Departamento de Fisiología, Facultad de Veterinaria Departmento de Biología Celular e Histología, Facultad de Medicina, Universidad de Murcia, Murcia, Spain.
Reproduction. 2010 Aug;140(2):259-72. doi: 10.1530/REP-10-0129. Epub 2010 May 25.
Sperm-mediated gene transfer (SMGT) is a method for the production of transgenic animals based on the intrinsic ability of sperm cells to bind and internalize exogenous DNA molecules and to transfer them into the oocyte at fertilization. Recombinase-A (RecA) protein-coated exogenous DNA has been used previously in pronuclear injection systems increasing integration into goat and pig genomes. However, there are no data regarding transgene expression after ICSI. Here, we set out to investigate whether the expression of transgenic DNA in porcine embryos is improved by recombinase-mediated DNA transfer and if it is possible to generate transgenic animals using this methodology. Different factors which could affect the performance of this transgenic methodology were analyzed by studying 1) the effect of the presence of exogenous DNA and RecA protein on boar sperm functionality; 2) the effect of recombinase RecA on in vitro enhanced green fluorescent protein (EGFP)-expressing embryos produced by ICSI or IVF; and 3) the efficiency of generation of transgenic piglets by RecA-mediated ICSI. Our results suggested that 1) the presence of exogenous DNA and RecA-DNA complexes at 5 microg/ml did not affect sperm functionality in terms of motility, viability, membrane lipid disorder, or reactive oxygen species generation; 2) EGFP-expressing embryos were obtained with a high efficiency using the SMGT-ICSI technique in combination with recombinase; however, the use of IVF system did not result in any fluorescent embryos; and 3) transgenic piglets were produced by this methodology. To our knowledge, this is the first time that transgenic pigs have been produced by ICSI-SGMT and a recombinase.
精子介导的基因转移 (SMGT) 是一种基于精子细胞结合和内化外源 DNA 分子并在受精时将其转移到卵母细胞中的固有能力来生产转基因动物的方法。以前，在原核注射系统中使用了带有重组酶-A (RecA) 蛋白的外源 DNA，从而增加了整合到山羊和猪基因组中的效率。然而，关于 ICSI 后转基因表达的情况还没有数据。在这里，我们着手研究重组酶介导的 DNA 转移是否可以提高猪胚胎中转基因 DNA 的表达，以及是否可以使用这种方法生成转基因动物。通过研究 1) 外源 DNA 和 RecA 蛋白的存在对猪精子功能的影响；2) 重组酶 RecA 对通过 ICSI 或 IVF 产生的体外增强型绿色荧光蛋白 (EGFP) 表达胚胎的影响；以及 3) 通过 RecA 介导的 ICSI 生成转基因猪的效率，分析了可能影响这种转基因方法性能的不同因素。我们的结果表明，1) 以 5μg/ml 存在的外源 DNA 和 RecA-DNA 复合物不会影响精子的运动能力、活力、膜脂质紊乱或活性氧的产生；2) 使用 SMGT-ICSI 技术结合重组酶可以高效获得 EGFP 表达的胚胎；然而，使用 IVF 系统不会产生任何荧光胚胎；并且 3) 可以通过这种方法产生转基因猪。据我们所知，这是首次通过 ICSI-SGMT 和重组酶生产转基因猪。
