Cij:CD-1 strain male mice were purchased from Charles River Laboratories (Charles River Japan Inc., Kanagawa, Japan). Busulfan (Sigma, St. Louis, MO) was injected i.p. into 4- to 6-wk-old mice at a dose of 35 mg/kg to destroy the spermatogenic cells. To study long-lasting expression of the transgene, mice were examined 3-4 wk after the treatment. All animal experiments conformed to the Guide for the Care and Use of Laboratory Animals (Mit-subisi Kasei Institute of Life Sciences Animal Care Committee, according to NIH #86-23). antibiotics levaquin
CMV-lacZ plasmid, which contains the Cytomegalovirus immediate early promoter/enhancer (CMV-IE) and the Escherichia coli lacZ gene, was purchased from Clontech (p-CMVp; Palo Alto, CA). Prm-lacZ plasmid contains the lacZ gene driven by the enhancer/promoter region (—560 to +30, transcription initiation site as +1) of the mouse Protamine-1 (Prm-1) gene, and the sequence for the nuclear transporting signal was placed at the 5′-terminus of the p-galactosi-dase coding sequence. Previous transgenic analyses revealed that a 113-base pair (bp) region between base pairs -150 and -37 of the Prm-1 gene is sufficient to confer postmeiot-ic spermatid-specific expression on a reporting gene. A 5′-flanking sequence of Prm-1 containing the enhancer region was isolated by polymerase chain reaction (PCR) amplification using 5′ -GTCTAGTAATGTCCAACACC and 5′-CCTGTGAGCAGGT GGAATTT as the primers and was used to construct Prm-lacZ plasmid.