Amini Hamid-Reza, Pakdel Abbas, Shahr-Babak Hossein Moradi, Eghbalsaied Shahin
Transgenesis Center of Excellence, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan, Iran; Department of Animal Science, University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran.
Department of Animal Science, Isfahan University of Technology, Isfahan, Iran.
Theriogenology. 2017 Mar 15;91:90-97. doi: 10.1016/j.theriogenology.2016.12.031. Epub 2016 Dec 26.
Chicken is a dual-purpose animal important from both agricultural and medical aspects. Even though significant improvements have been made in chicken transgenesis technologies, chicken genome manipulation has not been widely used in developmental biology. This study was aimed to evaluate chicken egg white nuclease properties and thereof plausibility of devising an in vivo transfection technology without causing physical damage to the embryo. First, the nuclease activity of egg albumen was assessed. The egg white nucleases were strongly active in degrading DNA and RNA. The egg white DNase activity was comparable to commercially available DNase-I. Nuclease activities were also assessed after heating, proteinase K, or EDTA treatment. Unlike proteinase K, both heating and EDTA were noticeably effective for the nuclease inactivation. Simultaneous application of lipoplex form of DNA (1 μg pDB2: 3 μl Lipofectamine2000) and EDTA showed a synergistic effect in protection against egg white nucleases. Finally, we injected the lipoplexes with or without EDTA close to the embryo at day0, but outside the embryonic epiblast. Implementation of a scrutinized PCR assay indicated that transfection took place only when EDTA was complemented to the lipoplexes. The transfection rate of day4 embryos and the hatched chicks were 54.5 and 30.0%, respectively. EGFP expression was detected in two out of three transgenic chicks. In conclusion, this study provided a detail analysis of chicken egg albumen nuclease properties and suggested the feasibility of developing a puncture-free handmade technology for transfection of the chicken embryo.
鸡是一种在农业和医学方面都很重要的两用动物。尽管鸡转基因技术已经取得了显著进步，但鸡基因组操作在发育生物学中尚未得到广泛应用。本研究旨在评估鸡卵清白酶的特性，以及设计一种对胚胎不造成物理损伤的体内转染技术的可行性。首先，评估了蛋清的核酸酶活性。卵清白酶在降解DNA和RNA方面具有很强的活性。卵清DNA酶活性与市售的DNA酶I相当。还在加热、蛋白酶K或EDTA处理后评估了核酸酶活性。与蛋白酶K不同，加热和EDTA对核酸酶失活都有显著效果。同时应用脂质体形式的DNA（1μg pDB2：3μl Lipofectamine2000）和EDTA在抵抗卵清白酶方面显示出协同作用。最后，我们在第0天在胚胎附近但在胚胎外胚层之外注射了含或不含EDTA的脂质体。实施严格的PCR检测表明，只有当EDTA添加到脂质体中时才会发生转染。第4天胚胎和孵化出的小鸡的转染率分别为54.5%和30.0%。在三只转基因小鸡中有两只检测到了EGFP表达。总之，本研究对鸡卵清核酸酶特性进行了详细分析，并提出了开发一种无穿刺的手工鸡胚胎转染技术的可行性。
