Relating Toxicity Toxicity to Transfection: Using Sphingosine To Maintain Prolonged Expression in Vitro
Relating Toxicity Toxicity to Transfection: Using Sphingosine To Maintain Prolonged Expression in Vitro
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DOI：
10.1021/mp500604r
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发表时间：
2015-01-01
影响因子：
4.9
通讯作者：
Anchordoquy, Thomas J.
中科院分区：
文献类型：
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作者：
Betker, Jamie L.;Anchordoquy, Thomas J.
阳离子试剂通常用于促进DNA递送，并且转染实验通常在确定最佳电荷比的细胞培养物中开始。虽然转染率通常在更高的+/-电荷比下增强，但通常不考虑在升高的电荷比下与更大量的阳离子组分相关的细胞毒性。此外，在典型的24-48小时转染实验中，阳离子脂质摄取对细胞活力的长期影响并不明显。在这项研究中，我们比较了阳离子脂质复合物的转染效率对培养细胞的短期和长期（7天）活力的影响。我们的研究结果表明，虽然暴露于DOTAP基脂质体24小时后毒性很小，但细胞活力继续下降，并最终在更长时间内损害报告基因表达。天然存在的阳离子两亲物鞘氨醇取代DOTAP大大降低了毒性，并允许长时间维持高表达。
Cationic reagents are commonly used to facilitate DNA delivery, and transfection experiments are typically initiated in cell culture where the optimal charge ratio is determined. While transfection rates are often enhanced at higher +/- charge ratios, the cellular toxicity associated with the greater amounts of cationic components at elevated charge ratios is often not considered. In addition, the prolonged effects of cationic lipid uptake on cell viability are not evident in a typical 24-48 h transfection experiment. In this study, we compare the transfection efficiency of cationic lipoplexes to effects on viability of cultured cells in both the short and long term (7 days). Our results indicate that, while minimal toxicity is evident 24 h after exposure to DOTAP-based lipoplexes, cell viability continues to decline and ultimately compromises reporter gene expression at longer times. Substitution of a naturally occurring cationic amphiphile, sphingosine, for DOTAP greatly reduces toxicity and allows high expression to be maintained over prolonged periods.