ES cell is a nearly totipotent cell line and highly useful for the preparation of a genetically modified animal and the like. For example, a chimeric animal can be produced by injecting ES cells, in which a particular gene has been destructed, into normal host blastocysts to be mixed with the cells of the host embryos and returning the mixture to a uterus, and a genetically modified animal, in which the particular gene has been destructed (knockout animal), can be produced by crossing the obtained chimeric animals or offsprings and selecting the animals born.
Rat is a mammal having a size more suitable for handling than mouse, and is one of the most useful experimental animals widely utilized in various fields including medicine. Therefore, the establishment of rat ES cells and the production of chimeric rat using the cells have been desired. However, no technique has been established so far for efficiently preparing a chimeric rat wherein an ES cell differentiates into germ cell lineage and genetic information from the ES cell is transmitted to the next generation without being influenced by the strain of the rat ES cell and the strain of the host embryo (i.e., germline-transmitted chimeric rat).
Regarding the preparation of a chimeric rat with confirmed germline transmission, reports were presented by a group of Qi-Long Ying et al. and a group of Austin Smith et al., respectively, in 2008 (patent document 1, non-patent documents 1 and 2). In the two reports, chimeric rats were prepared using rat ES cells; however, in both cases, a germline-transmitted chimeric rat was successfully prepared from only one combination out of a plurality of combinations of ES cell strains and host embryo strains. It has been suggested that in the routinized practice of preparing a knockout rat, it is important to realize the preparation of a chimeric rat using a wide variety of combinations of ES cell strains and host embryo strains, which poses a problem to be solved in the future.
Also, despite the fact that some actual cases of generating a germline-transmitted chimeric rat have been reported, although they are limiting, as stated above, no report is available on the establishment of gene-targeted genetically modified rats (e.g., knockout rats, knockin rats). This may be due to an influence of the quality of the starting rat ES cell.
While rat iPS cell capable of producing chimeras is established using MEK inhibitor, GSK3 inhibitor and type I TGFβ receptor Alk5 inhibitor (A-83-01), germline transmission has not been achieved (non-patent document 3). In addition, it has also been reported that a combination of MEK inhibitor and Alk5 inhibitor strikingly improves efficiency of iPS cell production from human fibroblasts (non-patent document 4).
In recent years, Watanabe et al. have found that Rho-binding kinase (Rho-associated kinase; ROCK) inhibitor Y-27632 blocks apoptosis of human ES cell, and induces growth after single cell separation by an enzyme treatment (non-patent document 5), and also, usefulness of ROCK inhibitor for human stem cell culture has been reported (patent document 2).