Quantitative real-time polymerase chain reaction (qRT-PCR) restriction fragment length polymorphism (RFLP) method for monitoring highly conserved transgene expression during gene therapy

Evaluation of the transfer efficiency of a rat heme oxygenase-1 (HO-1) transgene into mice requires differentiation of rat and mouse HO-1. However, rat and mouse HO-1 have 94% homology; antibodies and enzyme activity cannot adequately distinguish HO-1. We designed a quantitative real-time polymerase chain reaction (qRT-PCR) method to monitor HO-1 transcription relative to a housekeeping gene, GAPDH. The ratio of rat and mouse HO-1 mRNA could be estimated through restriction fragment length polymorphism (RFLP) analysis of the PCR products. In vitro, murine AML12 hepatocytes were transfected with rat HO-1. After 40 h, the total HO-1 mRNA was enriched 2-fold relative to control cells, and rat HO-1 comprised 84% of HO-1 cDNA. In vivo, the rat HO-1 transgene was cloned into a Sleeping Beauty transposase (SB-Tn) construct and was injected hydrodynamically into a mouse model of sickle cell disease (SCD). After 21 days, there was a 32% enrichment of HO-1 mRNA relative to control mice and the rat transgene comprised 88% of HO-1 cDNA. After 21 days, HO-1 protein expression in liver was increased 2.5-fold. In summary, qRT-PCR RFLP is a useful and reliable method to differentiate the transgene from host gene transcription, especially when the host and transgene protein are identical or highly homologous. This method has translational applications to the design, delivery, and monitoring of gene-therapy vectors.

Abbreviations: Alb, albumin, bp, base pair, DR, direct repeat, GAPDH, glyceraldehyde 3-phosphate dehydrogenase, HO-1, heme oxygenase-1, IR, inverted repeat, LRS, lactated Ringers solution, RFLP, restriction fragment length polymorphism, RT, reverse transcription, SB-Tn, Sleeping Beauty transposase, SCD, sickle cell disease, SFFV, spleen focus-forming virus, qRT-PCR, quantitative real-time polymerase chain reaction