建立稳定的精子载体法技术体系对家畜的转基因育种等研究具有重要意义.为获得公猪精子与DNA最佳共孵育时间,优化建立猪精子载体法技术体系,本研究通过定量PCR、荧光显微镜检测和精液常规分析方法等,研究分析猪精子与DNA不同共孵育时间对猪精子活力、活率、DNA转染率、吸附DNA和内化DNA量的影响.结果表明,多聚乙烯亚胺(polyethyleneimine,PEI)包裹的标记DNA与精子共孵育15、30、60和90 min后,随着孵育时间的延长,精子活力和活率有极显著下降(P＜0.01),而精子转染率呈极显著(P＜0.01)或显著(P＜0.05)上升,吸附外源DNA量和内化外源DNA量也呈上升趋势,但是30 min后不同孵育时间之间差异不显著(P＞0.05).此外,随着共孵育时间的延长,与精子转染率提高幅度相比,精子活力和活率的下降幅度更明显.综合考虑精子活力、活率、转染率、吸附DNA量和内化DNA量等参数,猪精子与外源DNA共孵育时间以30 min为最佳.%To obtain the optimism co-incubation time of boar sperm with DNA and set up the technique platform of swine sperm-mediated gene transfer, the Real time quantitative PCR, fluorescence microscope imaging and regular semen quality analysis were applied to detect the influence of incubation time of sperm with DNA on viability, motility, transfect efficiency, and the amount of adsorbed DNA and internalized DNA of sperm. The result revealed that, after 15, 30, 60, and 90 min co-incubation of the parceled and labeled DNA with sperm, the sperm motility and viability decreased extremely significantly with the increase of incubation time (P<0.0l), while the transfect efficiency increased extremely significantly (P<0.01) or significantly (P<0.05), as well as the amount of adsorbed DNA and internalized DNA increased, However, there was no difference among kicubation times (P>0.05) after 30 min incubation. In addition, compared with the increase of transfect efficiency of sperm, the decrease of motility and viability of sperm relatively was more obvious with the increase of co-incubation time. Overall, considering the parameters of motility, viability, transfect efficiency and the amount of adsorbed DNA and internalized DNA of sperm, the best co-incubation time of swine sperm with foreign DNA is 30 min.
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