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Gene transfection and expression in a primary culture of mammary epithelial cells isolated from lactating sows
Author(s) -
Sun Y.L.,
Lin C.S.,
Chou Y.C.
Publication year - 2005
Publication title -
cell biology international
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.932
H-Index - 77
eISSN - 1095-8355
pISSN - 1065-6995
DOI - 10.1016/j.cellbi.2005.03.021
Subject(s) - transfection , biology , green fluorescent protein , microbiology and biotechnology , gene expression , gene , reporter gene , expression vector , mammary gland , transgene , promoter , recombinant dna , biochemistry , genetics , cancer , breast cancer
Porcine mammary epithelial cells (PMECs) were isolated from lactating sow mammary glands and cultured on a matrix gel. Primary culture cells expressed significant amounts of the specific marker cytokeratin as determined by immunohistochemistry, and exhibited mammary‐specific functions, such as transcription of α‐lactalbumin, β‐casein and β‐lactoglobulin genes. They also formed mammospheres when the medium was supplemented with lactogenic hormones. The PMECs were used to study gene transfer and expression in vitro. A gene encoding enhanced green fluorescent protein (EGFP) was used as a reporter and two constructs were investigated, pEGFP‐N1 (a vector constructed with a CMV promoter followed by the EGFP gene) and pGB562/GFP (a mammary gland‐specific expression vector with regulatory sequences from the goat β‐casein gene linked to EGFP). The efficiency of DNA transfer into the cultured PMECs was about 20–30%. GFP expression in the pGB562/GFP‐transfected PMECs was markedly stimulated by prolactin supplements in the medium. The established PMECs maintained optimal gene expression from 1 to 20 passages and appeared to provide an efficient and convenient system for assessing the expression of transgenes containing mammary gland‐specific promoters.