Positively targeted ES cells were microinjected into C57BL6 blastocysts and transplanted into pseudopregnant recipients. Five highly chimeric mice (agouti coat color, three males and two females) were isolated and bred with C57BL/6J mice to generate germ-line transmission heterozygous mice of Abcg8 gene disruption. Heterozygous offspring mice were back-crossed to C57BL/6J mice (n > 5) to produce disrupted line and inter-crossed to generate knockout mice.