Isolation, characterization, and EGFP expression in the buffalo (Bubalus bubalis) mammary gland epithelial cell line

Abstract

This study was aimed to establish a buffalo mammary epithelial cells (BuMECs) line and maintain it for long-term by subculturing. BuMECs isolated from lactating buffalo mammary glands were cultured on a collagen matrix gel. BuMECs expressed significant amounts of the epithelial cell specific marker cytokeratin 18 as determined by immunohistochemistry. The BuMECs displayed monolayer, cobble-stone morphology, and formed lumen-, dome-, and duct-like structures. Furthermore, they were capable of synthesizing CSN2, BLG, ACACA, and BTN1A1, showed viability after thawing and expressed milk protein genes. The enhanced green fluorescent protein gene was transferred successfully into the BuMECs using lipofection method and the transfected cells could be maintained for long-term in culture by subculturing.

Differentiation dynamics of mammary epithelial stem cells from Korean holstein dairy cattle under ECM-free conditions

“…The RT-PCR results (Figure 12(A) and (B)) revealed the expression of all genes in isolated and cultured cells. Previous studies have also used the RT-PCR and Western blotting for the study of β-casein in cultured cells (Hu et al, 2009;Kaushik et al, 2013). Our Western blot results also showed the expression of β-casein in the cultured KHMESCs ( Figure 13).…”

“…Earlier studies have also reported the expression of some of these genes in bovines (Hu et al, 2009;Kaushik et al, 2013;Monzani et al, 2011). BTN1A1 is a milk fat globule transmembrane glycoprotein and mammary-specific protein in milk fat secretion, which dominantly expressed on the mammary epithelial cells particularly during final stage of pregnancy and lactation (Fox & McSweeney, 2006;Szyda, & Komisarek, 2007).…”

“…BTN1A1 is a milk fat globule transmembrane glycoprotein and mammary-specific protein in milk fat secretion, which dominantly expressed on the mammary epithelial cells particularly during final stage of pregnancy and lactation (Fox & McSweeney, 2006;Szyda, & Komisarek, 2007). ACACA (a cytosolic enzyme in the lactating mammary gland) (Larson & Smith, 1974) is also used as a marker of mammary epithelial cell (Kaushik et al, 2013) and provides cytosolic malonyl-CoA for fatty acid synthesis. Previous findings have confirmed that insulin is not essential only for the genes expression of the milk protein, but also stimulates and regulates milk protein synthesis (Menzies, Lefevre, Macmillan, & Nicholas, 2009).…”

“…The most recent methodologies, viz. morphology, histopathology (Alkafafy, Rashed, & Helal, 2012;Kaushik et al, 2013;Safayi et al, 2012), bromodeoxyuridine retention (Capuco, 2007), Hoechst dye-effluxing to identify side population (SP) properties and surface markers by flow cytometer or immunochemistry (Rauner & Barash, 2012;VanKeymeulen et al, 2011), are being used for the characterization of bovine mammary epithelial stem/progenitor cells. We isolated the Korean Hosltein mammary epithelial stem cells (KHMESCs) from Korean Holstein mammary tissue using collagenase.…”

Stem Cell Research: A Novel Boulevard towards Improved Bovine Mastitis Management

“…Delineation of mammary epithelial cell hierarchy, as perceived today, consists of MaSCs giving rise to uni-potent luminal-restricted progenitors that, in turn, differentiate into alveolar myoepithelial or secretory cells. According to recent studies, some in vitro and in vivo methods are currently being utilized to isolate, characterize and study stemness and progenitor activity of bovine putative mammary stem/progenitor cells, including morphology, histopathology 42, 56-59, bromodeoxyuridine retention 50, Hoechst dye-effluxing to identify side population (SP) properties and surface markers by flowcytometer or immunochemistry 23, 42.…”