Neurobiology Laboratory, Viral Vector Core, National Institute of Environmental Health Sciences, NIH/DHHS, 111 T.W. Alexander Drive, Research Triangle Park, N.C, 27709, USA.
Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, NIH/DHHS, 111 T.W. Alexander Drive, Research Triangle Park, N.C, 27709, USA.
Biochem Biophys Res Commun. 2020 May 21;526(1):85-90. doi: 10.1016/j.bbrc.2020.03.026. Epub 2020 Mar 17.
Gene delivery to fertilized eggs is often the first step in creation of transgenic animals, CRISPR knock-out, or early developmental studies. The zona pellucida, a hardened glycoprotein matrix surrounding the mammalian fertilized eggs, often complicates gene delivery by forming a barrier against transfection reagents and viruses. High efficiency techniques to perforate or penetrate the zona allow for access and gene delivery to fertilized eggs. However, these techniques often rely on highly skilled technologists, are costly, and require specialized equipment for micromanipulation, laser perforation, or electroporation. Here, we report that adenoassociated viruses (AAVs) with serotypes 1 or DJ can efficiently diffuse across the zona to deliver genes without any manipulations to fertilized eggs. We observe lowered rates of embryo development after treatment of embryos with all AAV serotypes. However, we were able to reduce adverse effects on embryo development by exposing embryos to AAVs at later stages of in vitro development. AAVs have low immune response and do not incorporate into their host chromosomes to cause insertional mutations. Hence, AAVs can serve as a highly effective tool for transient delivery of genes to fertilized mammalian eggs.
将基因递送到受精卵中通常是创建转基因动物、CRISPR 敲除或早期发育研究的第一步。卵透明带是一种围绕哺乳动物受精卵的硬化糖蛋白基质，它常常形成一道屏障，阻止转染试剂和病毒进入，从而使基因传递变得复杂。高效的打孔或穿透透明带技术可使基因进入和传递到受精卵。然而，这些技术通常依赖于技术熟练的技术人员，成本高昂，并且需要专门的设备进行微操作、激光穿孔或电穿孔。在这里，我们报告说，血清型 1 或 DJ 的腺相关病毒 (AAV) 可以有效地穿过透明带扩散，无需对受精卵进行任何操作即可传递基因。我们观察到，在用所有 AAV 血清型处理胚胎后，胚胎的发育率降低。然而，我们通过在体外发育的后期阶段使胚胎暴露于 AAV 来降低对胚胎发育的不利影响。AAV 具有低免疫原性，不会整合到宿主染色体中导致插入突变。因此，AAV 可以作为一种非常有效的工具，用于将基因瞬时递送到受精的哺乳动物卵中。
