Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Av. San Martín 4453, C1417 Buenos Aires, Argentina.
Theriogenology. 2010 Oct 1;74(6):922-31. doi: 10.1016/j.theriogenology.2010.04.017. Epub 2010 Jun 8.
In order to establish conditions for intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) in cattle, various aspects of fertilization and embryonic development were assessed after five activation treatments. Spermatozoa were co-incubated with pCX-EGFP (pCX-enhanced green fluorescent protein gene) plasmid and injected into metaphase II oocytes, which were then treated with ionomycin (Io), before further activation with the following agents: 6-dimethylaminopurine (Io-DMAP), additional Io plus DMAP (2Io-DMAP), Io alone (2Io), ethanol (Io-EtOH), or strontium chloride (Io-SrCl2). Fertilization rates at 16 h after ICSI, presence of a condensed spermatozoon head on Day 4 (Day 0 = ICSI), blastocyst and EGFP expression rates on Day 7, and Oct-4 pattern of Day 8 blastocysts were evaluated. Fertilization rates did not differ significantly among treatments. All (100%) of EGFP-positive embryos resulted from ICSI fertilization, whereas at least 60% of EGFP-negative embryos (>4 cells) had a condensed sperm head. Blastocyst rates after 2Io-DMAP were not significantly different from Io-DMAP or Io-EtOH, but they were higher than 2Io or Io-SrCl2 treatments (25.9, 18.7, 14.7, 9.4, and 10.9% respectively; P < 0.05). Transgene expression rates were higher for Io-DMAP, 2Io-DMAP and Io-SrCl2 than for 2Io and Io-EtOH (52.3, 53.0, 42.8, 28.2, and 29.4% respectively; P < 0.05). Over 80% of the blastocysts expressed egfp protein. In conclusion, ICSI-MGT was a powerful technique to produce bovine embryos that expressed the EGFP transgene. Moreover, the actual efficiency of ICSI-MGT could be readily evaluated by this method, which uses a marker expressed early in embryo development.
为了在牛体内建立胞质内精子注射介导的基因转移（ICSI-MGT）的条件，评估了五种激活处理后受精和胚胎发育的各个方面。精子与 pCX-EGFP（pCX-增强型绿色荧光蛋白基因）质粒共孵育，然后注射到中期 II 期卵母细胞中，再用离子霉素（Io）处理，然后用以下试剂进一步激活：6-二甲基氨基嘌呤（Io-DMAP）、额外的 Io 加 DMAP（2Io-DMAP）、Io 单独（2Io）、乙醇（Io-EtOH）或氯化锶（Io-SrCl2）。在 ICSI 后 16 小时评估受精率、第 4 天（第 0 天=ICSI）出现浓缩精子头的情况、第 7 天囊胚和 EGFP 表达率以及第 8 天囊胚的 Oct-4 模式。处理之间的受精率没有显著差异。所有（100%）的 EGFP 阳性胚胎均来自 ICSI 受精，而至少 60%的 EGFP 阴性胚胎（>4 个细胞）具有浓缩的精子头。2Io-DMAP 后的囊胚率与 Io-DMAP 或 Io-EtOH 无显著差异，但高于 2Io 或 Io-SrCl2 处理（分别为 25.9%、18.7%、14.7%、9.4%和 10.9%；P<0.05）。Io-DMAP、2Io-DMAP 和 Io-SrCl2 的转基因表达率高于 2Io 和 Io-EtOH（分别为 52.3%、53.0%、42.8%、28.2%和 29.4%；P<0.05）。超过 80%的囊胚表达 egfp 蛋白。总之，ICSI-MGT 是一种产生表达 EGFP 转基因的牛胚胎的强大技术。此外，通过这种方法可以很容易地评估 ICSI-MGT 的实际效率，该方法使用在胚胎发育早期表达的标记。
