Kumar Pramod R, Kumar Rakesh, Mitra Abhijit
Genome Analysis Laboratory, Animal Genetics Division, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P., India.
Genome Analysis Laboratory, Animal Genetics Division, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P., India.
Gene. 2016 Jan 15;576(1 Pt 3):505-11. doi: 10.1016/j.gene.2015.10.066. Epub 2015 Oct 30.
Current methods of transgenic animal production are afflicted by low efficiency and high cost. Recently, the electroporation aided sperm-mediated gene transfer (SMGT) emerges as a promising alternative with variable success rate. Among the domestic animal species, the electroporation-aided SMGT is less investigated in goats, except a few reports in which attempts have been made using the auto-uptake method of SMGT. In this study, we report an optimized electroporation condition for SMGT of caprine sperm cells. Results of this study demonstrated that electroporation of caprine sperm cells at 300 V for 200 mS in TALP medium allowed the maximum uptake of foreign DNA with minimum adverse effects on the vital semen parameters viz., progressive motility, viability, and membrane and acrosome integrity. Further, DNA binding assay revealed DNA uptake by 81.3% sperm cells when 1.0 μg of DNA was used under optimum electroporation conditions as compared to 16.5% on simple incubation. The qPCR analysis showed four-fold more (P<0.05) DNA uptake by sperm cells under electroporation than incubation. A similar cleavage rate was observed after IVF using either electroporated (23.20 ± 1.20) or non-electroporated (25.20 ± 2.41) sperm cells suggesting the absence of adverse effect of electroporation on the fertilizing ability. Out of the 116 embryos produced by electroporated sperm, five (4.31%) embryos showed the expression of the foreign gene. In conclusion, our results confirm that using optimized electroporation conditions, the caprine sperm cells can uptake foreign DNA effectively with minimum negative effect on the semen parameters and could produce transgenic embryos.
目前转基因动物的生产方法效率低下且成本高昂。最近，电穿孔辅助精子介导基因转移（SMGT）作为一种有前景的替代方法出现，成功率各不相同。在家畜物种中，电穿孔辅助SMGT在山羊中的研究较少，除了少数几篇使用SMGT自动摄取方法进行尝试的报告。在本研究中，我们报告了一种优化的山羊精子细胞SMGT电穿孔条件。本研究结果表明，在TALP培养基中以300 V电压对山羊精子细胞进行200毫秒的电穿孔，可使外源DNA摄取量最大，同时对精液重要参数（即渐进性活力、生存力、膜和顶体完整性）的负面影响最小。此外，DNA结合试验显示，在最佳电穿孔条件下使用1.0 μg DNA时，81.3%的精子细胞摄取了DNA，而简单孵育时这一比例为16.5%。qPCR分析表明，电穿孔处理的精子细胞摄取的DNA比孵育处理的多四倍（P<0.05）。使用电穿孔（23.20±1.20）或未电穿孔（25.20±2.41）的精子细胞进行体外受精后，观察到类似的裂解率，这表明电穿孔对受精能力没有负面影响。在由电穿孔精子产生的116个胚胎中，有5个（4.31%）胚胎显示出外源基因的表达。总之，我们的结果证实，使用优化的电穿孔条件，山羊精子细胞可以有效摄取外源DNA，对精液参数的负面影响最小，并能产生转基因胚胎。
