and gives consistently high levels of expression of the input DNA.