In order to evaluate the usefulness of a cloning technique to produce gene-manipulated mice for the field of laboratory animal science, we produced mice cloned from gene-targeted embryonic stem (ES) cells and examined the vertical transmission of a targeted gene to their progeny. Of 1257 eggs constructed by nuclear transfer using M-phase ES donor cells targeted with an oviduct-specific glycoprotein (OGP) gene, 990 formed a pseudo-pronucleus and a polar body after activation. Of 504 cloned embryos transferred into recipients, 20 live cloned pups (2%) were recovered by Caesarean section at 19.5 days of gestation. Fourteen of these cloned mice were studied. Genotyping of the OGP locus and 20 microsatellite loci showed that they were genetically identical to the OGP gene-targeted TT2 cells. Eight cloned pups grew into adults, of which 7 were male and 1 was female (missing the Y chromosome). Mating experiments using the cloned mice were carried out. Of 89 F1 mice produced from the mating of cloned and C57BL/6J mice, 50 had the targeted OGP gene heterozygously. Thirty-seven F2 mice from 4 pairs of the OGP-/+ mice were composed of 9 OGP-/-, 18 OGP-/+, and 10 OGP+/+. Moreover, 26 offspring of one pair of the cloned mice were composed of 10 OGP-/-, 12 OGP-/+, and 4 OGP+/+. These offspring were fertile and transmitted the mutant OGP gene to the next generation. Comparison of these results with those of germline chimeric mice indicates that gene-targeted mice can be produced at least one generation earlier by nuclear transfer than by the conventional methods. In addition, the targeted OGP gene was constantly transmitted to the progeny of the gene-targeted mice. Cloning techniques are potentially a more efficient way to generate gene-manipulated mice for laboratory animal science, although such techniques include many unresolved problems, such as low production efficiency, and selection of a cell source for gene manipulation among others.