ES cells (129/SvEvTac-cell line) were electroporated with the linearized targeting vector DNA and cultured on sub-confluent embryonic fibroblasts. Transfected cell colonies were selected by culturing cells in medium containing 200 μg/mL G418 (Life Technologies, Rockville, MD, USA). The ES cells were screened for homologous recombination by PCR with the forward primer, neoF (5'-GGGTCGTTTGTTCGGATCAA-3') from neo cassette, and reverse primer intron 6R (5'-ACCAGTTGGTCCTAGCTCGA-3'), which is located outside the targeting construct in mouse Abcg8 intron 6. Positively targeted ES cell clones were confirmed by Southern blotting, using ES cell DNA digested by BamHI and a probe comprising of a 562 bp PCR fragment from partial intron 2 and exon 3 of Abcg8 gene.