[Expression of human multidrug resistance gene (mdr1) cDNA in murine ES cells and in chimeric mice].

Human multidrug resistance gene (mdr1) was introduced into mouse embryonic stem cells (ES-5 line) by calcium phosphate mediated transfection, and transfected ES-5 cells were then selected by stepwise increase in colchicine concentration (30, 50, 100, and 200 ng/ml respectively). Finally, we obtained 4 clones that could be stably grown in culture medium with colchicine at 200 ng/ml and designated as ES-mdr1 clones A, B, C, and D. Southern blot analysis of DNA from ES-mdr1 A and D cells digested by Hind III and hybridized with mdr1 cDNA 5 A probe was shown in Fig. 3. Characteristic 4.8 and 2.4 kb fragments of mdr1 gene were found as expected and their amplification under increased concentration of colchicine in culture medium was also evident from the figure. Slot blot and Northern analysis of total RNA and poly A+ RNA extracted from ES-mdr1 cells were shown in Fig. 4 and 5, demonstrating that ES-mdr1 cells could express mdr1 mRNA. Indirect immunofluorescence analysis with antibodies against p170 glycoprotein indicated that p170 protein translated from mdr1 mRNA was present at the surface of ES-mdr1 cells (Plate I, Fig. 2). The biological characteristics of ES-mdr1 cells cultured in medium containing 200 ng/ml colchicine were investigated. The cells maintained their undifferentiated morphology and grew in nests (Plate I, Fig. 1), like the parental ES-5 cells. When ES-mdr1 cells were cultured in suspension in vitro, these cells were still capable of producing simple and cystic embryoid bodies. ES-mdr1 cells injected subcutaneously into 129 mice formed tumor-like outgrowths giving a great variety of cell types (Plate I, Fig. 4). These results indicated that the integration and expression of human mdr1 gene and selection against colchicine did not affect the pluripotency of ES-mdr1 cells both in vitro and in vivo. However, ES-mdr1 cells, unlike their parental ES-5 cells, could no longer be induced to differentiate by either RA or HMBA (Plate I, Figs. 3a, 3b), indicating that the human mdr1 gene transfected ES cells had changed their competence of inducible response to differentiation in vitro. The details and possible significance of such change require further studies. From the above preliminary data, we are of the opinion that ES-mdr1 cells may serve as a model to study the mode of action of p170 glycoprotein at cellular level and to screen possible means to counteract the action of mdr1 gene.(ABSTRACT TRUNCATED AT 400 WORDS)