Gene transfection and expression in a primary culture of mammary epithelial cells isolated from lactating sows.
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.
Sun
Y L
YL
Division of Biotechnology, Animal Technology Institute Taiwan, P.O. Box 23, Chunan 350, Miaoli, Taiwan, ROC.
Lin
C S
CS
Chou
Y C
YC
D000818:Animals; D002364:Caseins / Q000235:genetics / Q000378:metabolism; D018929:Cell Culture Techniques; D002460:Cell Line / Q000502:physiology*; D003094:Collagen; D004338:Drug Combinations; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005260:Female; D015870:Gene Expression / Q000502:physiology*; D007150:Immunohistochemistry; D007633:Keratins / Q000032:analysis; D007768:Lactalbumin / Q000235:genetics / Q000378:metabolism; D007774:Lactation / Q000502:physiology*; D007782:Lactoglobulins / Q000235:genetics / Q000378:metabolism; D007797:Laminin; D008321:Mammary Glands, Animal / Q000187:drug effects / Q000378:metabolism*; D011388:Prolactin / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011509:Proteoglycans; D013552:Swine / Q000502:physiology*; D014162:Transfection
D002364:Caseins; D004338:Drug Combinations; D007782:Lactoglobulins; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D007633:Keratins; D011388:Prolactin; D003094:Collagen; D007768:Lactalbumin