Histone H1-mediated transfection:: Serum inhibition can be overcome by Ca2+ ions
Histone H1-mediated transfection:: Serum inhibition can be overcome by Ca2+ ions
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DOI：
10.1023/a:1007581700996
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发表时间：
2000-02-01
影响因子：
3.7
通讯作者：
Böttger, M
中科院分区：
文献类型：
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作者：
Haberland, A;Knaus, T;Böttger, M
目的.聚阳离子和阳离子脂质体基因转移的缺点之一是对血清敏感。基因治疗需要转染-DNA复合物对血清和血液具有抗性。由于Ca 2+已被证明是聚阳离子基因转移的有效辅因子，我们决定研究其在血清存在下对转染的影响。我们研究了荧光素酶基因（pCMV Luc）在人内皮细胞ECV 304上的转基因表达。发现HI和DOSPER介导的转染被高于10%浓度的血清抑制。如果在培养基中加入2 mM Ca 2+或2 mM Ca 2 +/0.1 mM氯喹替代转染混合物，并在孵育后在细胞上放置24小时，则即使是100%血清的抑制作用也被克服。高血清水平不会干扰H1和DOSPER-DNA复合物的结合和摄取，但会抑制后续步骤，如内体逃逸。新生磷酸钙微沉淀形式的Ca 2+和其他溶酶体溶解剂通过其融合和膜溶解活性促进内体/溶酶体释放。
Purpose. One of the drawbacks of polycationic and cationic liposomal gene transfer is its sensitivity to serum. Gene therapy requires the transfectant-DNA complex to be resistant to serum as well as blood. Since Ca2+ has proved to be an efficient cofactor of polycationic gene transfer, we decided to investigate its effects on transfection in the presence of serum.Methods. We studied transgene expression of luciferase gene (pCMV Luc) on ECV 304 human endothelial cells using I-Il histone and DOSPER as transfectants in the presence of 0-100% fetal calf serum.Results. Hi-and DOSPER-mediated transfection was found to be inhibited by serum above the concentration of 10%. If 2 mM Ca2+ or 2 mM Ca2+/0.1 mM chloroquine was included in the culture medium which replace the transfection mixture and was left on the cells for 24 hours postincubation, the inhibiting effect of even 100% serum was overcome.Conclusions. A high serum level does not interfere with binding and uptake of H1- and DOSPER-DNA complexes, but inhibits subsequent steps such as endosomal escape. Ca2+ in the form of nascent calcium phosphate microprecipitates and other lysosomolytical agents facilitate endosomal/lysosomal release by their fusigenic and membranolytic activity.