In order to inactivate the a-casein gene (Fig. 1a) a targeting construct was established based on previous experience with constructs used to inactivate the band c-casein genes [5, 27] . The targeting event removes the complete second exon which includes the translational start codon and the 15 amino acid signal peptide (Fig. 1b) . In 3 independent transfections into ES cells the targeting frequency was found to be around 3%. Successfully modified ES cells clones were identified using a PCR analysing the 59 end of the targeted gene (Fig. 1c) . The targeting event was then confirmed by Southern blot analysis assessing the 59 end of the targeted a-casein gene (Fig. 1d) . The integration event was also confirmed using PCR analysis and Southern blotting with primer combinations and probes specific for the 39 end (Fig. 1e) . Two targeted cell clones were grown up and injected into blastocysts. Animals carrying a homozygous deletion of the a-casein gene are phenotypically normal prior to lactation. Fig. 2a alongside a wild-type control milk sample. Milk from control animals displayed a series of protein bands, with the most prominent proteins corresponding to molecular weights previously observed for a-, band c-casein (with apparent molecular weights of 42, 30 and 25 kDa, respectively) [28] . The identity of the protein bands was confirmed using mass-spectrometry. Table 4 details the data for the 8 most prominent protein bands indicated in Fig. 2c . These findings were also supported using Western blot analyses for a-casein and b-casein ( Fig. 3a and 3b ). Milk from acasein deficient mice shows some characteristic protein bands which are absent from milk of wild-type or heterozygous mice (Fig. 2d) . Mass-spectrometry analysis was used to identify some of these proteins. The major additional band with an apparent molecular weight of 78 kDa was identified as grp78/BiP. BiP is an ER resident protein which is involved in the assembly of multi-protein complexes like antibodies [29, 30] . The identity of the grp78/BiP protein was confirmed by Western blot analysis (Fig. 3c) . In addition two further ER-resident proteins, grp94 and PDIA6 (protein disulfide-isomerase associated protein 6), were detected in milk of the a-casein deficient mice (Fig. 2d, table 4) .