Department of Reproduction and Development, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran.
Mol Reprod Dev. 2010 Oct;77(10):868-75. doi: 10.1002/mrd.21228.
In relation to the growing recent interest in the establishment of sperm-mediated gene transfer (SMGT) technology as a convenient and effective method for the simple production of transgenic animals, in this study the possibility of using SMGT to produce transgenic caprine embryos was investigated for the first time. Buck sperm were directly incubated with different concentrations (0-500 ng) of pcDNA/his/Lac-Z plasmid and used for IVF or ICSI. Sperm used for ICSI were categorized into motile or live-immotile group before being injected into oocytes. In a separate experiment, dead sperm prepared by repeated freezing/thawing were used for DNA-incubation before ICSI. Sham injection was carried out by intracytoplasmic injection of approximately the same volume of media containing different doses of DNA using an ICSI needle. Transgene expression and transmission were detected by X-Gal staining and PCR analysis of developed embryos, respectively. A reasonable blastocyst rate was observed in all the groups. Only embryos in the sham group were negative for transgene transmission. Transgene expression was completely dependent on the delivery technique and status of sperm, and was only observed in the live-immotile and dead ICSI groups. The results of this study showed that the technique (IVF vs. ICSI vs. sham injection), sperm status (motile vs. live-immotile vs. dead) and to some extent DNA concentration affect embryo development, transgene transmission and expression.
关于建立精子介导的基因转移（SMGT）技术作为一种简便有效的生产转基因动物的方法，近来引起了越来越多的关注。在这项研究中，我们首次研究了使用 SMGT 生产转基因山羊胚胎的可能性。将不同浓度（0-500ng）的 pcDNA/his/Lac-Z 质粒直接与精子孵育，然后用于 IVF 或 ICSI。在将精子注入卵母细胞之前，根据精子的运动能力或死活能力，将用于 ICSI 的精子分为运动组或死活不运动组。在另一个实验中，使用反复冻融制备的死精子在进行 ICSI 之前进行 DNA 孵育。通过使用 ICSI 针将含有不同剂量 DNA 的介质注入到细胞质内，进行假注射。通过 X-Gal 染色和对发育胚胎的 PCR 分析，分别检测转基因的表达和传递。所有组均观察到了合理的囊胚率。仅在假注射组的胚胎中转基因传递呈阴性。转基因的表达完全取决于精子的传递技术和状态，仅在死活不运动的 ICSI 组和死精子 ICSI 组中观察到。本研究结果表明，该技术（IVF 与 ICSI 与假注射）、精子状态（运动与死活不运动与死）以及在一定程度上 DNA 浓度影响胚胎发育、转基因传递和表达。
