非病毒载体聚乙烯亚胺转基因因素的优化

聚乙烯亚胺属于阳离子多聚物,可浓缩DNA作为转基因非病毒载体。但其转染影响变量较多,如果不能充分控制将不能得到重复的、良好的结果。这里测定了聚乙烯亚胺转基因效率的影响因素,为合成更复杂的人工转基因载体创造条件。我们通过聚乙烯亚胺转染编码β-半乳糖苷酶的pSVβ质粒到CO S-7和N IH 3T 3细胞中,测定质粒因素、血清、细胞密度、操作方式以及转染复合物的保存因素对聚乙烯亚胺转基因效率的影响。结果表明:质粒中生物活性抑制剂明显降低转染效率,通过截流分子量3000或10000的超滤可以除去生活性抑制剂;断裂的质粒降低转染效率;培养液中的血清、白蛋白降低转染效率;细胞密度影响转染效率;PE I/DNA复合物与细胞作用8h后吸去,转染效果最优。冻存显著降低PE I/DNA转染复合物转染效率。聚乙烯亚胺可以作为合成新型非病毒载体的骨架,但必须控制有关因素才能发挥最佳的和可重复性的转染结果。

Gene Transfer by a Novel Nonviral Vector Polyethylenimine

This study inquired about the influences on the gene delivery efficiency of polyethylenimine. pSVbeta plasmids were transferred into COS-7 and NIH3T3 cells with polyethylenimine. Influences of plasmids factor, albumin, serum, cell density, operation methods and polyethylenimine/DNA preserve factors on transfection efficiency were investigated. Inhibitors of biological activities in plasmids could be removed by ultrafiltration with cutoff molecular weight of 3000 or 10000. Linear plasmids lowered transfection efficiency. Serum and albumin in the culture medium decreased the transfection efficiency of polyethylenimine/DNA. Cell density was associated with PEI/DNA transfection efficiency. Incubation of PEI/DNA complexes with the cells for 8 h and then aspiration for removal of the complexes could obtain an optimal transfection effect. Freezing of PEI/DNA complexes significantly decreased transfection efficiency. In conclusion, polyethylenimine could obtain optimal and reduplicate transfection results by controlling related factors.