A New Family of Murine Retroviral Vectors with Extended Multiple Cloning Sites for Gene Insertion

Abstract

Murine retroviral vectors with multiple unique cloning sites in the body and 3′ long terminal repeat (LTR) are described. The various alterations to the vectors include changing the gag+ start codon (AUG) to a stop codon (UAA), a deletion of 468 bp from the envelope region, and an additional 387-bp deletion of the promoter and enhancer sequences from the 3′ LTR. Multiple cloning sites in the body and 3′ LTR facilitate double-copy vector construction. The hygromycin resistance and luciferase genes were subcloned into the body and 3′ LTR to evaluate effects of vector modifications and effects of insert location (body vs. LTR and same orientation vs. reverse orientation with respect to the vector LTRs) on virus titer. The results indicate the modifications or insert position do not negatively influence potential vector titer and expression capacity. The described vectors have potentially useful characteristics for gene therapy studies. To develop a new family of retroviral vectors with the best features of earlier vectors, several changes were made in a Moloney murine leukemia virus (MuLV) backbone. The hygromycin resistance and firefly luciferase genes were subcloned into the new vectors to assess manipulation effects. The data showed no detrimental effects on the viral titer or expression capacity of the vectors. This new family of vectors has titer and expression properties desirable for gene therapy studies.