Transfection of Adipocytes and Preparation of Nuclear Extracts

The uniqueness of the adipose cell type must be considered before choosing a procedure for gene transfer into mature adipocytes. Because adipocytes isolated from adipose tissue (AT) are filled with so many lipids that cells cannot be plated on a culture support, because of their low density, and because such terminally differentiated cells do not divide, the procedure of choice for introducing DNA into fat cells is electroporation. The detailed protocol described below has been published in references (1 ,2 ). One must keep in mind however, that primary adipocytes isolated from the animal have a limited life-span in culture (not exceeding 1 wk), so that only transient transfection can be envisaged in such a system. When stable transfectants are needed, other cellular models must be used, such as established preadipose cell lines. Among the most popular, 3T3-L1, 3T3-F442A, and Ob17 can also be transfected by electroporation (3 ). A protocol adapted to these situations is also given. These techniques are simple to perform, but require special equipment for electric shock delivery. Finally, it is important to realize that only a small proportion of the cell population will incorporate foreign DNA, using these transfection procedures. High-efficiency gene transfer in mature adipocytes, using recombinant adenoviral systems is also possible (3 ,4 ), but will not be described here.