Gene expression in bovine mammary somatic cells isolated from milk*

Traditionally, in order to examine the expression of genes involved in mammary gland metabolism, RNA was prepared from mammary biopsies or from tissue samples taken at slaughter. The objective of this study was to validate the use of milk somatic cells as a model to study gene expression in the lactating bovine mammary gland. The expression pattern of milk protein and epithelial cell marker genes was similar between somatic cells and mammary tissue indicating that cells exfoliated into milk provide a viable non-invasive tissue source for gene profi ling in the bovine mammary gland.


INTRODUCTION
The physiological status of a cell or organism is, to a large extent, directed by the complement of genes expressed.Gene profi ling studies usually require tissues collected using invasive techniques such as biopsies or tissue samples taken at slaughter.Somatic cells present in milk provide a potential alternative non-invasive source of mammary tissue.They are present in the milk throughout lactation and consist primarily of leukocytes and epithelial cells exfoliated from the mammary epithelium.Boutinard et al. (2002) reported that somatic cells exfoliated from the mammary epithelium during the milking process provided an alternative non-invasive source of mammary tissue for gene expression profi ling in goats.This approach potentially offers signifi cant improvement over the use of biopsy and post-mortem sampling for assessing gene expression.Additionally, the use of milk somatic cells could allow repeated gene expression profi ling during the lactation period using the same animal.To date, this approach for bovine mammary gene expression studies has not been evaluated.Therefore, our hypothesis was that somatic cells isolated from milk would provide a useful alternative tissue source for gene expression profi ling in the bovine mammary gland.To validate the use of milk somatic cell RNA for gene expression profi ling in the bovine mammary gland, we compared the profi les of the major milk protein genes and selected epithelial cell markers between mammary tissue and milk somatic cells collected from cows treated with growth hormone (GH) and untreated control animals.

MATERIAL AND METHODS
Milk somatic cells were collected from 8 spring-calved, fi rst lactation (midlate) Jersey cows, randomly allocated to control (saline) and treatment (single subcutaneous injection of commercial GH; Lactatropin®, Elanco Animal Health).All cows were maintained under identical environment and management practices, fed a nutritionally balanced total mixed ration ad libitum and milked twice daily.After six days of treatment, all cows were milked and immediately euthanised.Total cellular RNA was isolated from milk somatic cells using Trizol (Invitrogen) and purifi ed using RNeasy columns (Qiagen).Only three cows per treatment group yielded suffi cient quantity of RNA from somatic cells to conduct the experiment.Total RNA was converted to fl uorescently-labelled cDNA (SuperScript cDNA Labelling System, Invitrogen) and hybridized to 23.000 bovine expressed sequence tag (EST) microarrays printed on glass slides.The slides were scanned using GenePix 5.0 software.Microarray data were normalized and analysed using REML (Baird et al., 2002(Baird et al., , 2003) ) and a list of genes differentially expressed in response to GH treatment were compared with expression profi les from post-mortem tissues from the same cows (McCoard et al., 2004, companion paper).
The ESTs were selected from AgResearch's proprietary database that represents approximately 80% of the bovine genome.The arrays used for the comparison of mammary tissue and somatic cells contained different sets of ESTs.However, both arrays contained ESTs representing the milk protein genes and epithelial cell markers described in this study.

RESULTS
Comparison of gene expression profi les showed that the expression of the genes encoding κ-casein, β-casein, α s1 -casein, α-lactalbumin were similar between somatic cells and post-mortem mammary tissue collected from the same animal (Table 1).However, β-lactoglobulin gene expression was down-regulated in somatic cells and up-regulated in mammary tissue.The pattern of expression of the genes encoding selected epithelial cell markers was similar between somatic cells and mammary tissue (Table 1).

DISCUSSION
This study demonstrates the functional use of RNA extracted from milk somatic cells for assessing gene expression profi les in the bovine mammary gland.It has been reported that somatic cells accurately refl ect events occurring in the goat mammary gland (Boutinard et al., 2002) but this is the fi rst study to validate the use of milk somatic cells for gene expression studies in the cow.Comparison of the expression profi le of milk protein genes between milk somatic cells and post-mortem tissues showed the same pattern in the two RNA samples.Miyoshi et al. (2002) and Tammen et al. (2003) reported that cytokeratin 5, cytokeratin 14 and nuclear factor-1 are good markers for mammary epithelial cells and can HAYASHI A.A. ET AL. be used as markers for differentiation of epithelial cells during carcinogenesis in humans.Our study confi rms that the pattern of gene expression for these and others epithelial cell markers and milk protein genes are similar between mammary tissue and milk somatic cells.

CONCLUSIONS
This study indicates that milk somatic cells accurately refl ect gene expression profi les in lactating mammary tissue.This method is a signifi cant improvement over the use of biopsy samples and allows easy and repetitive sampling without damage to the mammary gland.Thus, gene expression profi ling, as determined via RNA from somatic cells may provide a convenient means to more dynamically characterize events within the bovine mammary gland.

Table 1 .
Effect of GH on milk protein and epithelial cell marker genes expression relative to controls (P<0.05) in simultaneously collected post-mortem mammary tissue and milk somatic cells