SUMMARY Rats have important advantages over mice as an experimental system for physiological and pharmacological investigations. The lack of rat embryonic stem (ES) cells has restricted the availability of transgenic technologies to create genetic models in this species. Here, we show that rat ES cells can be efficiently derived, propagated, and genetically manipulated in the presence of small molecules that specifically inhibit GSK3, MEK, and FGF receptor tyrosine kinases. These rat ES cells express pluripotency markers and retain the capacity to differentiate into derivatives of all three germ layers. Most importantly, they can produce high rates of chimerism when reintroduced into early stage embryos and can transmit through the germline. Establishment of authentic rat ES cells will make possible sophisticated genetic manipulation to create models for the study of human diseases.

The use of homologous recombination to modify genes in embryonic stem (ES) cells provides a powerful means to elucidate gene function and create disease models1. Application of this technology to engineer genes in rats has previously been impossible in the absence of germline competent ES cells in this species. We have recently established authentic rat ES cells2, 3. Here we report the generation of the first gene knockout rats using the ES cell-based gene targeting technology. We designed a targeting vector to disrupt the tumor suppressor gene p53 (also known as Tp53) in rat ES cells via homologous recombination. p53 gene-targeted rat ES cells can be routinely generated. Furthermore, the p53 gene-targeted mutation in the rat ES cell genome can transmit through the germline via ES cell-rat chimeras to create p53 gene knockout rats. The rat is the most widely used animal model other than humans in biological research4–7. The establishment of gene targeting technology in rat ES cells, in combination with advances in genomics and the vast amount of research data on physiology and pharmacology in this species, now provides a powerful new platform for the study of human disease.

Mouse embryonic stem cell (mESC) self-renewal can be maintained by activation of the leukaemia inhibitory factor (LIF)/signal transducer and activator of transcription 3 (Stat3) signalling pathway or dual inhibition (2i) of glycogen synthase kinase 3 (Gsk3) and mitogen-activated protein kinase kinase (MEK). Several downstream targets of the pathways involved have been identified that when individually overexpressed can partially support selfrenewal. However, none of these targets is shared among the involved pathways. Here, we show that the CP2 family transcription factor Tfcp2l1 is a common target in LIF/ Stat3-and 2i-mediated self-renewal, and forced expression of Tfcp2l1 can recapitulate the self-renewal-promoting effect of LIF or either of the 2i components. In addition, Tfcp2l1 can reprogram post-implantation epiblast stem cells to naı¨ve pluripotent ESCs. Tfcp2l1 upregulates Nanog expression and promotes self-renewal in a Nanogdependent manner. We conclude that Tfcp2l1 is at the intersection of LIF-and 2i-mediated self-renewal pathways and plays a critical role in maintaining ESC identity. Our study provides an expanded understanding of the current model of ground-state pluripotency.