Increased gene dosage for β- and κ-casein in transgenic cattle improves milk composition through complex effects

Abstract

We have previously generated transgenic cattle with additional copies of bovine β- and κ casein genes. An initial characterisation of milk produced with a hormonally induced lactation from these transgenic cows showed an altered milk composition with elevated β-casein levels and twofold increased κ-casein content. Here we report the first in-depth characterisation of the composition of the enriched casein milk that was produced through a natural lactation. We have analyzed milk from the high expressing transgenic line TG3 for milk composition at early, peak, mid and late lactation. The introduction of additional β- and κ-casein genes resulted in the expected expression of the transgene derived proteins and an associated reduction in the size of the casein micelles. Expression of the transgenes was associated with complex changes in the expression levels of other milk proteins. Two other major milk components were affected, namely fat and micronutrients. In addition, the sialic acid content of the milk was increased. In contrast, the level of lactose remained unchanged. This novel milk with its substantially altered composition will provide insights into the regulatory processes synchronizing the synthesis and assembly of milk components, as well as production of potentially healthier milk with improved dairy processing characteristics.

Figure 1. Two-dimensional electrophoresis of mammary secretion from a transgenic (TG3) and a control (WT) cow.

Mammary secretion was collected from a TG3 and a wild type cow, firstly as one-year old heifers after hormonal induction of lactation (induced), and then from the same animals at peak lactation after a natural calving (natural). Milk proteins were separated by isoelectric point (pI) in the first dimension and molecular weight (MW) in the second dimension. The gels were stained for protein with Coomassie blue and sections of each gel are shown in which the major caseins (as marked) are resolved. The white centred arrow indicates the exogenously expressed A3 isoform of β-casein. The black centred arrow indicates the exogenously expressed B isoform of κ-casein. Shown are representative gels which have been cropped for improved clarity. The full size gels are presented in Supplementary Fig. S2.

Figure 2. Concentration of total β-casein and κ-casein in TG3 and control milk.

Shown are the β-casein and κ-casein levels found in a 24 h milk sample of seven transgenic TG3 (black) and six wild type control (red) cows at early, mid, peak and late lactation.

Figure 3. Casein micelle diameter in milk from transgenic and control cows.

The size of the casein micelles was measured by photon correlation spectroscopy in whole milk (solid bars) and skim milk (open bars) produced in a natural lactation by three transgenic (TG3-1, -2, -3; black) and four wild type control (WT-1, -2, -3, -4; red) cows.

Figure 4. Concentration of major milk components in milk from transgenic and control cows.

Shown are the results for the milk components (a) protein, fat and lactose; (b) the minerals calcium, magnesium and zinc and (c) the endogenous milk proteins α-casein, α-lactalbumin and β-lactoglobulin in milk samples from seven transgenic TG3 (black) and six wild type control (red) cows at early, mid, peak and late lactation.

Figure 5. DIGE analysis of milk proteins from transgenic and control cows.

Milk from transgenic as well as wild type cows was collected at peak of a natural lactation and the proteins from each cow were individually labelled with Cy5 and Cy3, respectively. Shown is the Cy5 (TG3, red), Cy3 (wild type, green), and a merged color-coded C5/Cy3 signal obtained from a section of a representative gel loaded with WT and TG3 milk. The different isoforms of the milk proteins that were quantified have been labelled in the merged panel. αS1: αS₁-casein; αS2: αS₂-casein, isoforms 1–6; βCN: β-casein, isoforms A1-A3; κCN: κ-casein, isoforms A1-A5, B1-B5, BX1-BX3, BX?; αLac: α-lactalbumin; BLG: β-lactoglobulin. The gels are cropped for improved clarity and the full size gels are presented in Supplementary Fig. S3.

Figure 6. Sialic acid content in milk from transgenic and control cows.

Shown is the level of sialic acids associated with milk proteins from two wild type (WT1, WT2) and three transgenic cows (TG3 1–3). Neu5Gc is represented as solid bars in light red and grey, Neu5Ac as red and black solid bars and total sialic acid as red and black open bars for wild type and transgenic samples, respectively.