PMC:13917 / 2077-7888 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/13917","sourcedb":"PMC","sourceid":"13917","source_url":"http://www.ncbi.nlm.nih.gov/pmc/13917","text":"Introduction\nWith each successive pregnancy, the mammary gland completes a cycle of growth, functional differentiation and involution. These processes are of great importance in biology in their own right, but they also provide an example of how proliferation, differentiation and apoptosis are integrated into the organization of a complex three-dimensional tissue unit, whose function changes with time. The growth and development of ductal and alveolar structures during pregnancy is dependent on the interaction between the epithelial cells and stromal components of the mammary fat pad and requires the concerted actions of both peptide and steroid hormones, and cell-cell and cell-substratum interactions [1]. The necessity for these complex structural and hormonal interactions provide a challenge for the development of in vitro models for molecular studies that accurately mimic the differentiation and death of mammary epithelial cells.\nA variety of mammary culture systems have been developed to facilitate studies on the regulation of gene transcription in the mammary gland. Whole organ and explant cultures have been of value in identifying the role of specific hormones in both the growth and differentiation of mammary tissue and the induction of milk protein gene expression [2]. These cultures have a limited lifespan, however, and are not useful for studies at the cellular level. Epithelial cells can be isolated from mammary tissue, maintained in culture and induced to differentiate with lactogenic hormones. The use of such primary cultures has demonstrated the importance of the cellular substratum in the differentiation process [3]. A major drawback of this system, in addition to the short lifespan of the cells, is the considerable amount of starting material required.\nSpontaneously immortalized cell lines have arisen from prolonged culture of primary epithelial cells in low serum (2%). Many of these established mammary epithelial cell lines have proved to be useful tools in molecular and bio-chemical studies. They include EpH4 cells [4] and the COMMA-1D cell line, one of the most widely used in vitro mammary systems, which exhibits mammary-specific functional differentiation when exposed to lactogenic hormones and extracellular matrix (ECM) [5]. Subclones of COMMA-1D include HC11 and CID9 [6,7]. HC11 cells have been widely used by us, and others, for studies on transcriptional regulation of milk protein gene expression [8,9], whereas CID9 cells have been extensively used to demonstrate the role of ECM in milk protein gene expression [7]. Furthermore, a pure epithelial population, SCp2, has been derived from CID9 [10].\nIn our laboratory, we are particularly interested in the signalling pathways that regulate gene expression in the differentiating and involuting mouse mammary gland. Despite the undoubted value of these culture systems, expression of transgenes in vivo does not always recapitulate expression observed in culture. This reflects the complex requirements for mammary epithelial cell differentiation and apoptosis. There is a need, therefore, for mammary epithelial cell lines that more accurately mimic the developing and involuting gland. Such cells should preferably be immortalized by a conditional (ie reversible) mechanism (in contrast to currently available lines) and be able to be genetically modified.\nIn order to achieve this, we adopted a modification of the procedure used to generate 'immortomouse', a line of transgenic mice that harbour a temperature-sensitive variant of an immortalizing gene, SV40 T-Ag, which is expressed from a constitutive promoter H2Kb [11]. Although the 'immortomouse' shows thymic hyperplasia, these transgenic mice undergo normal development and have proven to be a useful source of material to establish cell lines from tissues that have previously been refractory to culturing [12]. Our attempts to establish mammary epithelial lines from these mice were unsuccessful, however. This may be due to insufficient levels of T-Ag being expressed in the mammary epithelial compartment to immortalize these cells or the presence of T-Ag in the other mammary compartments, thereby allowing the preferential immortalization of fibroblasts and other stromal cell types.\nWe therefore decided to refine this approach and target expression of the thermolabile T-Ag mutant specifically to the mammary epithelium of transgenic mice. Targeted expression can be achieved using either the mouse mammary tumour virus long terminal repeats or a milk protein gene promoter. We chose to use the promoter of the sheep milk protein gene encoding β-lactoglobulin (BLG), because BLG is less dependant than WAP on the transgene genomic integration site for its expression [13] BLG transgenes are expressed at low levels in the mammary glands of virgin mice, whereas expression is regulated during pregnancy and lactation with a similar expression profile to that of β-casein [14]. Therefore, it is likely that BLG expression occurs in dividing cells early in the differentiation pathway, a critical factor in establishing cultures from early stages of mammary gland development. Such cultures could contain epithelial stem cells because these are known to be distributed throughout the ductal tree [15]. We report herein the isolation and characterization of a stable line of mammary epithelial cells, named KIM-2, from mid-pregnant mammary glands of one line of mice with a low transgene copy number. Importantly, this cell line has a phenotypically normal epithelial morphology at 37°C and permits the analysis of the complex processes of differentiation and apoptosis in vitro. Moreover, we provide evidence that this line is susceptible to genetic manipulation, thus making available a resource for analysis of genetic function.","divisions":[{"label":"Title","span":{"begin":0,"end":12}}],"tracks":[{"project":"Colil","denotations":[{"id":"T11","span":{"begin":2330,"end":2331},"obj":"3416834"},{"id":"T12","span":{"begin":2332,"end":2333},"obj":"2251252"},{"id":"T13","span":{"begin":1293,"end":1294},"obj":"3886667"},{"id":"T14","span":{"begin":4752,"end":4754},"obj":"1520282"},{"id":"T15","span":{"begin":4955,"end":4957},"obj":"1718646"},{"id":"T1","span":{"begin":5278,"end":5280},"obj":"9550724"},{"id":"T2","span":{"begin":3638,"end":3640},"obj":"1711218"},{"id":"T3","span":{"begin":3884,"end":3886},"obj":"7678459"},{"id":"T4","span":{"begin":1655,"end":1656},"obj":"3467345"},{"id":"T5","span":{"begin":2281,"end":2282},"obj":"6587390"},{"id":"T6","span":{"begin":2463,"end":2464},"obj":"7877621"},{"id":"T7","span":{"begin":2465,"end":2466},"obj":"2643093"},{"id":"T8","span":{"begin":2579,"end":2580},"obj":"2251252"},{"id":"T9","span":{"begin":712,"end":713},"obj":"7001510"},{"id":"T10","span":{"begin":2069,"end":2070},"obj":"2466037"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/docs/sourcedb/PubMed/sourceid/"}],"attributes":[{"subj":"T11","pred":"source","obj":"Colil"},{"subj":"T12","pred":"source","obj":"Colil"},{"subj":"T13","pred":"source","obj":"Colil"},{"subj":"T14","pred":"source","obj":"Colil"},{"subj":"T15","pred":"source","obj":"Colil"},{"subj":"T1","pred":"source","obj":"Colil"},{"subj":"T2","pred":"source","obj":"Colil"},{"subj":"T3","pred":"source","obj":"Colil"},{"subj":"T4","pred":"source","obj":"Colil"},{"subj":"T5","pred":"source","obj":"Colil"},{"subj":"T6","pred":"source","obj":"Colil"},{"subj":"T7","pred":"source","obj":"Colil"},{"subj":"T8","pred":"source","obj":"Colil"},{"subj":"T9","pred":"source","obj":"Colil"},{"subj":"T10","pred":"source","obj":"Colil"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"Colil","color":"#ec93d1","default":true}]}]}}