Department of Tissue Dynamics and Regeneration, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany.
Department of Bioengineering, Stanford University, Stanford, CA, USA.
Methods Mol Biol. 2023;2680:263-275. doi: 10.1007/978-1-0716-3275-8_17.
Planarians have become a powerful model system for stem cell research and regeneration. While the tool kit for mechanistic investigations has been steadily expanding over the last decade, robust genetic tools for transgene expression are still lacking. We describe here methods for in vivo and in vitro mRNA transfection of the planarian species Schmidtea mediterranea. These methods utilize the commercially available TransIT-mRNA transfection reagent to efficiently deliver mRNA encoding a synthetic nanoluciferase reporter. Using a luminescent reporter overcomes the bright autofluorescent background of planarian tissues and allows quantitative measurements of protein expression levels. Collectively, our methods provide the means for heterologous reporter expression in planarian cells and the basis for future development of transgenic techniques.
涡虫已经成为干细胞研究和再生的强大模式生物系统。尽管过去十年中,用于机制研究的工具包一直在稳步扩展,但用于转基因表达的稳健遗传工具仍然缺乏。我们在这里描述了用于扁形动物物种 Schmidtea mediterranea 的体内和体外 mRNA 转染的方法。这些方法利用市售的 TransIT-mRNA 转染试剂,高效地递送电编码合成纳米荧光素酶报告基因的 mRNA。使用发光报告基因克服了扁形动物组织的强自发荧光背景,并允许对蛋白质表达水平进行定量测量。总的来说,我们的方法为在扁形动物细胞中异源报告基因表达提供了手段,并为未来转基因技术的发展奠定了基础。