Inclusion of high molecular weight dextran in calcium phosphate-mediated transfection significantly improves gene transfer efficiency
Inclusion of high molecular weight dextran in calcium phosphate-mediated transfection significantly improves gene transfer efficiency
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DOI：
10.1170/t810
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发表时间：
2007-01-01
影响因子：
1.6
通讯作者：
Lu, Y.
中科院分区：
文献类型：
--
作者：
Wu, C.;Lu, Y.
以磷酸钙为基础的哺乳动物细胞转染法是一种广泛应用的基因转移技术。为了促进这种基因转移方法的效率，对几种多糖化合物进行了测试和评估，以提高DNA的转染率。以HIV-1来源的慢病毒载体质粒为基因转移指示剂，我们证明了在2倍的Hepes缓冲盐水(HBS)中添加0.6-1.2%的高分子葡聚糖-500可使转染率提高50%以上(反映在GFP阳性细胞的数量上)，并使慢病毒载体颗粒的滴度增加更多(最多4倍)。当使用较高分子量的葡聚糖制剂取代葡聚糖-500，以及当葡聚糖与另一种聚阳离子化合物聚布林结合使用时，这种转染率的提高进一步增强。对转基因细胞的检测表明，葡聚糖对细胞的存活和生长没有明显的不良影响。我们的数据代表了第一个表明葡聚糖可用于增强磷酸钙介导的基因转移的报告；这可能在使用瞬时转染技术产生高滴度病毒载体方面的应用中有用。
Calcium phosphate-based mammalian cell transfection is a widely used gene transfer technology. To facilitate the efficiency of this gene transfer method, several polysaccharide compounds were tested and evaluated for their effectiveness in enhancing DNA transfection. Using a HIV-1-derived lentivirus vector plasmid as a gene transfer indicator, we demonstrated that the addition of high molecular weight dextran-500 at 0.6-1.2% in the 2x Hepes buffered saline (HBS) increased transfection efficiency by over 50% ( as reflected by the number of GFP-positive cells) and increased the titer of resulting lentivirus vector particles even more (up to 4-fold). This enhancement of transfection efficiency was further increased when higher molecular weight dextran formulations were used in place of dextran-500, and also when dextran was used in combination with polybrene, another polycationic chemical compound. Examination of transfected cells showed that dextran had no apparent adverse effect on cell viability and growth. Our data represent the first report showing that dextran can be used to enhance calcium phosphate-mediated gene transfer; this may be useful in applications for the generation of high-titer virus vector stocks using transient transfection technology.