title: A simple PCR-based strategy for estimating species-specific contributions in chimeras and xenografts.
abstract: Many tissue-engineering approaches for repair and regeneration involve transplants between species. Yet a challenge is distinguishing donor versus host effects on gene expression. This study provides a simple molecular strategy to quantify species-specific contributions in chimeras and xenografts. Species-specific primers for reverse transcription quantitative real-time PCR (RT-qPCR) were designed by identifying silent mutations in quail, duck, chicken, mouse and human ribosomal protein L19 (RPL19). cDNA from different pairs of species was mixed in a dilution series and species-specific RPL19 primers were used to generate standard curves. Then quail cells were transplanted into transgenic-GFP chick and resulting chimeras were analyzed with species-specific primers. Fluorescence-activated cell sorting (FACS) confirmed that donor- and host-specific levels of RPL19 expression represent actual proportions of cells. To apply the RPL19 strategy, we measured Runx2 expression in quail-duck chimeras. Elevated Runx2 levels correlated with higher percentages of donor cells. Finally, RPL19 primers also discriminated mouse from human and chick. Thus, this strategy enables chimeras and/or xenografts to be screened rapidly at the molecular level.
authors.affiliation: Department of Orofacial Sciences, University of California at San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA.
authors.forename: Erin L
authors.initials: EL
authors.lastname: Ealba
authors.forename: Richard A
authors.initials: RA
authors.lastname: Schneider
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002645:Chickens; D002678:Chimera / Q000235:genetics*; D017931:DNA Primers; D004372:Ducks; D006801:Humans; D051379:Mice; D016133:Polymerase Chain Reaction; D011784:Quail; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012269:Ribosomal Proteins / Q000235:genetics*; D013045:Species Specificity; D014183:Transplantation, Heterologous
chemical_list: D017931:DNA Primers; D012269:Ribosomal Proteins
keywords: Quail-chick chimeras; RPL19; Species-specific primers