title: 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.
authors.affiliation: Department Medicine, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Carol M
authors.initials: CM
authors.lastname: Bruzzone
authors.forename: John D
authors.initials: JD
authors.lastname: Belcher
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Schuld
authors.forename: Kristal A
authors.initials: KA
authors.lastname: Newman
authors.forename: Julie
authors.initials: J
authors.lastname: Vineyard
authors.forename: Julia
authors.initials: J
authors.lastname: Nguyen
authors.forename: Chunsheng
authors.initials: C
authors.lastname: Chen
authors.forename: Joan D
authors.initials: JD
authors.lastname: Beckman
authors.forename: Clifford J
authors.initials: CJ
authors.lastname: Steer
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Vercellotti
mesh_terms: D054458:Amplified Fragment Length Polymorphism Analysis / Q000379:methods*; D000755:Anemia, Sickle Cell / Q000235:genetics / Q000628:therapy*; D000818:Animals; D004195:Disease Models, Animal; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D015971:Gene Expression Regulation, Enzymologic; D022761:Gene Transfer, Horizontal; D015316:Genetic Therapy; D005822:Genetic Vectors; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics* / Q000378:metabolism; D051547:Heme Oxygenase-1 / Q000235:genetics* / Q000378:metabolism; D022781:Hepatocytes / Q000201:enzymology; D008297:Male; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D012150:Polymorphism, Restriction Fragment Length; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D019076:Transgenes
chemical_list: D008565:Membrane Proteins; D012333:RNA, Messenger; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; C496542:Hmox1 protein, mouse; C491523:Hmox1 protein, rat