Gene transfection and expression in a primary culture of mammary epithelial cells isolated from lactating sows

Abstract

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 alpha-lactalbumin, beta-casein and beta-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 beta-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.