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

Abstract

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.

Fig 1. Mouse and rat DNA sequence differences provide an opportunity to distinguish rat from mouse HO-1 by RFLP

Primers were made from areas of concordance such that the same primer pair amplifies both native and transgene, generating a single mass product of 212 bp. The PCR products can be distinguished by RFLP. A discordant type II restriction endonuclease site (ApaI) was identified in the cDNA of rat, but not mouse, HO-1 that generates cDNA fragments of 92 and 120 bp in the rat. Species mismatches between rat and mouse are underlined in the mouse DNA sequence. The PCR primers are shown next to the arrows at the 5′ and 3′ ends.

Fig 2. RFLP analysis distinguishes RT-PCR products of HO-1 from rat and mouse

The HO-1 RT-PCR cDNA products from rat and mouse liver can be distinguished after digestion with ApaI restriction enzyme. Without ApaI digestion (-ApaI), both the rat and the mouse generate a cDNA with 212 bp. After digestion with ApaI (+ApaI), the mouse HO-1 cDNA remains 212 bp, but the rat HO-1 cDNA generates fragments of 92 and 120 bp. RFLP reactions contained 400 ng of cDNA PCR products that were incubated at 37°C with (+ApaI) and without (-ApaI) ApaI enzyme. Reaction products were separated by 2% agarose gel electrophoresis with ethidium bromide UV illumination. The data presented are representative of more than 3 experiments.

Fig 3. The rat HO-1 transgene is transcribed in transfected murine AML12 hepatocytes and can be differentiated from mouse HO-1 by RFLP analysis of RT-PCR products of HO-1

(A) Type II restriction endonuclease digestion of HO-1 RT-PCR products with ApaI allows determination of the relative contributions of the rat HO-1 transgene cDNA and the endogenous mouse HO-1 gene. AML12 cells were transfected using lipofectamine with either a rat HO-1 transgene or an irrelevant transgene. RNA was isolated from AML12 cells 40 h after transfection. RT-PCR was used to make HO-1 cDNA. The cDNA was digested with ApaI restriction enzyme. The resulting products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. Lane 1 is loaded with cDNA from murine AML12 hepatocytes transfected with a rat HO-1 transgene. Lane 2 contains cDNA from AML12 hepatocytes transfected with an irrelevant transgene. Lane 3 contains cDNA from non-transfected AML12 cells. Lane 4 contains control HO-1 cDNA from rat liver. (B) AML12 cells transfected with a rat HO-1 transgene had more HO-1 protein expression on a Western blot than cells transfected with an irrelevant gene or non-transfected cells. Lanes 1-4 of the Western blot, transfected as described in (A), were loaded with 22 μg of AML12 protein. Lane 5 contains recombinant rat HO-1 protein (400 ng). The data presented are representative of 2 experiments.

Fig 4. The rat HO-1 transgene is transcribed in mouse liver and can be differentiated from mouse HO-1 by RFLP analysis of HO-1 cDNA

A Sleeping Beauty transposase (SB-Tn) was utilized to deliver the rat HO-1 gene to S+S-Antilles sickle mice. The SB-Tn plasmid containing the rat HO-1 gene was delivered by hydrodynamic injection of 12.5 μg or 25 μg of SB-Tn-HO-1 plasmid DNA in 2.5 ml of sterile lactated Ringers solution (LRS) into the tail veins of the mice. Control sickle mice were injected with LRS containing no DNA. After either 96 hours (12.5 μg DNA injected) or 21 days (25 μg DNA injected), livers were harvested and RNA was isolated for RT-PCR RFLP analysis of HO-1 transcription and microsomes were isolated for Western blot analysis of HO-1 protein expression. (A) RFLP digestion with ApaI of the HO-1 cDNA from these livers shows an enrichment of rat HO-1 cDNA (92 and 120 bp) relative to mouse HO-1 cDNA (212 bp) in the mice injected with SB-Tn-HO-1 at both 96 hours and 21 days after injection. There is only mouse HO-1 cDNA (212 bp) in the mice injected with LRS only and the mouse injected with hemin. Each lane in (A) was loaded with 100 μg of ApaI-digested HO-1 RT-PCR cDNA. The bar graph in (A) summarizes the RFLP data (n=3 gels/sample) and shows the mean (± SD) percentage of rat HO-1 transgene in mouse liver HO-1 RNA. (B) Western blots for HO-1 expression in these livers shows mice injected with SB-Tn-HO-1 express more HO-1 in the liver at both 96 hours and 21 days after injection than mice injected with LRS only. Each lane of the Western blot was loaded with 6 μg of liver microsomal protein. Bands for HO-1 (32 kDa) and GAPDH are shown in (B) from a representative Western blot. As a positive control for HO-1 protein expression in the Western, a C57BL/6 mouse was injected with hemin (40 μmol/kg i.p.) for 3 consecutive days and the liver was removed 16 h after the third injection (Hemin). The bar graph in (B) summarizes the Western blot quantitation (n=3 blots/sample) and shows the mean (± SD) HO-1 protein expression in the mouse livers.