VERSATILE RETROVIRUS VECTOR SYSTEMS FOR REGULATED GENE-EXPRESSION IN-VITRO AND IN-VIVO

Background: Several plasmid DNA-based mammalian expression systems hav e recently been developed which make it possible to manipulate gene ex pression ria the administration of exogenous agents. In order to exten d the application of these systems, we have developed retroviral vecto rs which allow for the controlled expression of inserted genes both in vitro and in vivo. Materials and Methods: Two vector strategies which make use of the tetracycline-regulated gene expression system describ ed by Gossen and Bujard were evaluated. In a first strategy, one virus was generated which encoded the tTa or rtTA transactivator gene produ ct, and a second virus was generated in which expression of the gene o f interest was dependent upon tetracycline-responsive transcriptional control elements placed either within the viral LTR or within the prov iral transcriptional unit. In a second vector strategy, both component s of the tet-regulatable system were incorporated into a single provir al genome in such a way that expression of both the transgene and the transactivator gene product were under control of tet-regulatable cont rol elements. Results: Both vector strategies resulted in the ability to regulate the expression of inserted genes. In one single virus conf iguration, gene expression could be regulated over 100x and the level of gene expression in the induced state was comparable to or greater t han that achieved with standard LTR-based vectors. The use of differen t deletions in the viral LTR made it possible to generate a number of vectors which provide for a fourfold range of levels of expression of inserted genes in tile induced state. Studies in mice with transduced cells demonstrated that gene expression could be induced in vivo by ma nipulation of tetracycline for at least 48 days. Conclusions: The avai lability of highly transmissible, regulatable retroviral vectors shoul d greatly facilitate studies in which it is of interest to manipulate the expression of specific genes in vitro or in vivo.