Martin Negin P, Myers Page, Goulding Eugenia, Chen Shih-Heng, Walker Mitzie, Porter Thomas M, Van Gorder Lucas, Mathew Amanda, Gruzdev Artiom, Scappini Erica, Romeo Charles
Neurobiology Laboratory, National Institute of Environmental Health Sciences;
Comparative Medicine Branch, National Institute of Environmental Health Sciences.
J Vis Exp. 2018 Nov 1(141). doi: 10.3791/58327.
Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the host chromosome and is passed on to progeny. Thus, they are ideal vectors for creation of stable cell lines, in vivo delivery of indicators, and transduction of single cell fertilized eggs to create transgenic animals. However, mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against lentiviral gene delivery. Lentiviruses are too large to penetrate the zona and are typically delivered by microinjection of viral particles into the perivitelline cavity, the space between the zona and the embryonic cells. The requirement for highly skilled technologists and specialized equipment has minimized the use of lentiviruses for gene delivery to mouse embryos. This article describes a protocol for permeabilizing the mouse fertilized eggs by perforating the zona with a laser. Laser-perforation does not result in any damage to embryos and allows lentiviruses to gain access to embryonic cells for gene delivery. Transduced embryos can develop into blastocyst in vitro, and if implanted in pseudopregnant mice, develop into transgenic pups. The laser used in this protocol is effective and easy to use. Genes delivered by lentiviruses stably incorporate into mouse embryonic cells and are germline transmittable. This is an alternative method for creation of transgenic mice that requires no micromanipulation and microinjection of fertilized eggs.
慢病毒是将基因导入哺乳动物细胞的有效载体。转导后，慢病毒基因组稳定整合到宿主染色体中并传递给后代。因此，它们是创建稳定细胞系、体内递送指示剂以及转导单细胞受精卵以创建转基因动物的理想载体。然而，小鼠受精卵和早期胚胎受到透明带的保护，透明带是一种糖蛋白基质，形成了阻止慢病毒基因递送的屏障。慢病毒太大，无法穿透透明带，通常通过将病毒颗粒显微注射到卵周腔（透明带与胚胎细胞之间的空间）来递送。对高技能技术人员和专业设备的要求使得慢病毒用于向小鼠胚胎递送基因的应用受到了限制。本文描述了一种通过用激光在透明带上打孔使小鼠受精卵通透化的方案。激光打孔不会对胚胎造成任何损伤，并允许慢病毒进入胚胎细胞进行基因递送。转导后的胚胎可以在体外发育成囊胚，如果植入假孕小鼠体内，则可以发育成转基因幼崽。本方案中使用的激光有效且易于使用。通过慢病毒递送的基因稳定整合到小鼠胚胎细胞中并且可以种系传递。这是一种创建转基因小鼠的替代方法，不需要对受精卵进行显微操作和显微注射。
