1. Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Cell J. 2013 Winter;14(4):270-5. Epub 2013 Feb 20.
Electroporation is the most common method used for the transfection of DNA. Although this method has been attributed for various cell using different buffer compositions, the effects of DNA concentration on the transfection efficiency has not yet been studied. Here the correlation between the efficiency of electroporation reaction and increments of DNA concentration has been investigated. Following this investigation, a study was set out to produce a transgenic goat which is capable of secreting recombinant human coagulation factor IX in its milk.
In this experimental study, a linearized recombinant vector (pBC1) entailing human coagulation factor IX (5.7 kb) cDNA was introduced into goat fetal fibroblast cells (three sub passages) via electroporation in four separate experiments (while other variables were kept constant), beginning with 10 µg DNA per pulse in the first experiment and increments of 10 µg/pulse for the next three reactions. Thereafter, polymerase chain reaction (PCR)-positive cell culture plates were diluted by several factors for further analysis of the transfected wells. Subsequently, positive cells were isolated for fluorescence in situ hybridization. Logistic regression model was used for data analyzing. Significance was defined as p< 0.05.
The results showed no significant difference among three first concentrations except for group 1 (10 µg/pulse) and group 3 (30 µg/pulse), but there was a significant difference between these groups and the fourth group (p<0.05), as this group (40 µg/pulse) statistically showed the highest rate of transfection. As the fluorescence in situ hybridization (FISH) results indicated the transgene was integrated in a single position in PCR positive cells.
Increasing amount of using the vector 40µg/pulse efficiently increased the number of transfected cells. Besides of voltage and buffer strength which had been previously shown to play a critical role in electroporation efficiency, our results showed an increase in DNA concentration could affect an exponential surge in the electroporation efficiency.
电穿孔是最常用的 DNA 转染方法。虽然这种方法已被用于不同缓冲液组成的各种细胞，但 DNA 浓度对转染效率的影响尚未得到研究。本研究旨在探讨电穿孔反应效率与 DNA 浓度增加之间的相关性。在此基础上，开展了一项研究，旨在生产能够在其乳汁中分泌重组人凝血因子 IX 的转基因山羊。
在这项实验研究中，通过电穿孔将包含人凝血因子 IX（5.7kb）cDNA 的线性化重组载体（pBC1）导入山羊胎儿成纤维细胞（三个亚代）中，在四个独立的实验中（其他变量保持不变），从第一个实验的每个脉冲 10µg DNA 开始，接下来的三个反应每个脉冲增加 10µg。然后，用几个因素稀释 PCR 阳性细胞培养板，以进一步分析转染孔。随后，对阳性细胞进行荧光原位杂交分离。使用逻辑回归模型进行数据分析。p<0.05 为有统计学意义。
结果表明，除第 1 组（10µg/脉冲）和第 3 组（30µg/脉冲）外，前 3 个浓度之间无显著差异，但这两组与第 4 组（40µg/脉冲）之间有显著差异（p<0.05），因为这组的转染效率最高。荧光原位杂交（FISH）结果表明，转基因整合到 PCR 阳性细胞的单一位置。
使用载体 40µg/脉冲增加剂量可有效增加转染细胞的数量。除了先前已显示在电穿孔效率中起关键作用的电压和缓冲液强度外，我们的结果还表明，DNA 浓度的增加会影响电穿孔效率的指数增长。
