Objective To establish clean homozygote Neo r trangenic mouse strain.Methods Ten clean offsprings of Neo r transgenic mice free of 10 microorganisms infection were obtained through bio cleaning of embryo transfer. Among them five mice were identified as Neo r positive by PCR and Southern blot. The positive mice were bred by brother sister breeding methods to produce homozygotes. Results Four homozygote Neo r transgenic mice were selected through PCR, Southern blot and breeding experiment to form Neo r transgenic mouse strain. The mouse embryonic fibroblast (MEF) prepared from fetuses of female mouse mated with male Neo r transgenic mouse showed resistance against G418 as high as 1 mg/ml in the culture medium. This Neo r MEF can be used as feed cell layer when ES cell gene targeting is conducted. Conclusion The above results show that we obtained clean animals through embryo transfer, a genetical homozygote Neo r transgenic mouse strain.