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首页> 外文期刊>Molecular biotechnology >A comparison of calcium phosphate coprecipitation and electroporation. Implications for studies on the genetic effects of DNA damage.
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A comparison of calcium phosphate coprecipitation and electroporation. Implications for studies on the genetic effects of DNA damage.

机译：磷酸钙共沉淀和电穿孔的比较。对DNA损伤的遗传效应研究的意义。

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摘要

Plasmid-based transfection assays provide a rapid means to measure homologous and nonhomologous recombination in mammalian cells. Often it is of interest to examine the stimulation of recombination by DNA damage induced by radiation, genotoxic chemicals, or nucleases. Transfection is frequently performed by using calcium phosphate coprecipitation (CPP), because this method is well suited for handling large sample sets, and it does not require expensive reagents or equipment. Alternative transfection methods include lipofection, microinjection, and electroporation. Since DNA strand breaks are known to stimulate both homologous and nonhomologous recombination, the induction of nonspecific damage during transfection would increase background recombination levels and thereby reduce the sensitivity of assays designed to detect the stimulation of recombination by experimentally induced DNA damage. In this article, we compare the stimulatory effects of nuclease-induced double-strand breaks (DSBs) on homologous and nonhomologous recombination for molecules transfected by CPP and by electroporation. Although electroporation yielded fewer transfectants, both nonhomologous and homologous recombination were stimulated by nuclease-induced DSBs to a greater degree than with CPP. Ionizing radiation is an effective agent for inducing DNA strand breaks, but previous studies using CPP generally showed little or no stimulation of homologous recombination among plasmids damaged with ionizing radiation. By contrast, we found clear dose-dependent enhancement of recombination with irradiated plasmids transfected using electroporation. Thus, electroporation provides a higher signal-to-noise ratio for transfection-based studies of damage-induced recombination, possibly reflecting less nonspecific damage to plasmid DNA during transfection of mammalian cells.
机译：基于质粒的转染试验提供了一种快速的方法来测量哺乳动物细胞中的同源和非同源重组。通常有必要检查辐射，遗传毒性化学物质或核酸酶诱导的DNA损伤对重组的刺激。转染通常使用磷酸钙共沉淀（CPP）进行，因为该方法非常适合处理大型样品，并且不需要昂贵的试剂或设备。备选的转染方法包括脂质转染，显微注射和电穿孔。由于已知DNA链断裂会刺激同源和非同源重组，因此在转染过程中诱导非特异性损伤会增加背景重组水平，从而降低旨在通过实验诱导的DNA损伤检测重组刺激的检测灵敏度。在本文中，我们比较了核酸酶诱导的双链断裂（DSB）对CPP和电穿孔转染的分子的同源和非同源重组的刺激作用。尽管电穿孔产生的转染子较少，但是与CPP相比，核酸酶诱导的DSB刺激非同源和同源重组的程度更高。电离辐射是诱导DNA链断裂的有效试剂，但是先前使用CPP的研究通常显示在电离辐射损坏的质粒之间很少或没有刺激同源重组。相比之下，我们发现用电穿孔转染的辐照质粒的重组具有明显的剂量依赖性。因此，电穿孔为基于转染的损伤诱导的重组研究提供了更高的信噪比，可能反映了哺乳动物细胞转染过程中对质粒DNA的非特异性损伤较少。

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