The combination of somatic cell nuclear transfer (SCNT) and transgenic technology leads to the production of transgenic cloned animals, wherein the preparation of competent transgenic donor cells is the pivotal upstream step. The purpose of this study was to establish an efficient procedure to prepare human lactoferrin (hLTF) transgenic donor cells for SCNT. Thus, two cell culture systems were employed: caprine mammary epithelial cells (for evaluation of the hTLF transgenic expression in vitro), and fetal-derived fibroblast cells (for identification of competent transgenic donor cells). Induced by hormonal signals, recombinant hLTF was detected in the supernatant of transfected mammary epithelial cells by Western blot. Reliable hLTF transgenic fibroblast cell clones were identified by screening with multiple PCR amplification, EGFP fluorescence, and chromosomal counting (32.5+/-2.3%). This study may provide an effective upstream system to prepare SCNT donor cells for the production of human recombinant pharmaceuticals from the milk of transgenic animals.