Commonly disrupted pathways in brain and kidney in a pig model of systemic endotoxemia.

Sepsis is a life-threatening state that arises due to a hyperactive inflammatory response stimulated by infection and rarely other insults (e.g., non-infections tissue injury). Although changes in several proinflammatory cytokines and signals are documented in humans and small animal models, far less is known about responses within affected tissues of large animal models. We sought to understand the changes that occur during the initial stages of inflammation by administering intravenous lipopolysaccharide (LPS) to Yorkshire pigs and assessing transcriptomic alterations in the brain, kidney, and whole blood. Robust transcriptional alterations were found in the brain, with upregulated responses enriched in inflammatory pathways and downregulated responses enriched in tight junction and blood vessel functions. Comparison of the inflammatory response in the pig brain to a similar mouse model demonstrated some overlapping changes but also numerous differences, including oppositely dysregulated genes between species. Substantial changes also occurred in the kidneys following LPS with several enriched upregulated pathways (cytokines, lipids, unfolded protein response, etc.) and downregulated gene sets (tube morphogenesis, glomerulus development, GTPase signal transduction, etc.). We also found significant dysregulation of genes in whole blood that fell into several gene ontology categories (cytokines, cell cycle, neutrophil degranulation, etc.). We observed a strong correlation between the brain and kidney responses, with significantly shared upregulated pathways (cytokine signaling, cell death, VEGFA pathways) and downregulated pathways (vasculature and RAC1 GTPases). In summary, we have identified a core set of shared genes and pathways in a pig model of systemic inflammation.

Building the vertebrate codex using the gene breaking protein trap library.

One key bottleneck in understanding the human genome is the relative under-characterization of 90% of protein coding regions. We report a collection of 1200 transgenic zebrafish strains made with the gene-break transposon (GBT) protein trap to simultaneously report and reversibly knockdown the tagged genes. Protein trap-associated mRFP expression shows previously undocumented expression of 35% and 90% of cloned genes at 2 and 4 days post-fertilization, respectively. Further, investigated alleles regularly show 99% gene-specific mRNA knockdown. Homozygous GBT animals in ryr1b, fras1, tnnt2a, edar and hmcn1 phenocopied established mutants. 204 cloned lines trapped diverse proteins, including 64 orthologs of human disease-associated genes with 40 as potential new disease models. Severely reduced skeletal muscle Ca2+ transients in GBT ryr1b homozygous animals validated the ability to explore molecular mechanisms of genetic diseases. This GBT system facilitates novel functional genome annotation towards understanding cellular and molecular underpinnings of vertebrate biology and human disease.

The human genome counts over 20,000 genes, which can be turned on and off to create the proteins required for most of life processes. Once produced, proteins need move to specific locations in the cell, where they are able to perform their jobs. Despite striking scientific advances, 90% of human genes are still under-studied; where the proteins they code for go, and what they do remains unknown. Zebrafish share many genes with humans, but they are much easier to manipulate genetically. Here, Ichino et al. used various methods in zebrafish to create a detailed 'catalogue' of previously poorly understood genes, focusing on where the proteins they coded for ended up and the biological processes they were involved with. First, a genetic tool called gene-breaking transposons (GBTs) was used to create over 1,200 strains of genetically altered fish in which a specific protein was both tagged with a luminescent marker and unable to perform its role. Further analysis of 204 of these strains revealed new insight into the role of each protein, with many having unexpected roles and localisations. For example, in one zebrafish strain, the affected gene was similar to a human gene which, when inactivated, causes severe muscle weakness. These fish swam abnormally slowly and also had muscle problems, suggesting that the GBT fish strains could 'model' the human disease. This work sheds new light on the role of many previously poorly understood genes. In the future, similar collections of GBT fish strains could help researchers to study both normal human biology and disease. They could especially be useful in cases where the genes responsible for certain conditions are still difficult to identify.