Silencing of transduced genes hampers production of transgenic mice using retroviral vectors. We show stable expression of the enhanced green fluorescent protein (EGFP) gene in chimeric mice generated from retrovirally transduced embryonic stem cells. The vector was a murine stem cell virus-typed retroviral vector (GCDsap) in which the long terminal repeat and primer-binding site were derived from a PCC4 cell-passaged myeloproliferative sarcoma virus and the endogenous retrovirus dl587rev, respectively. To increase the viral titer, the vector was packaged with vesicular stomatitis virus G protein, which allowed concentration of the virus into pellets followed by resuspension in serum-free medium. In chimeric mice, EGFP was detected in various tissues including hematopoietic cells, neurons, cardiac muscle, and intestine. Furthermore, high expression was maintained in the progeny of these mice, suggesting successful germline transmission of active proviruses. Although the proportion of EGFP-expressing cells and the mean intensity of EGFP expression varied among tissues and mice, 100% of peripheral blood leukocytes expressed EGFP in mice carrying a single provirus copy, as well as in their progeny. Therefore, the gene transfer system described here provides a useful tool not only to generate transgenic animals but also to manipulate human embryonic stem cells..