Cell Signaling &Nanobiotechnology Laboratory, Department of Biochemistry &Immunology, Federal University of Minas Gerais, Belo Horizonte, Brazil.
Nanocell Institute, Divinópolis, MG, Brazil.
Sci Rep. 2017 Mar 20;7:44317. doi: 10.1038/srep44317.
Microinjection is commonly performed to achieve fish transgenesis; however, due to difficulties associated with this technique, new strategies are being developed. Here we evaluate the potential of lentiviral particles to genetically modify Nile tilapia cells to achieve transgenesis using three different approaches: spermatogonial stem cell (SSC) genetic modification and transplantation (SC), in vivo transduction of gametes (GT), and fertilised egg transduction (ET). The SC protocol using larvae generates animals with sustained production of modified sperm (80% of animals with 77% maximum sperm fluorescence [MSF]), but is a time-consuming protocol (sexual maturity in Nile tilapia is achieved at 6 months of age). GT is a faster technique, but the modified gamete production is temporary (70% of animals with 52% MSF). ET is an easier way to obtain mosaic transgenic animals compared to microinjection of eggs, but non-site-directed integration in the fish genome can be a problem. In this study, PI3Kc2α gene disruption impaired development during the embryo stage and caused premature death. The manipulator should choose a technique based on the time available for transgenic obtainment and if this generation is required to be continuous or not.
显微注射通常用于实现鱼类转基因；然而，由于该技术存在困难，新的策略正在被开发。在这里，我们评估了慢病毒颗粒将尼罗罗非鱼细胞进行基因修饰以实现转基因的潜力，使用了三种不同的方法：精原干细胞（SSC）基因修饰和移植（SC）、体内转导配子（GT）和受精卵转导（ET）。使用幼虫的 SC 方案会产生持续产生修饰精子的动物（80%的动物具有 77%的最大精子荧光 [MSF]），但这是一个耗时的方案（尼罗罗非鱼的性成熟需要 6 个月）。GT 是一种更快的技术，但修饰配子的产生是暂时的（70%的动物具有 52%的 MSF）。与卵的显微注射相比，ET 是获得嵌合转基因动物的一种更简单的方法，但在鱼类基因组中的非定点整合可能是一个问题。在这项研究中，PI3Kc2α 基因缺失破坏了胚胎阶段的发育并导致了早逝。操作者应根据获得转基因的可用时间以及是否需要连续几代来选择技术。
