The formation of chimaeras by injecting embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) into embryos is a standard method of verifying cell pluripotency. Moreover, the use of genetically modified ESCs or iPSCs in this procedure is a powerful way to elucidate gene function and generate transgenic animals. Although ESC-based technology is being gradually supplanted by genome editing tools, such as clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) [[1], [2], [3]], in some cases, it remains the method of choice. Limitations in knock-in fragment size, notable off-target mutation risks [4,5], and mosaicism in founder generations [6] are just some drawbacks of the CRISPR/Cas9 method, which complicate the analyses in transgenic animals and favour ESC-mediated gene targeting. The conventional method involves introducing modified ESCs or iPSCs into the diploid blastocysts. In this case, pluripotent cells integrate into the inner cell mass (ICM) of the blastocyst, thereby co-existing with the host cells in the epiblast (EPI) and finally contributing to three primary germ layers: the ectoderm, endoderm, and mesoderm. Consequently, ESCs must compete with the host embryo cells for the developmental niche to populate the EPI and settle the germline. Transfer of these chimaeric blastocysts always results in the birth of chimaeric offspring, thus, to achieve genetically modified mice, extensive breeding of chimaeras followed by genotyping to identify the germline transmission is needed. A slightly improved method relies on injecting diploid ESCs or iPSCs into tetraploid blastocysts [[7], [8], [9]]. The combination of tetraploid embryos with diploid pluripotent ESCs results in the elimination of tetraploid cells from the EPI lineage at the early postimplantation stages of development [[10], [11], [12], [13], [14], [15], [16], [17], [18]], thus leading to the generation of entirely ESC-derived newborns. Although this tetraploid complementation method allows the direct production of viable and fertile ESC- or iPSC-derived mice, it creates the risk of generating 2n↔4n chimaeras [9,10,14,15,19,20]. Moreover, mice produced by this method display nonspecific lethality and congenital abnormalities, which complicates the phenotypic analysis [10,21,22]. Attempts to overcome this obstacle, such as employing as a host an ICM-deficient 4n blastocyst [19] or a blastocyst in which the forming endogenous germ cells are selectively ablated [23], failed to produce healthy animals with minimal financial and time commitments.

It has been shown that injecting ESCs into the host embryo at the 4- or 8-cell stage results in the more efficient production of F0 ESC mice with full germline transmission compared to the tetraploid complementation method [[24], [25], [26]]. Activating ESCs to synchronise them in the log phase of growth before injection and applying medium optimised for the survival of both the ESCs and embryos additionally increased the number of live animals with high ESC contribution; however, the efficiency still did not reach 100% [27].

Based on our previous results demonstrating that the FGF4/MAPK signalling pathway underlies communication between ESCs and host cells in chimaeric embryos [28], we hypothesized that the ability to steer the fate of cells in the mouse chimaeric embryo and thus increase the competition of ESCs in the EPI could additionally contribute to the optimisation of this procedure. In this work, we cultured chimaeric embryos in the presence of FGF4, which has been demonstrated to be a key regulator of the primitive endoderm (PrE) vs EPI cell fate within the ICM [[29], [30], [31], [32], [33], [34], [35]]. As a result, we obtained all embryos with a pure, ESC-derived EPI and confirmed their full developmental potential (the ability to give rise to normal animals) by transfer to recipients. This culture modification increased the efficiency of yielding fully ESC-derived newborns to 100%. However, this approach has its limitations, as it requires the use of fully pluripotent ESCs able to give rise to individuals demonstrating germline contribution, without the support of host cells,.