Heterozygous mice were fertile and gave rise to normal litter sizes. Breeding heterozygous animals led to wild-type, heterozygous and homozygous knockout mice in the expected Mendelian distribution (data not shown). Although not quantitative, Northern analyses showed that while the knockout mice showed no detectable mRNA for Abcg8, expression of Abcg5 appeared unaltered (Figure 1c). To exclude the possibility of alternative splicing with the production of a non-functional but truncated Abcg8/sterolin-2 protein that may serve as a chaperone for Abcg5/sterolin-1, RT-PCR was performed on cDNA reverse transcribed from total liver RNA from Abcg8-deficient mice. No mRNA for Abcg8/sterolin-2 was detected containing any sequences downstream of exon 4 in the Abcg8-deficient mice, whether primers were located in exons 4 and 13, exons 9 and 13 or exons 10 and 13 (Figure 1d,1e). This demonstrates that if an alternatively spliced message could potentially code for a truncated protein, it is not detectable in the Abcg8-deficient mice. The membrane-spanning domains of Abcg8/sterolin-2 are encoded by exons 9–13.