Dundas, Sinclair Robertson; (1990) A study of rat mammary parenchyma: cell sorting and clonal characterisation of luminal and myoepithelial cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Rat mammary gland cell biology has been studied using a novel approach. Parenchymal cell types have been isolated and characterised by clonal behaviour in culture. Culture conditions with which normal mammary cells from adult virgin rats could be cloned with high efficiency were established. Several morphologically distinctive colony types were observed, and cells have been cloned from isolated ducts and alveoli. Luminal and myoepithelial cells were isolated from mammary gland using fluorescence activated cell sorting (FACS). Cells were flow sorted on the basis of the differential expression of two membrane antigens - a 70KD luminal-specific antigen , and neutral endopeptidase EC 3.4.24.11 (NEP/CALLA), which is expressed by myoepithe­ lial cells in the intact gland. Sorted cells were then separately cloned so that the in situ origin of differ­ ent clone types could be unequivocally determined. Monoclonal antibodies recognising cell-type specific cytokeratin polypeptides were used to confirm clonal phenotypes derived from sorted cells. Primary clones have been studied with a panel of cell type-specific markers at various stages of growth, using indirect immunofluorescence and flow cytometry. In order to determine the number of different stable phenotypes and follow their epithelial differentiation status, primary clones have been re-cloned and the phenotypes of progeny colonies examined. These studies demonstrated the existence of distinct multipotent clone types capable of generating cells of both luminal and myoepithelial phenotypes in culture. Responses of the different clones to various growth factors and hormones have been examined. Continuous cell lines have been established from both sorted luminal and myoepithelial cells by retroviral trans­ duction of a recombinant construct encoding a tempera­ ture-sensitive non-DNA binding SV40 large T protein. The behaviour of these lines has been compared with sorted primary cultures, and the organotypic differen­ tiation of both examined in fat pad transplants. The results are analysed in the context of the mechanisms and cellular interactions controlling growth and dif­ ferentiation of the mammary parenchyma in situ.

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