Leung Stephen Wan, Cheng Po-Ching, Chou Chih-Ming, Lin Chi, Kuo Yu-Chieh, Lee Yu-Lin Amy, Liu Cheng-Yang, Mi Fwu-Long, Cheng Chia-Hsiung
Department of Radiation Oncology, Yuan's General Hospital, Kaohsiung 80249, Taiwan.
Department of Molecular Parasitology and Tropical Diseases, School of Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan.
Int J Biol Macromol. 2022 Jan 1;194:384-394. doi: 10.1016/j.ijbiomac.2021.11.080. Epub 2021 Nov 22.
Many challenges, such as virus infection, extreme weather and long cultivation periods, during the development of fish larvae have been observed, especially in aquaculture. Gene delivery is a useful method to express functional genes to defend against these challengers. However, the methods for fish larvae are insufficient. In our earlier report, low-molecular-weight chitosan (LMWCS) showed a strong positive charge and may be useful for polyplex formulation. Herein, we present a simple self-assembly of LMWCS polyplexes (LMWCSrNPs) for gene delivery into zebrafish larvae. Different weight ratios of LMWCS/gamma-polyglutamic acid (γ-PGA)/plasmid DNA were analyzed by gel mobility assay. Delivery efficiency determined by green fluorescent protein (GFP) expression in zebrafish liver (ZFL) cells showed that delivery efficiency at a weight ratio of 20:8:1 was higher than others. Zeta potential and transmission electron microscopy (TEM) analysis showed that the round shape of the particle size varied. In our earlier reports, IRF9S2C could induce interferon-stimulated gene (ISG) expression to induce innate immunity in zebrafish and pufferfish. Further delivery of pcDNA3-IRF9S2C-HA plasmid DNA into ZFL cells and zebrafish larvae by LMWCSrNP successfully induced ISG expression. Collectively, LMWCSrNP could be a novel gene delivery system for zebrafish larvae and might be used to improve applications in aquaculture.
在鱼类幼体发育过程中，已经观察到许多挑战，如病毒感染、极端天气和较长的养殖周期，尤其是在水产养殖中。基因递送是一种表达功能基因以抵御这些挑战的有用方法。然而，针对鱼类幼体的方法并不充分。在我们早期的报告中，低分子量壳聚糖（LMWCS）显示出强正电荷，可能对多聚体形成有用。在此，我们展示了一种简单的LMWCS多聚体（LMWCSrNPs）自组装方法，用于将基因递送至斑马鱼幼体。通过凝胶迁移试验分析了LMWCS/γ-聚谷氨酸（γ-PGA）/质粒DNA的不同重量比。通过斑马鱼肝脏（ZFL）细胞中绿色荧光蛋白（GFP）表达确定的递送效率表明，重量比为20:8:1时的递送效率高于其他比例。zeta电位和透射电子显微镜（TEM）分析表明，颗粒尺寸的圆形有所不同。在我们早期的报告中，IRF9S2C可诱导干扰素刺激基因（ISG）表达，从而在斑马鱼和河豚中诱导先天免疫。通过LMWCSrNP将pcDNA3-IRF9S2C-HA质粒DNA进一步递送至ZFL细胞和斑马鱼幼体中，成功诱导了ISG表达。总体而言，LMWCSrNP可能是一种用于斑马鱼幼体的新型基因递送系统，可用于改善水产养殖中的应用。
