Functional analysis and transcriptional output of the Göttingen minipig genome

Abstract

BackgroundIn the past decade the Göttingen minipig has gained increasing recognition as animal model in pharmaceutical and safety research because it recapitulates many aspects of human physiology and metabolism. Genome-based comparison of drug targets together with quantitative tissue expression analysis allows rational prediction of pharmacology and cross-reactivity of human drugs in animal models thereby improving drug attrition which is an important challenge in the process of drug development.ResultsHere we present a new chromosome level based version of the Göttingen minipig genome together with a comparative transcriptional analysis of tissues with pharmaceutical relevance as basis for translational research. We relied on mapping and assembly of WGS (whole-genome-shotgun sequencing) derived reads to the reference genome of the Duroc pig and predict 19,228 human orthologous protein-coding genes. Genome-based prediction of the sequence of human drug targets enables the prediction of drug cross-reactivity based on conservation of binding sites. We further support the finding that the genome of Sus scrofa contains about ten-times less pseudogenized genes compared to other vertebrates. Among the functional human orthologs of these minipig pseudogenes we found HEPN1, a putative tumor suppressor gene. The genomes of Sus scrofa, the Tibetan boar, the African Bushpig, and the Warthog show sequence conservation of all inactivating HEPN1 mutations suggesting disruption before the evolutionary split of these pig species. We identify 133 Sus scrofa specific, conserved long non-coding RNAs (lncRNAs) in the minipig genome and show that these transcripts are highly conserved in the African pigs and the Tibetan boar suggesting functional significance. Using a new minipig specific microarray we show high conservation of gene expression signatures in 13 tissues with biomedical relevance between humans and adult minipigs. We underline this relationship for minipig and human liver where we could demonstrate similar expression levels for most phase I drug-metabolizing enzymes. Higher expression levels and metabolic activities were found for FMO1, AKR/CRs and for phase II drug metabolizing enzymes in minipig as compared to human. The variability of gene expression in equivalent human and minipig tissues is considerably higher in minipig organs, which is important for study design in case a human target belongs to this variable category in the minipig. The first analysis of gene expression in multiple tissues during development from young to adult shows that the majority of transcriptional programs are concluded four weeks after birth. This finding is in line with the advanced state of human postnatal organ development at comparative age categories and further supports the minipig as model for pediatric drug safety studies.ConclusionsGenome based assessment of sequence conservation combined with gene expression data in several tissues improves the translational value of the minipig for human drug development. The genome and gene ...

The genomic organization and expression pattern of the low-affinity Fc gamma receptors (FcγR) in the Göttingen minipig

“…The Göttingen minipig genome draft generated by Heckel et al ( 2015 ) based on Sus scrofa 10.2 was used as a reference genome. Known sequences of FCGR2B and FCGR3A were blasted (Altschul et al 1990 ) against whole genome shotgun-sequencing data of the Göttingen minipig (accession: AOCR01000000) and the Wuzishan minipig (accession: AJKK01000000) to identify overlapping contigs (contiguous sequences).…”

“…Genes above and below the black line are encoded at the forward strand and the reverse strand, respectively. The sequence from the initial minipig genome draft containing FCGR2B (Heckel et al 2015 ) is represented by a gray line. Yellow and blue lines represent whole genome shotgun contigs of the Göttingen minipig and the Wuzishan minipig, respectively.…”

“…In addition, the number of low-affinity FcγRs existing in the minipig and the allocation of the FCGR genes in the corresponding locus of the Göttingen minipig genome was not conclusively determined. The latest version of the Göttingen minipig genome was generated by Heckel et al by mapping of the whole genome-sequencing data on the Duroc pig genome Sus scrofa 10.2 (Heckel et al 2015 ). There, FCGR2B was the only gene annotated in the low-affinity FCGR locus.…”

A porcine model of osteosarcoma

“…Probe selection and array design is described elsewhere. 59 In all, 200 ng double-stranded cDNA was Cy3-labelled using the 'One colour DNA Labelling Kit' (Roche NimbleGen, Madison, WI, USA). A minimum of three technical replicates of each sample were loaded, and raw data intensities were collected using a NimbleGen MS200 scanner.…”

The porcine translational research database: a manually curated, genomics and proteomics-based research resource

“…For genes that were determined to be missing from build 10.2 (Additional file 1) (and for the mis-assembled or duplicated gene artifacts (Additional file 1), we also constructed templates from de novo assemblies derived from Illumina 80 bp reads of the pig alveolar macrophage transcriptome (Dawson, unpublished results) using the de novo assembly algorithm of CLC Genomics Workbench using word size of 20 and a bubble size of 50. When necessary, predicted templates (from bovine or human sequences) were supplemented with porcine expressed sequence tag (EST) assemblies, single ESTs and portions of the published Tibetan (Bioproject # PRJNA291130), Wuzhishan (Bioproject # PRJNA144099), Goettingen (Bioproject # PRJNA291011) [29], Jinhua, Meishan, Bamei, Large White, Berkshire, Hampshire, Pietrain, Landrace, Rongshang and Duroc (Bioproject # PRJNA309108) porcine genomes [30]. ESTs were assembled using CAP3 (http://doua.prabi.fr/software/cap3).…”