A BAC containing the entire Kiss1 coding region with approximately 109 kb of upstream sequence and 69 kb of downstream sequence (RP24-299B2) was modified by ET-cloning recombineering methods. A DNA fragment encoding cre recombinase followed by the SV40 polyadenylation signal and an Frt-flanked kanamycin resistance marker was inserted at the translational start site of Kiss1. The resistance marker was deleted by arabinose induction of Flp recombinase in bacterial cells during cloning. The purified transgenic construct was injected into one-cell stage embryos from C57BL/6 mice. Three lines (J2-4, J2-4, and J2-6) were generated from the RP24-299B2 construct. J2-4 exhibited cre activity in known areas of Kiss1 mRNA expression and was analyzed in detail.