Iqbal Khursheed, Barg-Kues Brigitte, Broll Sandra, Bode Jürgen, Niemann Heiner, Kues Wilfried
Institute of Farm Animal Genetics, Friedrich-Loeffler-Institute Biotechnology, Mariensee, Neustadt, Germany.
Biotechniques. 2009 Nov;47(5):959-68. doi: 10.2144/000113270.
Injection of linearized DNA constructs into the pronuclei of fertilized mammalian eggs is a standard method for producing transgenic embryos and animals. Here, we show that injection of covalently closed circular (ccc) plasmids into the cytoplasm of fertilized bovine and murine eggs is a highly efficient and simple alternative for ectopic expression of foreign DNA in embryos. A broad range of plasmids could be successfully expressed in preimplantation stages, including plasmids and minicircles with a scaffold/matrix attachment region (S/MAR), conventional plasmids, and bacterial artificial chromosomes (BACs). Although the foreign DNA plasmids are mainly maintained as episomal entities during preimplantation development, they accurately behave like nuclear DNA. Onset of transcription of an Oct4 promoter-controlled marker gene coincided with the species-specific time points of major embryonic genome activation, and could be modulated by in vitro DNA-methylation. This approach allows an experimental access to reprogramming events in early mammalian embryos.
将线性化的DNA构建体注射到受精的哺乳动物卵原核中是生产转基因胚胎和动物的标准方法。在此，我们表明将共价闭合环状（ccc）质粒注射到受精的牛和鼠卵细胞质中是在胚胎中异位表达外源DNA的一种高效且简单的替代方法。多种质粒能够在植入前阶段成功表达，包括带有支架/基质附着区域（S/MAR）的质粒和微型环、传统质粒以及细菌人工染色体（BAC）。尽管外源DNA质粒在植入前发育过程中主要以附加体形式存在，但它们的行为与核DNA精确相似。Oct4启动子控制的标记基因的转录起始与主要胚胎基因组激活的物种特异性时间点一致，并且可以通过体外DNA甲基化进行调节。这种方法为研究早期哺乳动物胚胎中的重编程事件提供了实验途径。
