Gene expression profiling of porcine cells and tissues by microarray analysis

Pig to human xenotransplantation represents an ambitious venture that requires, besides evasion of rejection mechanisms and physiological incompatibilities, the generation of pathogen‐free pigs as donors for well characterized xenografts to warrant medicinal products that do comply with statutory regulatory demands [1–3]. The publication of a high quality draft sequence for the pig genome (Sus scrofa) and a series of accompanying reports for the first time offered the feasibility of whole genome expression profiling of porcine tissues and cells [4–8]. The SFB TR CRC 127 project Z2 “Microbiological Safety including Virological Safety” is based on microbial profile analysis of porcine tissues in order to prevent zoonotic events, including infection by porcine endogenous retroviruses (PERV) [9]. The project comprises the detection and characterization of potential pathogens as well as the investigation of the microbial influence on the transcriptional status of tissues and cells. Hence, specific expression patterns, e.g. up‐regulation of antiviral host factors or cell cycle/apoptotic regulators, may also provide information on ongoing or precedent events that may have impact on tissues/cells quality and therefore its suitability as xenografts. We use microarray technology for monitoring viability of tissues and cells as well as their microbial/viral status. An Agilent based, 60K DNA microarray representing 25,415 different genes of the recently published Sus scrofa genome (NCBI Sus scrofa 10.2‐assembly) was generated [10]. The microarray was specified for German Landrace and Göttingen Minipig, amongst other pig species, by hybridizing complex RNA samples generated from five different pig organs and blood as well as chromosomal porcine DNA to highlight non expressed genes. Four Diagnostic PERV sequences for pro/pol (all classes of PERV), env (to differentiate between PERV‐A, ‐B and ‐C) as well as 15 human transgenes such as CD59 (human complement regulatory protein), DAF (Decay accelerating factor or CD55), human A20 (hA20) and others were included. In total, the microarray displays 25,434 genes each represented by up to three different 60‐mer oligonucleotides. To reveal functionality of the microarray the transcriptional status of ST‐IOWA cells freshly infected with molecularly cloned virus PERV‐C (1312) [11] was monitored. Total mRNA levels at day 7, 28 and 56 post infection were compared with naive uninfected cells. All samples were tested in triplicates and the relative signal intensity of hybridized probes was compared. Special attention was given to antiviral host factors such as APOBEC and tetherin of which involvement as antiviral factors on PERV expression has been demonstrated [12–14]. Constitutively expressed housekeeping genes, i.e. porcine glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), beta actin and cyclophilin A, respectively, were used as controls [15, 16]. The presented microarray supports the safety and quality by monitoring the transcriptional status of xenotransplants. References [1] EMEA/CHMP/CPWP/83508/2009. Guideline on Xenogeneic Cell‐Based Medicinal Products. [2] U.S. Department of Health and Human Services. Food and Drug Administration. Center for Biologics Evaluation and Research (CBER). February 2002. Draft Guidance for Industry: Precautionary Measures to Reduce the Possible Risk of Transmission of Zoonoses by Blood and Blood Products from Xenotransplantation Product Recipients and Their Intimate Contacts. [3] U.S. Department of Health and Human Services. Food and Drug Administration. Center for Biologics Evaluation and Research (CBER). April 2003. 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