Department of Biology, Stanford University, Stanford, CA, United States of America.
Department of Biology, De Anza College, Cupertino, CA, United States of America.
PLoS One. 2023 Aug 17;18(8):e0289361. doi: 10.1371/journal.pone.0289361. eCollection 2023.
Electroporation is an increasingly common technique used for exogenous gene expression in live animals, but protocols are largely limited to traditional laboratory organisms. The goal of this protocol is to test in vivo electroporation techniques in a diverse array of tadpole species. We explore electroporation efficiency in tissue-specific cells of five species from across three families of tropical frogs: poison frogs (Dendrobatidae), cryptic forest/poison frogs (Aromobatidae), and glassfrogs (Centrolenidae). These species are well known for their diverse social behaviors and intriguing physiologies that coordinate chemical defenses, aposematism, and/or tissue transparency. Specifically, we examine the effects of electrical pulse and injection parameters on species- and tissue-specific transfection of plasmid DNA in tadpoles. After electroporation of a plasmid encoding green fluorescent protein (GFP), we found strong GFP fluorescence within brain and muscle cells that increased with the amount of DNA injected and electrical pulse number. We discuss species-related challenges, troubleshooting, and outline ideas for improvement. Extending in vivo electroporation to non-model amphibian species could provide new opportunities for exploring topics in genetics, behavior, and organismal biology.
电穿孔是一种在活体动物中用于外源基因表达的越来越常见的技术，但方案在很大程度上仅限于传统的实验室生物。本方案的目的是在各种热带青蛙物种的幼体中测试体内电穿孔技术。我们探索了来自毒蛙科（Dendrobatidae）、隐匿林蛙科（Aromobatidae）和玻璃蛙科（Centrolenidae）三个科的五种物种的组织特异性细胞中的电穿孔效率。这些物种以其多样化的社会行为和有趣的生理学特征而闻名，这些特征协调了化学防御、警戒色和/或组织透明度。具体来说，我们研究了电脉冲和注射参数对质粒 DNA 在幼体中物种和组织特异性转染的影响。在转染编码绿色荧光蛋白（GFP）的质粒后，我们发现 GFP 荧光在大脑和肌肉细胞中很强，并且随着注射的 DNA 量和电脉冲数的增加而增加。我们讨论了与物种相关的挑战、故障排除，并提出了改进的思路。将体内电穿孔技术扩展到非模式两栖动物物种可能为探索遗传学、行为和机体生物学的主题提供新的机会。
