Generation of Transgenic Xenopus laevis: I. High-Speed Preparation of Egg Extracts.
Generation of Transgenic Xenopus laevis: I. High-Speed Preparation of Egg Extracts.
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DOI：
10.1101/pdb.prot4838
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发表时间：
2007-09
期刊：
影响因子：
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通讯作者：
Shoko Ishibashi;K. Kroll;E. Amaya
中科院分区：
文献类型：
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作者：
Shoko Ishibashi;K. Kroll;E. Amaya
简介 在两栖动物胚胎发育的后期专门操纵基因需要精细控制表达的时间和地点。这些协议描述了为非洲爪蟾开发的基于核移植的高效转基因方法。该方法能够在非洲爪蟾胚胎中稳定表达克隆基因产物。由于转基因在受精前整合到基因组中，因此产生的胚胎不是嵌合的，从而无需培育下一代以获得非嵌合转基因动物。该过程基于限制性酶介导的整合（REMI），可分为三个部分：（I）卵子提取物的高速制备，（II）精子核制备和（III）核移植。该协议描述了鸡蛋提取物的高速制备方法。简而言之，制备了粗制的细胞生长抑制因子 (CSF) 停滞的卵提取物（即，减数分裂中期停滞的细胞质）。通过添加钙将这些提取物驱动到细胞周期的间期阶段，并进行高速离心以获得更纯的细胞质部分。该部分促进精子核膨胀，但不促进 DNA 复制。通过将鸡蛋提取物添加到反应中，精子染色质部分解凝，促进质粒 DNA 整合到基因组中。
INTRODUCTIONManipulating genes specifically during later stages of amphibian embryonic development requires fine control over the time and place of expression. These protocols describe an efficient nuclear-transplantation-based method of transgenesis developed for Xenopus laevis. The approach enables stable expression of cloned gene products in Xenopus embryos. Because the transgene integrates into the genome prior to fertilization, the resulting embryos are not chimeric, eliminating the need to breed to the next generation to obtain nonmosaic transgenic animals. The procedure is based on restriction-enzyme-mediated integration (REMI) and can be divided into three parts: (I) high-speed preparation of egg extracts, (II) sperm nuclei preparation, and (III) nuclear transplantation. This protocol describes the method for the high-speed preparation of egg extracts. Briefly, a crude, cytostatic factor (CSF)-arrested egg extract (i.e., cytoplasm arrested in meiotic metaphase) is prepared. These extracts are driven into the interphase stage of the cell cycle by addition of calcium, and high-speed centrifugation is performed to obtain a purer cytoplasmic fraction. This fraction promotes swelling of sperm nuclei, but does not promote DNA replication. By adding the egg extract to the reaction, the sperm chromatin partially decondenses, facilitating integration of plasmid DNA into the genome.