Comprehensive Assessment of Milk Composition in Transgenic Cloned Cattle

<div><p>The development of transgenic cloned animals offers new opportunities for agriculture, biomedicine and environmental science. Expressing recombinant proteins in dairy animals to alter their milk composition is considered beneficial for human health. However, relatively little is known about the expression profile of the proteins in milk derived from transgenic cloned animals. In this study, we compared the proteome and nutrient composition of the colostrum and mature milk from three lines of transgenic cloned (TC) cattle that specifically express human α-lactalbumin (TC-LA), lactoferrin (TC-LF) or lysozyme (TC-LZ) in the mammary gland with those from cloned non-transgenic (C) and conventionally bred normal animals (N). Protein expression profile identification was performed, 37 proteins were specifically expressed in the TC animals and 70 protein spots that were classified as 22 proteins with significantly altered expression levels in the TC and C groups compared to N group. Assessment of the relationship of the transgene effect and normal variability in the milk protein profiles in each group indicated that the variation in the endogenous protein profiles of the three TC groups was within the limit of natural variability. More than 50 parameters for the colostrum and mature milk were compared between each TC group and the N controls. The data revealed essentially similar profiles for all groups. This comprehensive study demonstrated that in TC cattle the mean values for the measured milk parameters were all within the normal range, suggesting that the expression of a transgene does not affect the composition of milk.</p> </div

Graphical representation of the PCA results for the nutrient profiles of the colostrum and mature milk from TC, C, and N animals.

<p>Graphical representation of the PCA results for the nutrient profiles of the colostrum and mature milk from TC, C, and N animals.</p

Differentially expressed protein spots with great than 2-fold changes (<i>p</i>≤0.05) in the TC and the C group compared to N group.

<p>The red and green arrows indicate the protein spots that were significantly up- and downregulated, respectively, by at least 2-fold.</p

List of the proteins differentially expressed in the transgenic and cloned groups compare to normal group.

*<p>Proteins not identified by MALDI-TOF/TOF; ^†Upregulated proteins showing greater than 2-fold changes in the TC (LZ, LA and LF) and C groups compared to N group (<i>p</i>≤0.05); ^‡No function annotation; ^§Downregulated proteins showing greater than 2-fold changes in the TC and C groups compared to N group (<i>p</i>≤0.05).</p

List of the proteins specifically expressed in the transgenic groups.

*<p>Proteins not detected by LC-MS/MS in the transgenic groups (LZ, LA and LF); ^†Proteins detected by LC-MS/MS; ^‡No function annotation.</p

Comparisons of the major whey proteins and immune-associated proteins in the colostrum and mature milk from the TC, C, and N animals.

<p>Values are means ± SD and * indicate significant difference between the TC, C and N groups.</p

Functional distribution and enrichment of specifically expressed proteins classified according to cellular component (A), biological process (B) and molecular function (C) in the TC groups, respectively.

<p>The red curves indicate the degree of functional enrichment of a particular gene.</p

Comparisons of the proximates in the colostrum and mature milk from TC, C, and N animals.

<p>(A) The proximates of the colostrum from each group. (B) The proximates of the mature milk from each group. Values are means ± SD and * indicate significant difference between the TC, C and N groups.</p

Venn diagrams representation of whey protein profiles comparison of the TC-LZ (A), TC-LA (B), TC-LF (C) and all TC (D) groups were created with respect to the C and N groups, respectively.

<p>The gray ellipse indicates the proteins reported to be present in bovine milk in previously studies. The blue, green and red circles indicate the protein sets of the TC, C and N groups, respectively.</p

The intra- and inter-group relationships of the differentially expressed proteins of the TC, C and N groups.

<p>(A) HCA of differentially expressed proteins. (B) PCA of differentially expressed proteins.</p