Vichera G, Moro L, Salamone D
Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires, Buenos Aires, Argentina.
Reprod Domest Anim. 2011 Apr;46(2):214-20. doi: 10.1111/j.1439-0531.2010.01642.x.
Transgenic animals constitute an important tool with many biotechnological applications. Although there have been advances in this field, we propose a novel method that may greatly increase the efficiency of transgenic animal production and thereby its application. This new technique consists of intracytoplasmic injection of liposomes, in bovine oocytes and zygotes, to introduce exogenous DNA. In the first experiment, we evaluated embryo development and EGFP expression in In Vitro Fertilization (IVF) embryos injected with different concentrations of exogenous DNA-liposome complexes (0.5, 5, 50, 500 ng pCX-EGFP/μl). The highest EGFP-embryos rates were obtained using 500 ng pCX-EGFP/μl. In the second experiment, we evaluated embryo development and EGFP expression following the injection of DNA-liposome complexes into pre-fertilized oocytes and presumptive zygotes, 16 and 24 h post-fertilization. Approximately 70% of the cleaved embryos and 50% of the blastocysts expressed EGFP, when egfp-liposome was injected 16 h post-fertilization. The percentages of positive embryos for the 24-h post-fertilization and pre-fertilization groups were 30.1 and 6.3, respectively. Blastocysts that developed from injected zygotes were analysed by PCR, confirming the presence of transgene in all embryos. Finally, we examined the embryo development and EGFP expression of parthenogenetic embryos that resulted from the injection of egfp-liposome complexes into pre-activated oocytes, and 3 and 11 h post-activated oocytes. The group with the highest expression rate (48.4%) was the one injected 3 h post-activation. In summary, this study reports the efficient, reproducible and fast production of IVF and parthenogenetic embryos expressing EGFP, by the intracytoplasmic injection of liposomes to introduce the foreign DNA.
转基因动物是一种具有多种生物技术应用的重要工具。尽管该领域已取得进展，但我们提出了一种新方法，它可能会大大提高转基因动物的生产效率，进而扩大其应用范围。这项新技术包括在牛卵母细胞和受精卵中进行脂质体胞质内注射，以导入外源DNA。在第一个实验中，我们评估了注射不同浓度外源DNA-脂质体复合物（0.5、5、50、500 ng pCX-EGFP/μl）的体外受精（IVF）胚胎的发育情况和EGFP表达。使用500 ng pCX-EGFP/μl时获得了最高的EGFP胚胎率。在第二个实验中，我们评估了在受精后16小时和24小时将DNA-脂质体复合物注射到受精前的卵母细胞和假定受精卵中后的胚胎发育和EGFP表达。当在受精后16小时注射egfp-脂质体时，约70%的分裂胚胎和50%的囊胚表达EGFP。受精后24小时组和受精前组的阳性胚胎百分比分别为30.1%和6.3%。对注射受精卵发育而来的囊胚进行PCR分析，证实所有胚胎中都存在转基因。最后，我们检查了将egfp-脂质体复合物注射到预激活卵母细胞以及激活后3小时和11小时的卵母细胞中所产生的孤雌胚胎的发育情况和EGFP表达。表达率最高的组（48.4%）是激活后3小时注射的组。总之，本研究报告了通过脂质体胞质内注射导入外源DNA，高效、可重复且快速地生产表达EGFP的IVF和孤雌胚胎。
