In order to assess whether the reduction in protein and RNA expression is correlated with overall morphological changes, sections of mammary tissue were obtained from animals at peak lactation and analysed by haematoxylin/eosin (H&E) staining and immuno-histochemistry (Fig. 6 ). As expected a-casein expression was clearly detectable in tissue from wild-type control mice and heterozygous mice (Fig. 6a) . In contrast no a-casein protein could be detected in sections of a-casein deficient mice (Fig. 6a) . As expected sections analysed with pre-immune serum did not show any a-casein specific staining (Fig. 6a) . No gross morphological alterations were detected in the sections of the different genotypes (Fig. 6b) . This suggests that the absence of a-casein, although critical for overall milk composition, does not significantly impact on the survival of the mammary gland. To address this question further we measured the expression and activity of capase proteins in the mammary gland. Firstly we assessed the presence of cleaved caspase 3 in the protein extracts derived from control mice, heterozygous mice and a-casein deficient mice. No cleaved caspase 3 could be detected (Fig. 7a) . In contrast cleaved caspase 3 protein with the expected molecular weight of 19 kDa was readily detected in the extracts of RAW264 cells treated with 10 mM staurosporin (Fig. 7a) . Similarly, no specific signals for cleaved caspase 3 could be detected in immuno-histochemical analyses of tissue sections. In addition, caspase 3 and caspase 7 activities was measured in cytoplasmic protein extracts derived from mammary tissue of control, heterozygous and a-casein deficient mice. No significant differences in caspase 3 and 7 were detected (Fig. 7b) . In contrast a significant increase in caspase activity could be detected in RAW cells treated with 10 mM staurosporin (Fig. 7b) . cDNAs derived from representative samples of control and acasein deficient mice were analysed for characteristic gene expression changes related to apoptosis. Potential changes in candidate genes were assessed using a PCR array containing 84 apoptosis related genes. The expression of 16 genes was changed significantly (11 down-regulated and 5 up-regulated in a-casein deficient mice vs. control). Five genes, which were changed by more than 7 fold (1 down-regulated and 4 up-regulated), were further analysed in cDNAs derived from 3 control, 5 heterozygous and 5 a-casein deficient animals (Fig. 7c) . Only 3 genes showed consistent and significant expression differences between control and a-casein deficient animals. Expression of the anti-apoptotic genes survivin (baculoviral inhibitor of apoptosis repeat-contain- ing-5; Birc5) and nucleolar protein 3 (Nol3/ARC: nuclear apoptosis repressor with caspase recruitment domain) was increased by 22 and 2.5 fold respectively. In contrast expression of another anti-apoptotic gene, TNF receptor-associated factor 1 (Traf1), was reduced to 5% of control levels in a-casein deficient mice.