A temperature-sensitive mutant of the myeloproliferative sarcoma virus, altered by a point mutation in the mos oncogene, has been modified as a selectable retroviral vector.

Abstract

The myeloproliferative sarcoma virus (MPSV) is a mos-oncogenic retrovirus which induces an acute myeloproliferative disease in adult mice. The isolation and molecular cloning of two mutants of MPSV temperature sensitive (ts) for mos transformation (Kollek et al., J. Virol. 50:717-724, 1984) have been described previously. In this report, we describe the biological activity of these clones, the molecular basis of the ts lesion of one clone, and the construction of a selectable vector based on the MPSV ts genome. Both molecular clones, ts159 and ts124, proved to have retained the ts phenotype, the former being tighter for the induction and maintenance of the transformed phenotype. A single transition (G----A) at position 1888 in the mos coding region, resulting in the change of Gly to Arg at position 307, was responsible for the ts phenotype of clone ts159. Substitution of sequences carrying this mutation with the corresponding sequences of the wild-type virus generated a virus that was ts for transformation. Insertion of the dominant selectable marker gene for geneticin resistance (neor) into ts159 did not disrupt mos expression or its ts phenotype. neor-ts159 facilitates the study of mos action by allowing the selection of infected cells at the nonpermissive temperature before mos transformation has been induced. Furthermore, infected cells which show no obvious phenotype alteration due to mos expression can be identified by their Neor phenotype.