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

BMC Biology

Methodology Article

Imre Ocsovszki¹  Anna Molnár²  Virág Éva Dani²  Anna Georgina Kopasz²  Andrea Nagy²  Liza Hudoba²  László Géza Puskás²  Ildikó Fekete²  Lajos Mátés²  Péter Germán²  Dávid Zsolt Pusztai³  Andrea Bakné Drubi³  Gergely Imre³  Réka Karkas⁴  Khaldoon Sadiq Ahmed Abdullah⁴  Farkas Sükösd⁵  Gabriella Pankotai-Bodó⁵  András Kriston⁶  Ede Migh⁶  Péter Horváth⁷ 

[1] Department of Biochemistry, University of Szeged, Szeged, Hungary;Institute of Genetics, Biological Research Centre, Szeged, Hungary;Institute of Genetics, Biological Research Centre, Szeged, Hungary;Doctoral School of Biology, University of Szeged, Szeged, Hungary;Institute of Genetics, Biological Research Centre, Szeged, Hungary;Doctoral School of Multidisciplinary Medical Sciences, University of Szeged, Szeged, Hungary;Institute of Pathology, University of Szeged, Szeged, Hungary;Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre, Szeged, Hungary;Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre, Szeged, Hungary;Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland;

关键词: Fah;    Sleeping Beauty;    Somatic transgenesis;    In vivo gene silencing;    Tumor model;   

DOI  :  10.1186/s12915-022-01262-x

 received in 2021-08-25, accepted in 2022-02-22,  发布年份 2022

来源: Springer

【 摘 要 】

BackgroundUnderstanding 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.ResultsWe 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.ConclusionsWe 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.

【 授权许可】

CC BY   
© The Author(s) 2022

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