影响猪精子介导基因转移效果的因素研究

Factors Affecting Porcine Sperm Mediated Gene Transfer

为优化猪精子载体法技术参数,利用荧光定量PCR、荧光显微镜检测和精液常规检测方法,分析不同DNA转染剂、不同孵育温度和不同形态DNA对猪精子转染外源DNA的影响。结果表明,PEI和TransFast转染剂能极显著提高猪精子转染效果,且PEI优于TransFast,而NanoFect转染剂包裹DNA不能转染精子。随着转染温度的升高,精子的活力和活率均明显下降,而转染率和内化外源DNA量基本保持稳定,而吸附外源DNA量呈下降趋势。环状DNA和线性化DNA在与精子共孵育后,两者的精子活力、活率、转染率和内化外源DNA量差异均不显著,精子吸附线性化DNA量极显著高于环状DNA量。综合分析表明,外源DNA形态对猪精子转染效果无显著影响;PEI和TransFast能够显著提高猪精子转染效果;共孵育温度以17℃为最佳,本结果为开展精子载体法制备转基因猪研究提供了基础试验依据。

To optimize the technical parameters of porcine sperm mediated gene transfer, the real-time PCR, fluorescence microscope imaging and regular semen quality analysis were applied to detect the influ- ence of DNA transfect reagent, incubation temperature and DNA form on the transfection of porcine sperm with foreign DNA. The result revealed that PEI and TransFast significantly improved the transfection of porcine sperm with DNA, and PEI was better than TransFast, but NanoFect blocked the transfection com- pletely. With the increase of incubation temperature, the viability, motility, and the amount of binding DNA of sperm decreased significantly, while the transfection efficiency and amount of internalized DNA were not affected. The difference of viability, motility, and amount of internalized DNA was not signifi- cant between circular and linear DNA incubated sperm, whereas the amount of binding DNA of sperm incubated with linear DNA was extremely higher than that of circular DNA. It could be concluded that the form of DNA had no significant influence on the transfection of porcine sperm, PEI and TransFast improved the transfection of porcine sperm significantly, and the best incubation temperature for porcine sperm is 17 ℃. This study provided basic data for further porcine transgenesis research with sperm media- ted gene transfer.