Genetic Manipulation of Ruminant Mammary Epithelial Cells in Primary Culture

Wells, Michelle (2000) Genetic Manipulation of Ruminant Mammary Epithelial Cells in Primary Culture. PhD thesis, University of Glasgow.

Abstract

Much work has been carried out into the control of milk protein secretion in rodents, but relatively little is known about ruminant species. Practical, financial and statistical constraints preclude studies in vivo, while no satisfactory in vitro model currently exists. In addition, the cloning of milk protein genes and the development of transgenic technology have opened up new opportunities for directing the expression of foreign proteins in milk. Gene constructs are traditionally tested in mice, but results cannot necessarily be extrapolated to other species. The aim of this project was to establish a ruminant cell culture system suitable for the study of mammary gland function, and the evaluation of transgenes prior to the generation of transgenic livestock. Mammary epithelial cells were isolated from sheep or goats in the final trimester of pregnancy by enzymatic digestion with collagenase and hyaluronidase. Cells were fractionated by Percoll density gradient centrifugation. Cell typing of isolated cells showed that fraction 3 consisted predominantly of epithelial cells, with small numbers of myoepithelial and fibroblastic cells present. Culture of these cells on extracellular matrix derived from the Engelbreth-Holm-Swarm mouse sarcoma resulted in the formation of three-dimensional structures, termed mammospheres, and induction of milk protein secretion. Cells were found to be responsive both to substratum and to the lactogenic hormone prolactin, suggesting that this model may provide the most useful in vitro system to date. Cells were also successfully differentiated following recovery from liquid nitrogen, although synthetic and secretory activity was approximately 60% of that of freshly prepared cells. Transfection of primary ruminant epithelial cells following optimisation, resulted in transfection efficiencies of ~0.3%>. This low transfection rate necessitated extended passaging of cells. Investigation of differentiation following passaging showed that sheep cells did not secrete milk proteins after passage 2, while in goat cells beta-lactoglobulin, alphas1-casein, and more importantly, alpha-lactalbumin were detected at late passage (passage 5-7), although in declining amounts. In addition, sheep cells were increasingly susceptible to the harvesting procedure, while this had no discernible detrimental effect on goat cells. Cell typing of passaged cells showed a loss of myoepithelial and fibroblastic cell types, with increasing passage, concomitant with the loss of secretory function. Expression of reference constructs in ruminant mammary epithelial cells resulted in the successful detection of only one foreign product, human alpha1-antitrypsin (AAT). This construct was known to be expressed at very high levels (30 mg/ml) in vivo. Although post-translational processing was not investigated, resolution of proteins by SDS-PAGE and immunoblotting detected a single band running at the expected position of human AAT, when cells were cultured on EHS matrix in the presence of prolactin. Detection of other transgene products was unsuccessful. This culture system provides a good model for the study of ruminant mammary gland function. In particular, the ability of cells to differentiate after cryopreservation increases feasibility and provides consistent starting material for large numbers of experiments. If the problems of loss of differentiation with passaging and/or low transfection efficiencies can be resolved, the manipulation or insertion of genes provides new opportunities to study mammary function or to evaluate transgenes prior to the generation of transgenic animals.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Lynn Finch
• Keywords: Genetics
• Date of Award: 2000
• Depositing User: Enlighten Team
• Unique ID: glathesis:2000-76282
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 16:10
• Last Modified: 19 Nov 2019 16:10
• URI: https://theses.gla.ac.uk/id/eprint/76282