Production efficiency of 5xFAD Tg rats.

In the conventional method of producing transgenic (Tg) animals, donor DNA is microinjected into the pronuclei of zygotes using a sharp glass needle. However, this approach is generally inefficient as it requires highly skilled microinjection techniques to ensure zygote survival and the transgene is incorporated into only a small proportion of the offspring. In contrast, methods based on piggyBac transposase (PBase) enables more efficient insertion of DNA into the genome and generation of Tg animals. The use of piggyBac transposase have also been examined in rats→ However, this method has not yet been fully optimized or properly characterized. In this study, we examined the microinjection of PBase mRNA and donor plasmid DNA into the pronuclei of rat zygotes using piezo-assisted microinjection. This approach resulted in high survival rates and enabled the efficient generation of Tg rats, even with long donor DNA. When the zygotes were microinjected using Piezo, over 70% were viable, and after embryo transfer, over 80% of the pups carried the transgene. Furthermore, we confirmed germline transmission to the F1 and F2 generations. We also attempted to generate a rat model of Alzheimer’s using this method→ However, the protein was not detected despite mRNA expression, and the phenotype was not observed in behavioral tests. Although the generation of Alzheimer’s disease model remains a challenge, our findings show that piggyBac transposase mRNA combined with piezo-assisted microinjection represents a simple and efficient method for producing Tg rats, even with long donor DNA.传统制备转基因（transgenic, Tg）动物的常规方法，是借助尖锐玻璃针将供体DNA显微注射至受精卵的原核内。然而该方法普遍效率低下：不仅需要操作者掌握高精尖的显微注射技术以保障受精卵存活，且仅有少量子代能够成功整合外源转基因。与之相对，基于piggyBac转座酶（PBase）的方法可实现DNA向基因组的高效插入，进而更高效地生成转基因动物。此前已有研究在大鼠中探索了piggyBac转座酶的应用，但该方法尚未得到充分优化与系统表征。本研究中，我们采用压电辅助显微注射技术，将PBase mRNA与供体质粒DNA注射至大鼠受精卵的原核内。该策略可实现较高的受精卵存活率，即便使用长度较长的供体DNA，也能高效制备转基因大鼠。当通过压电装置完成显微注射后，超过70%的受精卵可保持存活；经胚胎移植后，超过80%的新生幼崽携带目标转基因。此外，我们还验证了该方法可实现生殖系传递至F1及F2代。我们还尝试利用该技术构建阿尔茨海默病（Alzheimer’s disease）大鼠模型，但尽管检测到了目标mRNA的表达，却未检测到对应的蛋白，且行为学实验中未观察到预期表型。尽管阿尔茨海默病动物模型的构建仍是一项挑战，但我们的研究结果表明，将piggyBac转座酶mRNA与压电辅助显微注射技术相结合，是一种简便高效的大鼠转基因动物制备方案，即便供体DNA片段较长也可适用。