Ball B A, Sabeur K, Allen W R
Department of Population Health and Reproduction, University of California-Davis, Davis, CA 95616, USA.
Equine Vet J. 2008 Jan;40(1):76-82. doi: 10.2746/042516407X235786.
Sperm-mediated gene transfer has been reported as a method for production of transgenic animals in a variety of species, and this technique represents a possible method for production of transgenic equids.
To evaluate the uptake of exogenous DNA (enhanced green fluorescent protein; pEGFP) by equine spermatozoa and to assess the ability of transfected spermatozoa to introduce this transgene into early equine embryos.
To evaluate incorporation of pEGFP into equine spermatozoa, washed spermatozoa were incubated with 32P-pEGFP, with or without lipofection. Spermatozoa were also transfected with fluorescently-labelled DNA (Alexa647-pEGFP) and changes in sperm viability and DNA uptake were assessed. Mares were inseminated with pEGFP-transfected spermatozoa and embryos recovered. Expression of pEGFP was assessed by epifluorescence microscopy of embryos, and the presence of pEGFP DNA and mRNA was assessed by PCR and RT-PCR, respectively.
Liposome-mediated transfection increased the incorporation of 32P-pEGFP into spermatozoa compared to controls. Flow cytometric evaluation of spermatozoa after transfection with Alexa647-pEGFP revealed a linear increase in the proportion of live, Alexa647+ spermatozoa with increasing DNA concentrations. After insemination with transfected spermatozoa, 8 embryos were recovered. There was no evidence of EGFP expression in the recovered embryos; however, PCR analysis revealed evidence of the pEGFP transgene in 2 of 5 embryos analysed.
The incorporation of exogenous DNA by equine spermatozoa was enhanced by liposome-mediated transfection and this did not adversely affect sperm viability, acrosomal integrity or fertility. Although the EGFP transgene was detected in a proportion of Day 7-10 embryos, there was no evidence of expression of EGFP in these embryos.
Sperm-mediated gene transfer offers a potential technique for the generation of transgenic equids.
精子介导的基因转移已被报道为一种在多种物种中生产转基因动物的方法，该技术是生产转基因马科动物的一种可能方法。
评估马精子对外源DNA（增强型绿色荧光蛋白；pEGFP）的摄取，并评估转染精子将该转基因导入早期马胚胎的能力。
为评估pEGFP整合到马精子中的情况，将洗涤后的精子与32P-pEGFP一起孵育，有无脂质体转染。精子还用荧光标记的DNA（Alexa647-pEGFP）进行转染，并评估精子活力和DNA摄取的变化。用pEGFP转染的精子对母马进行授精并回收胚胎。通过胚胎的落射荧光显微镜评估pEGFP的表达，分别通过PCR和RT-PCR评估pEGFP DNA和mRNA的存在情况。
与对照组相比，脂质体介导的转染增加了32P-pEGFP整合到精子中的量。用Alexa647-pEGFP转染后对精子进行流式细胞术评估显示，随着DNA浓度增加，活的Alexa647+精子的比例呈线性增加。用转染的精子授精后，回收了8个胚胎。在回收的胚胎中没有EGFP表达的证据；然而，PCR分析显示在分析的5个胚胎中有2个存在pEGFP转基因的证据。
脂质体介导的转染增强了马精子对外源DNA的整合，且这并未对精子活力、顶体完整性或生育力产生不利影响。尽管在一部分第7 - 10天的胚胎中检测到了EGFP转基因，但在这些胚胎中没有EGFP表达的证据。
精子介导的基因转移为生成转基因马科动物提供了一种潜在技术。
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