Abstract:
Producing chimaeras constitutes the most reliable method of verifying the pluripotency of newly established cells. Moreover, forming chimaeras by injecting genetically modified embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) into the embryo is part of the procedure for generating transgenic mice, which are used for understanding gene function. Conventional methods for generating transgenic mice, including the breeding of chimaeras and tetraploid complementation, are time-consuming and cost-inefficient, with significant limitations that hinder their effectiveness and widespread applications. In the present study, we modified the traditional method of chimaera generation to significantly speed up this process by generating mice exclusively derived from ESCs. This study aimed to assess whether fully ESC-derived mice could be obtained by modulating fibroblast growth factor 4 (FGF4) levels in the culture medium and changing the direction of cell differentiation in the chimaeric embryo. We found that exogenous FGF4 directs all host blastomeres to the primitive endoderm fate, but does not affect the localisation of ESCs in the epiblast of the chimaeric embryos. Consequently, all FGF4-treated chimaeric embryos contained an epiblast composed exclusively of ESCs, and following transfer into recipient mice, these embryos developed into fully ESC-derived newborns. Collectively, this simple approach could accelerate the generation of ESC-derived animals and thus optimise ESC-mediated transgenesis and the verification of cell pluripotency. Compared to traditional methods, it could speed up functional studies by several weeks and significantly reduce costs related to maintaining and breeding chimaeras. Moreover, since the effect of stimulating the FGF signalling pathway is universal across different animal species, our approach can be applied not only to rodents but also to other animals, offering its utility beyond laboratory settings.
摘要:
产生嵌合体构成了验证新建立的细胞的多能性的最可靠的方法。此外,通过将遗传修饰的胚胎干细胞(ESCs)或诱导的多能干细胞(iPSCs)注入胚胎来形成嵌合体是产生转基因小鼠的过程的一部分,用于理解基因功能。产生转基因小鼠的常规方法,包括嵌合体和四倍体互补的育种,既耗时又成本效益低,具有显著的局限性,阻碍了它们的有效性和广泛应用。在本研究中,我们改进了传统的嵌合体生成方法,通过生成完全来源于ESCs的小鼠,显著加快了这一过程.这项研究旨在评估是否可以通过调节培养基中的成纤维细胞生长因子4(FGF4)水平并改变嵌合胚胎中细胞分化的方向来获得完全ESC衍生的小鼠。我们发现外源性FGF4将所有宿主卵裂球引导到原始内胚层命运,但不影响ESCs在嵌合胚胎外胚层中的定位。因此,所有FGF4处理的嵌合胚胎都含有一个完全由ESCs组成的外胚层,转移到受体小鼠中后,这些胚胎发育成完全ESC衍生的新生儿。总的来说,这种简单的方法可以加速ESC衍生动物的产生,从而优化ESC介导的转基因和细胞多能性的验证.与传统方法相比,它可以将功能研究加速数周,并显著降低与维持和繁殖嵌合体相关的成本。此外,由于刺激FGF信号通路的作用在不同的动物物种中是普遍的,我们的方法不仅适用于啮齿动物,也适用于其他动物,提供超越实验室设置的实用程序。