We designed primers from areas of concordance such that the same primer pair amplifies both mouse and rat HO-1 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 the transgene (rat), but not the host (mouse). The ApaI restriction enzyme recognizes the palindrome GGGCCC. The mouse sequence at the same location is GGGCCT which is not recognized by ApaI. Digestion of rat HO-1 cDNA with ApaI generates fragments of 92 and 120 bp. The mouse HO-1 cDNA remains undigested by ApaI with a length of 212 bp.
Mouse and rat DNA sequence differences provide an opportunity to distinguish rat from mouse HO-1 by RFLP
In data not shown, RNA isolated from rat and mouse livers had 11.8- and 17.6-fold more GAPDH than HO-1, respectively, by qRT-PCR. In contrast, rat and mouse spleens had approximately equal quantities of HO-1 and GAPDH by qRT-PCR. The HO-1 qRT-PCR products from rat and mouse liver could be distinguished after digestion with an ApaI (). Both mouse and rat cDNA generate 212 bp PCR products without ApaI digestion. The mouse HO-1 cDNA remains 212 bp after ApaI digestion, but the rat HO-1 cDNA generates equimolar fragments of 92 and 120 bp.
RFLP analysis distinguishes RT-PCR products of HO-1 from rat and mouse
To further test the method in vitro, murine AML12 hepatocytes were transfected with a pORF rat HO-1 plasmid. RNA was isolated from AML12 cells after 40 h. HO-1 and GAPDH cDNAs were made by qRT-PCR. In data not shown, non-transfected AML12 cells demonstrated 3-fold more GAPDH mRNA than HO-1 mRNA by qRT-PCR. In contrast, AML12 cells transfected with the rat HO-1 transgene demonstrated 1.5-fold higher GAPDH mRNA than HO-1, consistent with a doubling in HO-1 transcription in HO-1 transfected cells. RFLP analysis was used to determine the relative contributions of rat HO-1 transgene and host mouse/AML12 HO-1. Both rat and mouse HO-1 bands can be seen at 212, 120 and 92 bp in ApaI digested cDNA from cells transfected with rat HO-1 (, lane 1). The mouse product is 212 bp and the rat products are 92 and 120 bp. AML12 cells transfected with an irrelevant transgene (lane 2) and non-transfected AML12 cells (lane 3) display only the mouse HO-1 band at 212 bp. Control HO-1 cDNA made from rat liver RNA and digested with ApaI displays only the rat bands at 92 and 120 bp (lane 4). Using the bands in lane 1, we calculated the rat transgene comprised 84.3% of HO-1 cDNA and the host HO-1 comprised 15.7%. The irrelevant transgene (lane 2) did not induce the endogenous mouse HO-1. A Western blot of HO-1 demonstrates the presence of an HO-1 band at 32 kDa (). AML12 cells transfected with a rat HO-1 plasmid had 2.3-fold more HO-1 protein expression than cells transfected with an irrelevant gene.
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
To demonstrate applicability of the qRT-PCR RFLP method to monitoring gene transfer in vivo
, a Sleeping Beauty
-Tn), was utilized to deliver the rat HO-1 gene to S+S-Antilles sickle mice by hydrodynamic injection (15
). Control mice were injected with LRS containing no DNA. Mouse livers were removed and frozen for analysis either 96 hours or 21 days after injection. In data not shown, qRT-PCR was used to measure GAPDH/HO-1 mRNA ratios in mouse liver RNA. After 96 hours and 21 days, respectively, there was a 1.2- and 1.3-fold enrichment of HO-1 mRNA in mice injected with SB
-Tn-HO-1 DNA relative to LRS control mice. RFLP analysis of mouse liver HO-1 cDNA showed the rat transgene comprised 82 - 88% of HO-1 cDNA (92 and 120 bp) and the host HO-1 (212 bp) comprised 12 - 18%. after 96 hours and 21 days, respectively, in the mice injected with SB
-Tn-HO-1 (). There was no rat HO-1 cDNA seen on the agarose gel in the mice injected with LRS only or hemin. The mouse injected with 12.5 μg of SB
-Tn-HO-1 plasmid DNA showed 2.6-fold greater HO-1 protein expression in liver after 96 hours on Western blot relative to a control mouse injected with LRS only (). Similarly, a mouse injected with 25 μg of SB
-Tn-HO-1 plasmid DNA had 2.5-fold greater HO-1 protein expression in liver after 21 days than a control mouse injected with LRS (). A mouse injected with hemin, a potent inducer of HO-1, showed 2.1-fold greater HO-1 protein expression in liver after 16 hours relative to a control mouse injected with LRS at 96 hours ().
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