We here show that infection with a lentiviral vector carrying three mouse reprogramming factors (Oct4, Klf4, and Sox2) and culture in the presence of two kinds of kinase inhibitors (MEK inhibitor and GSK3 inhibitor) permit efficient establishment and maintenance of riPSCs from REFs. Established riPSCs possess all the key features of rESCs, such as expression of pluripotency markers Oct4, Nanog, and E-ras, long-term self-renewal, the capacity to differentiate into derivatives of all three germ layers, and, most importantly, the ability to produce chimerism with high efficiency and to contribute to transmission through the germline.
MiPSCs generated by introducing four reprogramming factors (Oct3/4, Sox2, Klf4, and c-Myc) are able to produce germline-competent chimeras.
However, a drawback in this four-factor system is reactivation of the c-Myc retrovirus, which increases tumorigenicity in chimeric mice and their progeny. On the other hand, no tumors were observed in mice derived from iPSCs generated by introducing three factors (Oct3/4
, and Klf4
. Therefore, we used three factors (Oct3/4
, and Sox2
), eliminating c-Myc, with a special emphasis on escape from tumor development. As expected, we observed no tumorigenesis in chimeric rats or their progeny, with monitoring for over six months.
Two main factors can be conceived to affect the successful generation of germline chimeras. One is the conditions under which riPSCs are generated and cultured. In recent studies by Ping Li et al., rESCs were generated using serum-free N2B27 medium containing mLIF and a combination of two or three kinase inhibitors (2i/3i)  
. RiPSCs also have been generated using knockout DMEM medium containing knockout serum replacement (KSR), 2i, A83-01 and mLIF 
. Because successful germline transmission was reported only in the rESCs study, we generated riPSCs using N2B27 medium and the 2i/3i culture method. The other is the interaction between iPSC strain and host blastocyst strain. In this study, high efficiency of germline transmission was achieved by injection of WI riPSCs into WI blastocysts (donor cell : host blastocyst combination WI : WI) and of DA riPSCs into WI blastocysts (DA : WI). Other studies have generated rESC germline chimeras in the combinations DA : F344 [16,19], WI :WI and DA/WI 
, and WI-LEA : WI, WI : WI, WI : LEA, and LEA : WI 
. So far, only a few combinations of iPSCs strains and blastocyst strains have successfully generated germline chimeras. Although we could not obtain offspring from DA riPSCs, we may need to test different strains of blastocysts for more efficient germline transmission.
As in our previous report of interspecific chimeras 
, the riPSCs generated in this study were able to contribute interspecifically to chimera generation and interspecifically to germ cell lineages, indicating that these riPSCs were highly reprogrammed.
Taken together, our results demonstrate that rat somatic cells can be reprogrammed to ground state with germ line competence by transduction of only three reprogramming factors (Oct3/4, Klf4, and Sox2). We believe that these riPSCs will open a new area of studies using the rat as a useful animal model.