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首页> 外文期刊>Biotechnology Progress >Serum Starvation Improves Transient Transfection Efficiency in Differentiating Embryonic Stem Cells
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Serum Starvation Improves Transient Transfection Efficiency in Differentiating Embryonic Stem Cells

机译：血清饥饿提高了分化胚胎干细胞的瞬时转染效率

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摘要

Control of genetic expression is a critical issue in the field of stem cell biology, where determining a cell fate or reprogramming adult somatic cells into pluripotent cells has become a common experimental practice. In turn, for these cells to have therapeutic clinical potential, techniques for controlling gene expression are needed that minimizes or eliminates the risk of oncogenesis and mutagenesis. Possible routes for achieving this outcome could come in the form of a transient nonviral gene delivery system. In this study, we improved the efficiency of transient gene delivery to differentiating murine embryonic stem (ES) cells via serum starvation for 3 days before transfection. The transient expression of a constitutively-controlled plasmid increased from ~50% (replated control) to ~83% when transfected after 3 days of serum starvation but decreased to ~28% when transfected after 3 days in normal high serum-containing media. When probed with a liver-specific reporter, Cyp7Al, expression increased from ~1.4% (replated control) to ~3.7% when transfected after 3 days of serum starvation but decreased to ~0.7% when transfected after 3 days in high serum-containing media. Cy3-tagged oligonucleotides were used to rapidly quantify DNA uptake and predict ultimate transfection efficiency. This study suggests that modifications in media serum levels before transfection can have a profound effect on improving nonviral gene delivery.
机译：遗传表达的控制是干细胞生物学领域中的一个关键问题，在干细胞生物学中，确定细胞命运或将成年体细胞重编程为多能细胞已成为一种常见的实验方法。反过来，为了使这些细胞具有治疗性的临床潜力，需要用于控制基因表达的技术，该技术可最小化或消除致癌和诱变的风险。实现这一结果的可能途径可以是瞬时非病毒基因递送系统的形式。在这项研究中，我们提高了转染前3天通过血清饥饿将瞬时基因传递到分化鼠胚胎干细胞的效率。在血清饥饿3天后转染时，组成型控制质粒的瞬时表达从〜50％（对照）增至〜83％，但在正常的高血清培养基中转染3天后，其瞬时表达降至〜28％。当用肝脏特异性报告基因Cyp7A1进行检测时，在血清饥饿3天后转染时，表达从〜1.4％（相对于对照）增至〜3.7％，但是在高血清培养基中转染3天后，表达降至〜0.7％。 。 Cy3标记的寡核苷酸用于快速定量DNA摄取并预测最终转染效率。这项研究表明，转染前培养基血清水平的改变可对改善非病毒基因的传递产生深远的影响。

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