Demirhan I, Hasselmayer O, Chandra A, Ehemann M, Chandra P
Gustav-Embden Center of Biological Chemistry, Department of Molecular Biology, Frankfurt University Medical School, Germany.
J Hum Virol. 1998 Nov-Dec;1(7):430-40.
We studied the gene transfer efficiency of lipofection reagents in comparison to DEAE-Dextran. DOTAP, Dosper, and Lipofectin have lower transfection efficiency; Lipofectamine has a 2.5-fold better efficiency compared with DEAE-Dextran. We report a novel and highly efficient DNA transfer system based on the DNA-binding proteins histone 3 and histone 4. We have transferred the HIV-1 tat gene and measured the transactivation of HIV-1 LTR by the transactivator protein, expressed in Jurkat cells. The HIV-1 LTR was linked to the CAT gene as a reporter. Compared to DEAE-Dextran-mediated transfection, histone-mediated transfection resulted in a sevenfold higher expression of the CAT gene. The maximum transfection efficiency mediated by histones is dependent on the relative concentration (DNA:histone ratio) and the incubation time. In a gel-retardation assay, an optimal complex formation was observed under the same conditions that allowed the highest transfection efficiency. This ability of histones to increase the delivery and transgenic expression of foreign DNA in eukaryotic cells is not simply due to the positive ionic character of the histone proteins. Polylysine, histone H1, and histone H2A were unable to mediate gene transfection in our system. Monoclonal antibodies that recognize antigenic determinant present on all five histone proteins (anti-histone, pan) were able to neutralize the transfection-enhancing potential of histone 3 and histone 4. However, anti-histone IgG enhanced the retardation of mobility of histone-DNA complexes. The results of this study allow us to conclude that histones H3 and H4 can catalyze gene transfer and gene expression in eukaryotic cells without any requirement for additional constituents. For this reason, we have termed the new gene-delivery system as histonefection.
我们研究了脂质转染试剂相对于DEAE-葡聚糖的基因转移效率。DOTAP、Dosper和Lipofectin的转染效率较低；Lipofectamine的效率比DEAE-葡聚糖高2.5倍。我们报告了一种基于DNA结合蛋白组蛋白3和组蛋白4的新型高效DNA转移系统。我们已导入HIV-1 tat基因，并在Jurkat细胞中测量了由反式激活蛋白对HIV-1 LTR的反式激活作用。HIV-1 LTR与作为报告基因的CAT基因相连。与DEAE-葡聚糖介导的转染相比，组蛋白介导的转染使CAT基因的表达提高了7倍。组蛋白介导的最大转染效率取决于相对浓度（DNA:组蛋白比例）和孵育时间。在凝胶阻滞试验中，在允许最高转染效率的相同条件下观察到了最佳复合物形成。组蛋白在真核细胞中提高外源DNA递送和转基因表达的这种能力并非仅仅归因于组蛋白的正离子特性。在我们的系统中，聚赖氨酸、组蛋白H1和组蛋白H2A无法介导基因转染。识别所有五种组蛋白上存在的抗原决定簇的单克隆抗体（抗组蛋白，泛）能够中和组蛋白3和组蛋白4的转染增强潜力。然而，抗组蛋白IgG增强了组蛋白-DNA复合物迁移率的阻滞。这项研究的结果使我们得出结论，组蛋白H3和H4可以在真核细胞中催化基因转移和基因表达，而无需任何其他成分。因此，我们将这种新的基因递送系统称为组蛋白转染。
