One of the limitations of transgenesis is low efficiency. In this study, we generated transgenic mice harboring the enhanced green fluorescent protein (EGFP) gene, under the control of chicken-beta-actin promoter and cytomegalovirus enhancer, using two approaches and compared their efficiencies. One involved culture of EGFP-injected embryos developing through EGFP-expressing "green" blastocysts, followed by their transfer to uterus. The second was oviductal-transfer of EGFP-injected-eggs. Embryo culture-based-transgenesis (ECBT) produced 100% transgenic mice, unlike the second approach. Moreover, ECBT required reduced number of recipients and markedly increased pregnancy rates. Of the nine founders, seven exhibited ubiquitous EGFP-expression, one (GU1) was a mosaic and the other (G18) was non- expressing. The molecular basis for this was attributed to repeat-induced gene silencing, since the G18 had a high copy number (similar or equal to99/genome) of the non-mutated and non-rearranged EGFP-transgene integrated at a single site. Our results show the superiority of ECBT over the conventional oviductal approach for generating transgenic "green" mice. (C) 2003 Elsevier Science (USA). All rights reserved.