Establishment of permanent brown adipocyte cell lines achieved by transfection with SV40 large T antigen and ras genes.

We established multiple clonal cell lines by transfection of primary rat fetal brown adipocytes (BAT) with constitutive or inducible gene constructs of SV40 large T antigen (SVLTag) alone or in combination with transforming or normal ras genes. While primary BAT cell cultures stop growing and expressing uncoupling protein (UCP) after several passages in culture, our transfected cell lines could be propagated indefinitely and kept properties specific of BAT cells. Interestingly, cells transfected with ras genes alone did not survive as permanent cell lines. In contrast, cells transfected with SVLTag alone or in combination with various ras genes could readily be established as continuous cell lines. BAT cells transfected with SVLTag alone showed fibroblastic, flat morphology and clear contact inhibition of growth. In contrast, cells cotransfected with SVLTag and ras genes were more fusiform and possessed increased proliferative capacity. Thus, cells cotransfected with normal ras reached higher cell density at confluency, while cells cotransfected with transforming ras were clearly refractile, lost contact inhibition of growth, and presented a transformed phenotype. These results are consistent with the notion that ras genes are inefficient at overcoming senescence of primary BAT cells and can only confer improved proliferative ability to cells that have already been immortalized by a cooperating SVLTag gene. RNA analysis showed that most transfected cell lines expressed UCP along with the exogenously transfected genes. This demonstrated that genuine BAT cells, and not other possible contaminating cell lineages, gave rise to our selected cell lines. The variety of cell lines generated may correspond to various stages of progression through BAT development and therefore be useful to characterize signaling pathways and metabolic regulation of this particular tissue.