High expression of the caprine β‐casein gene in transgenic mice

Abstract

An 18-kb caprine genomic DNA fragment, comprising the fl-casein transcription unit with about 3-kb 5' and 6-kb 3' flanking regions, was microinjected into fertilized one-cell murine eggs. All nine lines of transgenic mice obtained expressed the transgene in their mammary glands, as demonstrated by Northern blot analysis of mRNA in miscellaneous tissues, and qualitative and quantitative analysis of caprine 8-casein in milk, using SDS/PAGE, Western blotting and rocket immunoelectrophoresis.Two lines produced milk containing up to 21 -24 mg of the exogenous protein/ml, a yield which is roughly twice that found in goat milk. The yield reached at least 40mg/ml in some progeny of crossbred G1 transgenic mice. Thus, the investigated gene appears to be a good candidate for making hybrid constructs that might promote an efficient production of valuable foreign proteins in milk. footprinting, interference methylation, gel retardation and oligonucleotide competition techniques. A mammary-glandspecific nuclear factor [14] bound at both a strong and a weak site was identified, and the observed down-regulation of other complexes during induction may indicate a transcriptional derepression [14]. According to these results, about 2.6 kb of the 5' flanking region of the 8-casein gene [9, 121 may contain most essential regulatory elements mediating the hormonal and extracellular matrix effects. According to some authors [9,10], exon I and intron I may not be essential in that respect, whereas others [16] have stressed their importance.Expression of major milk-protein-encoding genes and derived hybrid constructs, has been also investigated in transgenic animals, mainly mice and to a lesser extent rabbit, swine and ruminants: fl-lactoglobulin [19 -241, whey acidic protein (WAP) [25 -341, a-lactalbumin [35-371, asl-casein [38] and fl-casein [39 -431. Overall, the exogenous protein yield in milk was rather low but expression was found to be mammarytissue-and stage-specific. However, it has been reported that milk-protein-encoding genes might also be normally active in some other tissues, since the cognate mRNAs were detected in cytotoxic T lymphocytes [44] and sebaceous glands of lactating mice [36].In the case of the rat /%casein transgene with 3.5 kb of 5' and 3.0 kb of 3' flanking region, the expression level was 0.01-1% of that of the cognate endogenous gene in the transgenic murine lines [39, 411. Similarly, only low levels of human interleukin-2, at most 430 ng/ml of milk in one female, were produced by transgenic rabbits [42] carrying a chimeric gene comprising 2 kb of the 5' flanking region and exon I of the rabbit fl-casein gene fused to the human interleukin-2 gene.In the present paper, we report the high stage-and mammary-tissue-specific expression of an 18-kb caprine (goat) pcasein transgene comprising 3 kb of 5' and 6 kb of 3' flanking region, investigated in nine lines of transgenic mice. The transgene was expressed in all lines, and in five of them, mice produced milk containing up to 3 mg, 12 mg, 14 mg, 21 mg and 24...

Whey Acidic Protein (WAP) Depresses the Proliferation of Mouse (MMT) and Human (MCF-7) Mammary Tumor Cells

“…For example, lactoferrin inhibits cell proliferation and suppresses mammary tumorigenesis in vivo; lactoferrin induces growth arrest from the G1 phase to the S phase along with the suppression of Cdk2 kinase activity [37]. On the other hand, overexpression of β-casein did not affect mammary morphogenesis in transgenic mice [38]. The present results, taken together with our previous findings [21,24], strongly suggest that WAP is an important factor in controlling the normal development of mammary glands in some species.…”

Role of amino acids in translational mechanisms governing milk protein synthesis in murine and ruminant mammary epithelial cells

“…Furthermore, the fact that transgenic mice with the same genes express these proteins at much higher levels suggests a quantitative limitation on protein synthesis in the bovine gland [Persuy et al, 1992;Rijnkels et al, 1995]. By contrast, nontransgenic cattle treated with insulin clamps and infused with casein as a source of amino acids (AA) exhibited a transient but significant change in milk protein production [Griinari et al, 1997].…”

Milk composition and lactation of beta-casein-deficient mice.

“…We and others have previously demonstrated the profound alteration of the protein composition of milk by expression of milk protein genes in transgenic mice (2)(3)(4), and the expression of human pharmaceutical proteins in the milk of transgenic mice, rabbits, goats, pigs and sheep has been demonstrated (5)(6)(7)(8)(9)(10)(11)(12)(13). In some cases very high yields of active human proteins have been obtained (6,8,(11)(12)(13), and this approach is being commercialized.…”