A versatile transposon-based technology to generate loss- and gain-of-function phenotypes in the mouse liver.

BACKGROUND: Understanding the contribution of gene function in distinct organ systems to the pathogenesis of human diseases in biomedical research requires modifying gene expression through the generation of gain- and loss-of-function phenotypes in model organisms, for instance, the mouse. However, methods to modify both germline and somatic genomes have important limitations that prevent easy, strong, and stable expression of transgenes. For instance, while the liver is remarkably easy to target, nucleic acids introduced to modify the genome of hepatocytes are rapidly lost, or the transgene expression they mediate becomes inhibited due to the action of effector pathways for the elimination of exogenous DNA. Novel methods are required to overcome these challenges, and here we develop a somatic gene delivery technology enabling long-lasting high-level transgene expression in the entire hepatocyte population of mice. RESULTS: We exploit the fumarylacetoacetate hydrolase (Fah) gene correction-induced regeneration in Fah-deficient livers, to demonstrate that such approach stabilizes luciferase expression more than 5000-fold above the level detected in WT animals, following plasmid DNA introduction complemented by transposon-mediated chromosomal gene transfer. Building on this advancement, we created a versatile technology platform for performing gene function analysis in vivo in the mouse liver. Our technology allows the tag-free expression of proteins of interest and silencing of any arbitrary gene in the mouse genome. This was achieved by applying the HADHA/B endogenous bidirectional promoter capable of driving well-balanced bidirectional expression and by optimizing in vivo intronic artificial microRNA-based gene silencing. We demonstrated the particular usefulness of the technology in cancer research by creating a p53-silenced and hRas G12V-overexpressing tumor model. CONCLUSIONS: We developed a versatile technology platform for in vivo somatic genome editing in the mouse liver, which meets multiple requirements for long-lasting high-level transgene expression. We believe that this technology will contribute to the development of a more accurate new generation of tools for gene function analysis in mice.

Structural and molecular features of intestinal strictures in rats with Crohn's-like disease.

AIM: To develop a new rat model we wanted to gain a better understanding of stricture formation in Crohn's disease (CD). METHODS: Chronic colitis was induced locally by the administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS). The relapsing inflammation characteristic to CD was mimicked by repeated TNBS treatments. Animals were randomly divided into control, once, twice and three times TNBS-treated groups. Control animals received an enema of saline. Tissue samples were taken from the strictured colonic segments and also adjacent proximally and distally to its 60, 90 or 120 d after the last TNBS or saline administrations. The frequency and macroscopic extent of the strictures were measured on digital photographs. The structural features of strictured gut wall were studied by light- and electron microscopy. Inflammation related alterations in TGF-beta 2 and 3, matrix metalloproteinases 9 (MMP9) and TIMP1 mRNA and protein expression were determined by quantitative real-time PCR and western blot analysis. The quantitative distribution of caspase 9 was determined by post-embedding immunohistochemistry. RESULTS: Intestinal strictures first appeared 60 d after TNBS treatments and the frequency of them increased up to day 120. From day 90 an intact lamina epithelialis, reversible thickening of lamina muscularis mucosae and irreversible thickening of the muscularis externa were demonstrated in the strictured colonic segments. Nevertheless the morphological signs of apoptosis were frequently seen and excess extracellular matrix deposition was recorded between smooth muscle cells (SMCs). Enhanced caspase 9 expression on day 90 in the SMCs and on day 120 also in myenteric neurons indicated the induction of apoptosis. The mRNA expression profile of TGF-betas after repeated TNBS doses was characteristic to CD, TGF-beta 2, but not TGF-beta 3 was up-regulated. Overexpression of MMP9 and down-regulation of TIMP1 were demonstrated. The progressive increase in the amount of MMP9 protein in the strictures was also obvious between days 90 and 120 but TIMP1 protein was practically undetectable at this time. CONCLUSION: These findings indicate that aligned structural and molecular changes in the gut wall rather than neuronal cell death play the primary role in stricture formation.

Alleviated mucosal and neuronal damage in a rat model of Crohn's disease.

AIM: To establish a rat model suitable to investigate the repetitive relapsing inflammations (RRI) characteristic to Crohn's disease. METHODS: Colitis was induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS). RRI were mimicked by repeating administrations of TNBS. Tissue samples were taken from control, once, twice and three times treated rats from the inflamed and adjacent non-inflamed colonic segments at different timepoints during the acute intestinal inflammation. The means of the ulcerated area were measured to evaluate the macroscopic mucosal damage. The density of myenteric neurons was determined on whole mounts by HuC/HuD immunohistochemistry. Heme oxygenase-1 (HO-1) expression was evaluated by molecular biological techniques. RESULTS: TNBS-treated rats displayed severe colitis, but the mortality was negligible, and an increase of body weight was characteristic throughout the experimental period. The widespread loss of myenteric neurons, and marked but transient HO-1 up-regulation were demonstrated after the first TNBS administration. After repeated doses the length of the recovery time and extent of the ulcerous colonic segments were markedly decreased, and the neuronal loss was on a smaller scale and was limited to the inflamed area. HO-1 mRNA level was notably greater than after a single dose and overexpression was sustained throughout the timepoints examined. Nevertheless, the HO-1 protein up-regulation after the second TNBS treatment proved to be transient. Following the third treatment HO-1 protein expression could not be detected. CONCLUSION: Experimentally provoked RRI may exert a protective preconditioning effect against the mucosal and neuronal damage. The persistent up-regulation of HO-1 mRNA expression may correlate with this.