ISOLATION AND ANALYSIS OF DIFFERENT SUBPOPULATIONS OF NORMAL HUMAN BREAST EPITHELIAL-CELLS EARLY AFTER THEIR INFECTION WITH A RETROVIRAL VECTOR ENCODING A CELL-SURFACE MARKER

The use of gene transfer procedures has greatly facilitated the invest igation of cell lineage relationships and other developmental processe s in a variety of primary tissues. In this report we describe the infe ction and selection of primary human breast epithelial cells using ret roviral vectors (Jzen-HSA-NEO and MSCV-HSA.NEO) containing the complet e 228 bp coding sequence of a murine cell surface marker (Heat Stable Antigen, HSA) as well as the neomycin resistance (neo(r)) gene. Expres sion of the transduced HSA gene was detectable using either flow cytom etry or immunohistochemistry after staining infected cells with an ant imurine HSA-specific antibody (M1/69). Expression of the transduced ne o(r) gene conferred resistance to G418. In initial experiments with th e MCF-7 breast cancer cell line, continued expression of both markers was demonstrated in a high proportion of cells for at least 4 weeks af ter their infection by positive M1/69 staining of cells that had been selected by prior incubation in G418. Evidence of gene transfer to ear ly stage (< 9 days old) primary cultures of normal human breast epithe lial cells (15 experiments with cells from 12 normal individuals) was also obtained using an infection protocol in which these cells were ex posed to helper-free viral supernatants (2 incubations, 4 to 6 hr each ) after being subcultured for 12 to 18 hr to increase their rate of pr oliferation, The presence of 5-50 % (mean = 26 %) HSA(+) cells was dem onstrated in these experiments within 5 days after their infection and the HSA(+) populations included both myoepithelial and luminal phenot ypes. The transduced (HSA(+)) cells within both of these subpopulation s could also be separately isolated by FAGS and subcultured, These res ults should provide an important starting point for future studies of genetically modified or marked primary human breast epithelial cell po pulations.