In Vitro Cell. Dev. Biol.--Animal 34:326-332, April 1998 © 1998 Societyfor In Vitro Biology 1071-2690/98 $05.00 + 0.00 ESTABLISHMENT AND INITIAL CHARACTERIZATION OF THE OVINE MAMMARY EPITHELIAL CELL LINE NISH NETA ILAN,ITAMAR BARASH,ELISHAGOOTWINE, AND MOSHESHANI 1 Institute of Animal Science, The Volcani Center, Bet Dagan 50250, Israel (Received 22 October 1996; accepted 28 August 1997) SUMMARY Analysis of the molecular mechanisms involved in the differentiation and formation of the charactoristic three-dimen- sional structures of the developing mammary gland of the major milk-producing livestock (ducts, end buds, and alveoli) requires in vitro model cell cultures. The few cell lines that have been established from dairy animals do not fully reproduce the entire program of mammary differentiation. Here we present the initial characterization of a unique mammary epithelial cell line derived spontaneously from midpregnant sheep (NISH). These cells form in vitro functional structures resembling ducts, lateral buds, and alveoli that secrete ~-lactoglobulin (BLG) in an ECM (extracellular matrix)-dependent manner. Interestingly, the presence of growth hormone dramatically increased BLG secretion from NISH cells cultured on ECM. It appears that GH is required not only to establish the structural organization but also is continuously needed to maintain BLG expression. Stable transfection of NISH ceils with BLG/Human Serum Albumin (HSA) hybrid gene constructs revealed that the relative level of expression was comparable to the in vivo secretion of HSA in transgenic mice carrying these gene sequences. No expression could be detected in ceils transfected with hybrid genes carrying either HSA cDNA or the entire HSA gene, and HSA expression was dependent on the presence of intronic sequences. These results demonstrate that NISH cells may prove a useful tool for studying the differentiation and orgauogenesis of mammary epithelial ceils under defined culture conditions. Furthermore, transfected NISH cells may be an alternative for the transgenic mouse model in evaluating the potential of gene constructs to he efficiently expressed in the mammary gland of transgenic farm animals. Key words: end-buds; alveoli; ~-lactoglobulin; extracellular matrix; transfections; transgenes. INTRODUCTION The mammary gland offers an attractive model for the study of cell proliferation, differentiation, organogenesis, and oncogenesis. Sev- enty years of study has provided little information about the stem cells responsible for forming the structures associated with all stages of the normal morphogenesis of the gland (ducts, end buds, alveoli) (25). Identification and characterization of mammary gland stem cells has important implications regarding the proliferative cell popula- tions in preneoplasias and neoplasias of the mammary gland and are therefore of special interest. Many tissue culture models (organ cultures, primary mammary epithelial cells and cell lines) have been established to further the understanding of the complex biology of the mammary gland (re- viewed in 5,18). For years, the only functional and well-characterized cell line available was the COMMA-1D cell line, originating from normal pregnant mice (5,7,25). When transplanted into gland-free mammary fat pads, these ceils were capable of producing normal mammary ducts, end buds, and alveoli. However, after Passage 11 these cells produced alveolar hyperplasias and mammary tumors fol- lowing transplantation into gland-free fat pads (25). The COMMA- 1D cell line, its subpopulations, and cell clones were used exten- sively to study the effect of lactogenic hormones and extracellular matrix (ECM) on milk protein gene expression (27). As these cells ~Towhomcorrespondenceshould be addressed. form only alveoli-like structures and not ducts or end buds when grown on an in vitro matrix, they cannot be used to study duct for- mation and branching. Although many other mammary epithelial cell lines have been established from human and rodents and added to our knowledge of mammary gland physiology, very few cell lines are available from the major milk producers, the farm animals. The es- tablishment and study of mammary epithelial cell lines from farm animals is of special interest because the rodent model does not accurately reflect udder development and physiology (1). No mam- mary epithelial cell lines have been established from sheep or goats, and only four bovine cell lines are currently available. These bovine lines show no (HH2A and BMEC + H) or low (BME UV) casein syn- thesis that was only marginally regulated by collagen (30). MACT-T cells express varying levels of casein, the expression of which is collagen dependent but prolactin independent (16,30). These cell lines are therefore unsuitable for the study of the effect either of the hormonal regulation of bovine casein expression or the effect of ECM on milk protein gene expression. Here we report the establishment of a functional ovine mammary epithelial cell line (NISH) which may represent the stem ceils of the mammary gland. When plated in or on top of an appropriate ECM, NISH cells secrete [3-1actoglobulin (BLG) and are capable of forming structures resembling ducts, lateral buds, and alveoli. Moreover, sta- ble transfection of these cells with BLG/HSA hybrid gene constructs revealed that the relative level of expression resembled the in vivo 326