Abstract
A novel fusion gene has been created in which the expression of a dominant selectable marker, the human multidrug resistance gene, is directly linked to the expression of human adenosine deaminase cDNA. The chimeric gene was inserted between the long terminal repeats of a Harvey murine sarcoma virus expression vector and used to transfect drug-sensitive human KB carcinoma cells. Transfectants were selected in increasing concentrations of colchicine and found to contain multiple copies of the intact fusion gene, which is stably and efficiently expressed. A membrane-associated 210-kDa human P-glycoprotein-adenosine deaminase fusion protein is synthesized which retains function of the multidrug transporter and also exhibits adenosine deaminase activity. The data indicate that the human multidrug resistance gene may be used as a dominant selectable marker to introduce other genes in the form of gene fusions into cultured cells.
Highlights
The creation of a functional MDR-ADA hybrid would be of potential use in gene therapy of this disorder
We report the DNA-mediated transfer of the chimeric gene into drug-sensitive human KB cells, which upon selection with colchicine produce a membrane-associated P-glycoprotein-ADA fusion protein. This protein confers the full phenotype of multidrug resistance to the cells andexhibits ADA activity which resultsin 2’deoxycoformycin resistance in the presence of toxic amounts of adenosine
Fusion Gene-During the last few years, many novel fusion proteins have been created by genetic engineering and shown to preserve the biological function and activity of their constituents (e.g. Mas et al, 1986;Williams and Neuberger, 1986; Chaudhary et al, 1987;Lorberboum-Galski et al, 1988).a functional protein has not previously been fused to a dominant selectable marker for use in gene transfer
Summary
Fusion Gene-During the last few years, many novel fusion proteins have been created by genetic engineering and shown to preserve the biological function and activity of their constituents (e.g. Mas et al, 1986;Williams and Neuberger, 1986; Chaudhary et al, 1987;Lorberboum-Galski et al, 1988).a functional protein has not previously been fused to a dominant selectable marker for use in gene transfer. We have designed a fusion protein involving human P-glycoprotein, the gene product of the multidrug resistance gene ( M D R l ) , and human ADA. Negative control cells received no DNA whereas positive control cells were transfected with pHaMDR (Ueda et al, 1987), which contains the full length human M D R l cDNA in the same Harvey murine sarcoma virus expression vector (see Fig. 1C). This plasmid had earlier been shown to confer the full phenotype of multidrug resistance to mouse and human cell lines (Ueda et al, 1987).
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