Direct Comparison of the Insulating Properties of Two Genetic Elements in an Adenoviral Vector Containing Two Different Expression Cassettes

Targeted gene expression can be achieved through the use of cell-selective promoters. However, when the expression cassette is delivered by an adenovirus, "promoter interference," resulting in the loss of specificity, has been reported. To overcome this problem, insulator elements (the bovine growth hormone transcription stop signal or HS4 chromatin insulators of the chicken β-globin locus) have been used. The present study examines the efficacy of these insulators elements, when two independent expression cassettes (one in which a strong, ubiquitous promoter drives the expression of the green fluorescent protein and another in which the"cancer-selective" ERBB2 promoter drives the expression of the herpes simplex virus thymidine kinase [HSVtk] gene) are incorporated into the same recombinant adenovirus. As expected, the presence of either insulator does not alter the expression of HSVtk in ERBB2-positive cells, measured through sensitization of the cells to ganciclovir. When ERBB2-negative cells were infected at a multiplicity of infection (MOI) of 10, the uninsulated virus sensitized ERBB2-negative cells to the same extent as it did for ERBB2-positive cells; partial sensitization was observed when transcriptional terminators were used, indicating a partial insulating effect; and complete insulation (no sensitization to ganciclovir) was observed when HS4 chromatin insulators were used. However, this complete insulation was lost when the MOI of virus was increased to 100. Our study demonstrates the possibility of insulating a conditionally expressed transgene in the vicinity of another expression cassette, but this insulating effect is lost when the multiplicity of infection is increased.