Scott Melissa A, Hu Yueh-Chiang
Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.
Methods Mol Biol. 2019;1874:169-178. doi: 10.1007/978-1-4939-8831-0_9.
Direct modification of the genome of the zygotes (i.e., one-cell embryos) by the CRISPR/Cas9-editing reagents, followed by embryo transfer to pseudopregnant females for live birth, has been the most effective method to generate laboratory rodent models for research. The method relies on proper delivery of the editing reagents into zygotes, which is commonly achieved by a standard or slightly modified pronuclear microinjection technique. In this chapter, we describe in detail an alternative delivery method, named piezo-driven cytoplasmic microinjection, which offers a superior embryo survival and birth rate. Because this method uses a much wider injection needle than that in pronuclear injection, it allows a larger volume of the editing materials to be transported into the zygotes, leading to an increase in the targeting efficiency. This also eliminates the clogging issues seen regularly in pronuclear injection. Moreover, Cytochalasin B that is used to soften zygotes during piezo-driven microinjection has been suggested a role in improving the knockin efficiency, which provides an additional benefit to use this injection method.
通过CRISPR/Cas9编辑试剂直接修饰受精卵（即单细胞胚胎）的基因组，随后将胚胎移植到假孕雌性动物体内以实现活体分娩，这一直是生成用于研究的实验啮齿动物模型的最有效方法。该方法依赖于将编辑试剂正确导入受精卵，这通常通过标准的或稍作修改的原核显微注射技术来实现。在本章中，我们将详细描述另一种导入方法，即压电驱动的细胞质显微注射，该方法能提供更高的胚胎存活率和出生率。由于此方法使用的注射针比原核注射中的针要粗得多，所以它能将更大体积的编辑材料导入受精卵，从而提高靶向效率。这也消除了原核注射中经常出现的堵塞问题。此外，在压电驱动显微注射过程中用于软化受精卵的细胞松弛素B被认为在提高敲入效率方面发挥了作用，这为使用这种注射方法提供了额外的益处。
