Sun Y L, Lin C S, Chou Y C
Division of Biotechnology, Animal Technology Institute Taiwan, P.O. Box 23, Chunan 350, Miaoli, Taiwan, ROC.
Cell Biol Int. 2005 Jul;29(7):576-82. doi: 10.1016/j.cellbi.2005.03.021.
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.
从泌乳母猪乳腺中分离出猪乳腺上皮细胞（PMECs），并在基质胶上进行培养。通过免疫组织化学检测，原代培养细胞表达大量特异性标志物细胞角蛋白，并表现出乳腺特异性功能，如α-乳白蛋白、β-酪蛋白和β-乳球蛋白基因的转录。当培养基中添加催乳激素时，它们还能形成乳腺球。PMECs被用于体外基因转移和表达的研究。编码增强型绿色荧光蛋白（EGFP）的基因用作报告基因，研究了两种构建体，pEGFP-N1（一种由CMV启动子后接EGFP基因构建的载体）和pGB562/GFP（一种具有与EGFP相连的山羊β-酪蛋白基因调控序列的乳腺特异性表达载体）。DNA转入培养的PMECs的效率约为20%-30%。培养基中添加催乳素可显著刺激pGB562/GFP转染的PMECs中GFP的表达。所建立的PMECs在传代1至20次时维持最佳基因表达，似乎为评估含有乳腺特异性启动子的转基因表达提供了一个高效便捷的系统。
