Nuclear-associated plasmid, but not cell-associated plasmid, is correlated with transgene expression in cultured mammalian cells
Nuclear-associated plasmid, but not cell-associated plasmid, is correlated with transgene expression in cultured mammalian cells
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DOI：
10.1006/mthe.2000.0054
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发表时间：
2000-04-01
影响因子：
12.4
通讯作者：
Giorgio, TD
中科院分区：
文献类型：
--
作者：
James, MB;Giorgio, TD
细胞内质粒在阳离子脂质体转染后迅速并入HeLa细胞的细胞核。与HeLa细胞相比，CV1细胞将质粒转移到细胞核的效率较低，表达的转基因也较少。转染cy3标记的pGreenLantern质粒（Cy3-pGL）后，对培养的HeLa和CV1细胞及其分离细胞核进行分析。流式细胞术用于检测编码CMV启动子驱动的绿色荧光蛋白的质粒的传递和转基因表达。转染过程中，HeLa细胞迅速融入质粒，转染后8小时Cy3-pGL阳性细胞最多达到80%。cy3 - pgl阳性的HeLa细胞平均含有约2470个质粒拷贝。8 h后，转染HeLa细胞分离的细胞核中有48%的质粒标记物阳性。与HeLa细胞相比，较少的CV1细胞和CV1细胞核掺入质粒DNA，其中36%的细胞在12 h后达到转染峰值，8 h后12%的细胞核达到转染峰值。然而，平均cy3 - pgl阳性的CV1细胞与平均阳性的HeLa细胞的总细胞质粒拷贝数没有显著差异。然而，CV1核的核相关质粒数量是HeLa核的一半。HeLa细胞比CV1细胞更有效地将质粒从细胞质转运到细胞核。本研究展示了使用一种新的定量方法来研究质粒从细胞质到细胞核的运输及其对转基因表达的影响。
Intracellular plasmid is rapidly incorporated into the nucleus of HeLa cells following cationic lipoplex transfection. CV1 cells are less effective in translocating plasmid to the nucleus and also express less transgene than HeLa cells. Cultured HeLa and CV1 cells and corresponding isolated nuclei were analyzed after transfection of a Cy3-labeled pGreenLantern plasmid (Cy3-pGL). Flow cytometry was used to measure both plasmid delivery and transgene expression from the plasmid encoding a CMV promoter-driven green fluorescent protein. During transfection, HeLa cells rapidly incorporated the plasmid, reaching a maximum of 80% Cy3-pGL positive cells 8 h posttransfection. The average Cy3-pGL-positive HeLa cell contained approximately 2470 plasmid copies. Forty-eight percent of the nuclei isolated from the transfected HeLa cells were positive for the plasmid marker after 8 h. In contrast to HeLa cells, fewer CV1 cells and CV1 nuclei incorporated plasmid DNA with peak transfection occurring after 12 h for 36% of the cells and after 8 h for 12% of the nuclei. However, the average Cy3-pGL-positive CV1 cell did not have a significantly different number of total cellular plasmid copies than the average positive HeLa cell. CV1 nuclei, however, had half as much nuclear associated plasmid as HeLa nuclei. HeLa cells are more efficient than CV1 cells at transporting plasmid from the cytoplasm to the nucleus. This study demonstrates the use of a novel quantitative method to study plasmid transport from the cytoplasm to the nucleus and the effect on transgene expression.