Use of an immortalized bovine mammary epithelial cell line (MAC-T) to measure the mitogenic activity of extracts from heifer mammary tissue: effects of nutrition and ovariectomy

Berry, S.D.K.; Nielsen, Morten Schak; Sejrsen, K.; Pearson, R.E.; Boyle, P.L.; Akers, R.M.

doi:10.1016/s0739-7240(03)00062-6

Cited by 14 publications

(11 citation statements)

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“…α-SMA it is a typical marker for myoepithelial and mesenchymal cells [ 16 , 19 ] which are also present in the MG. Considering flow cytometry light scattering properties of MAC-T and primary cells, respectively, and knowing that MAC-T cells retain milk secretory characteristics [ 10 , 25 , 26 ], we expected a lower mRNA and protein expressions of α-SMA in MAC-T than primary cells. Surprisingly, the expression of α-SMA in primary cells was lower than in MAC-T cells.…”

Section: Discussionmentioning

confidence: 99%

Can widely used cell type markers predict the suitability of immortalized or primary mammary epithelial cell models?

et al. 2016

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BackgroundMammary cell cultures are convenient tools for in vitro studies of mammary gland biology. However, the heterogeneity of mammary cell types, e.g., glandular milk secretory epithelial or myoepithelial cells, often complicates the interpretation of cell-based data. The present study was undertaken to determine the relevance of bovine primary mammary epithelial cells isolated from American Holstein (bMECUS) or Swiss Holstein–Friesian (bMECCH) cows, and of primary bovine mammary alveolar epithelial cells stably transfected with simian virus-40 (SV-40) large T-antigen (MAC-T) for in vitro analyses. This was evaluated by testing their expression pattern of cytokeratin (CK) 7, 18, 19, vimentin, and α-smooth muscle actin (α-SMA).ResultsThe expression of the listed markers was assessed using real-time quantitative PCR, flow cytometry and immunofluorescence microscopy. Characteristic markers of the mesenchymal (vimentin), myoepithelial (α-SMA) and glandular secretory cells (CKs) showed differential expression among the studied cell cultures, partly depending on the analytical method used. The relative mRNA expression of vimentin, CK7 and CK19, respectively, was lower (P < 0.05) in immortalized than in primary mammary cell cultures. The stain index (based on flow cytometry) of CK7 and CK19 protein was lower (P < 0.05) in MAC-T than in bMECs, while the expression of α-SMA and CK18 showed an inverse pattern. Immunofluorescence microscopy analysis mostly confirmed the mRNA data, while partly disagreed with flow cytometry data (e.g., vimentin level in MAC-T). The differential expression of CK7 and CK19 allowed discriminating between immortal and primary mammary cultures.ConclusionsThe expression of the selected widely used cell type markers in primary and immortalized MEC cells did not allow a clear preference between these two cell models for in vitro analyses studying aspects of milk composition. All tested cell models exhibited to a variable degree epithelial and mesenchymal features. Thus, based on their characterization with widely used cell markers, none of these cultures represent an unequivocal alveolar mammary epithelial cell model. For choosing the appropriate in vitro model additional properties such as the expression profile of specific proteins of interest (e.g., transporter proteins) should equally be taken into account.

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Section: Discussionmentioning

confidence: 99%

Can widely used cell type markers predict the suitability of immortalized or primary mammary epithelial cell models?

et al. 2016

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“…On the other hand, little is known about the suitability of tissue fragments from heifers in establishing primary cell cultures. Mammary epithelial cell cultures, especially organoids from heifer tissues, were analyzed in the past rather to examine the mitotic effects of cell proliferation (Berry et al 2003; Thorn et al 2008). In the current study, a very low level of CSN2 gene expression was detected in cultures obtained from all samples (heifers, lactation, and involution).…”

Section: Discussionmentioning

confidence: 99%

Bovine mammary epithelial cell cultures for the study of mammary gland functions

Jedrzejczak

Szatkowska

2013

In Vitro Cell.Dev.Biol.-Animal

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In the present study, the analysis of epithelial cells derived from various sources was undertaken, beginning from the mammary gland tissue through the primary cultures and their subsequent passages. The objective of the study was the comparative analysis of the stage in which the epithelial cells obtained from individuals in different lactation cycles and disparate phases of cell culture growth are the most suitable for morphological research and analysis of gene expression activity. The cultures of primary bovine mammary epithelial cells and passages were identified morphologically using immunocytochemical methods. After positive identification, real-time PCRs were performed for the analysis of the expression level of casein genes, whey protein genes, and butyrophilin gene. The most stable reference genes in real-time PCRs for the mammary gland tissue and cell cultures were also determined. Of the reference genes, the UXT and GAPDH genes appeared to be the most stable ones for the mammary gland tissue samples and epithelial cell cultures. The results obtained allowed concluding that the mammary gland samples collected from heifers constituted the most effective material for the initiation of primary cultures. The primary cultures formed characteristic for the mammary gland tissue dome structures, which images were obtained using confocal microscopy. The highest levels of expression of the CSN1S1, CSN1S2, CSN2, and CSN3 genes were detected in primary cultures. The levels of expression of whey protein genes (LALBA and BGL) were highest in the second passage. The most abundant expression of the BTN1A1 gene was observed in primary cultures and the third passage. On the basis of the whole experiment, it can be concluded that primary cultures and cells of the second passage derived from heifer individuals appeared to be the best materials for the analysis of mammary gland function and gene expression activity.

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“…Mammary epithelial cells are the functional unit of the mammary gland. MAC-T cells provide a useful tool in the evaluation of foreign gene expression and the assessment of mammary tissue-specific expression in vitro, as they retain a number of biochemical and morphological characteristics of the mammary gland in vivo (38). The unitary tet-on IFN-α/EPO induction system has mammary gland-specific and dox-inducible traits.…”

Section: Discussionmentioning

confidence: 99%

Generation of transgenic fibroblasts producing doxycycline-inducible human interferon-α or erythropoietin for a bovine mammary bioreactor

Kang

Hong

Hwang³

et al. 2012

Molecular Medicine Reports

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Abstract. Interferon α (IFN-α) is a cytokine, produced predominantly in immune cells in response to pathogens, which interferes with viral replication in host cells. Another cytokine hormone, erythropoietin (EPO), is synthesized in interstitial fibroblasts of the kidney and acts as a stimulator for the production of red blood cells. Importantly, the two cytokines have been used in the treatment of certain hematological malignancies, including renal anemia. In the production of recombinant proteins, a transgenic expression system in bovine species is an efficient strategy for pharmaceutical production. In the present study, recombinant constructs capable of producing recombinant human IFN-α and EPO proteins were established and were generated containing the mammary gland-specific αS1-casein promoter region (between -175 and +796 nt), as this promoter was revealed to have the highest level of activity in a previous promoter study. In order to minimize developmental toxicity by constitutive exogenous expression, a doxycycline (dox)-inducible system was introduced to the IFN-α/EPO-expressing constructs. Therefore, a unitary tetracycline (tet)-on the IFN-α/EPO vector was established, which combined a tet-on activator cassette controlled by the αS1-casein promoter, with a responder cassette encoding the IFN-α/EPO gene, controlled by the tetracycline response element (TRE) promoter. In these systems, the tet-controlled transactivator is affected by mammary gland-specific αS1-casein promoter, and binding of the transcriptional activator to the TRE results in transcription of the downstream IFN-α/EPO genes in the presence of dox. To assess this, the unitary tet-on IFN-α/EPO vector was introduced into a bovine mammary gland cell line (MAC-T), and the cells were then treated with 0.1-1 µg/ml dox. A marked increase was observed in the expression levels of IFN-α/EPO. In addition, bovine transgenic fibroblasts containing a mammary gland-specific and dox-inducible IFN-α/EPO construct were generated. These transgenic fibroblasts may provide a source for somatic cell nuclear transfer for the generation of transgenic cattle producing recombinant human IFN-α/EPO protein during lactation. IntroductionInterferons (IFNs) are glycoproteins produced by the immune system of host cells in response to infection of pathogens (1-4). Various forms of interferons have been evaluated as potential therapeutics in a number of solid tumors and hematological malignancies, including renal anemia, renal cell carcinoma, melanoma, acquired immune deficiency syndrome-associated Kaposi's sarcoma, follicular lymphoma, hairy cell leukemia and chronic myelogenous leukemia (1). INF-α and INF-β cytokines exert their antiviral function by eliciting antiviral activity from target cells and inducing apoptosis in infected cells. They also contribute to the immune response by activation of natural killer cells and macrophages (2-4). IFN-α was approved by the Food and Drug Administration as a treatment for hairy cell leukemia in 1986 (1) and, using a gene cloning ...

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Use of an immortalized bovine mammary epithelial cell line (MAC-T) to measure the mitogenic activity of extracts from heifer mammary tissue: effects of nutrition and ovariectomy

Cited by 14 publications

References 10 publications

Can widely used cell type markers predict the suitability of immortalized or primary mammary epithelial cell models?

Can widely used cell type markers predict the suitability of immortalized or primary mammary epithelial cell models?

Bovine mammary epithelial cell cultures for the study of mammary gland functions

Generation of transgenic fibroblasts producing doxycycline-inducible human interferon-α or erythropoietin for a bovine mammary bioreactor

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