Fusion and Hybridization of Marsupial and Eutherian Cells V. Development of Selective Systems

Abstract

The availability of systems which permit the selective elimination of marsupial cells from fused cultures is an essential requirement for the production of marsupial x eutherian somatic cell hybrids. Such hybrids have particular advantages for genetic studies of mammalian cells. We describe the isolation and characterization of several drug-resistant marsupial cell strains. We have selected strains resistant to concentrations of 10 p,g/ml of the purine analogues 8-azaguanine and 6-thioguanine. Several of these strains were found to be deficient in the enzyme hypoxanthine phosphoribosyltransferase and consequently sensitive to hypoxanthine--aminopterin-thymidine (HAT) selective medium. We have also isolated marsupial cell strains resistant to concentrations of 22p,g/ml of the thymidine analogue 5-bromodeoxyuridine. These strains were thymidine kinase deficient and HAT sensitive. Drug resistance was a stable characteristic maintained for many generations in the absence of the drug. However, inhibition of growth of these drug-resistant strains was strongly density dependent, a factor that caused difficulties in the selection of hybrids. We have also developed selective systems which exploit differences between marsupial and eutherian cells in sensitivity to growth in ouabain, and in adhesiveness and other growth properties. Marsupial cells were found to be naturally much more sensitive to ouabain than rodent cells, a phenomenon that should be useful in the selection of marsupial x rodent cellular hybrids. We discuss a number of difficulties associated with the derivation and use of variant marsupial cell strains.