The standard protocol for generating mutant mice from heterozygous targeted ES cells currently is time-consuming and needs two breeding steps and, in some cases, risks the failure of germline transmission. In contrast, the tetraploid embryo complementation is a time-saving single step procedure escaping from the chimera mouse. Hybrid ES cell lines always have higher generation frequency because of their genetic heterozygosities, which also limit their application. However, inbred ES cell lines often fail to generate ES mice. Judged by microsatellite DNA marker analysis, the completely ES cell derived mice (ES mice) were efficiently produced with two inbred ES cell lines (SCR012 and CJ7) by microinjecting the ES cells into tetraploid blastocysts generated by electrofusion. As a result, after electrofusion, about (93.01±1.37)% of 2-cell stage embryos fused and (82.49±2.08)% of them developed to blastocysts after 48 h in vitro culture. The efficiency of generating newborn ES mice of wild type SCR012 cells was higher (8.3%) than that of CJ7 (2.7%). In the meanwhile, adult and fertile mutant ES mice with two gene knock-out ES cells-18 KO (derived from CJ7) and F8 KO (derived from SCR012) were generated. Interestingly, the generation frequency of 18 KO ES mice was even higher (8.1%) than wild-type CJ7 ES mice, indicating that the development potential among ES cell clones was significantly different from each other and the decline of development potential was not equal among ES cell clones during ES cell passage in vitro culture. The F8 KO mice had indistinguishable bleeding phenotype, which is identical to that of mice derived from breeding of chimeric mice. These results suggested that this technique can be used as a powerful approach to produced gene targeted mice efficiently.