Quantitative RT-PCR to evaluate in vivo expression of multiple transgenes using a common intron

An assay measuring RNA expression levels of a gene-encoded therapeutic must distinguish between endogenous mRNA and mRNA transcribed from the transgen e. Specificity for the delivered transgene is especially critical when the treatment involves genes that are expressed in the target tissue. To facili tate uniform detection of transgene RNA without interference from endogenou s mRNA, we have engineered expression vectors that include a 5' untranslate d region (5' UTR) containing a synthetic intron (PGL3). The synthetic intro n splice junction was the target sequence for a quantitative reverse transc ription CRT)-PCR assay utilizing Taq-Man(R) technology. In this study, we d emonstrate that a quantitative RT-PCR assay designed to recognize an engine ered intron splice site in the 5' UTR of expression constructs effectively measures the expression level of in vivo-delivered gene therapeutics.