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

Abstract

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.

Keywords: Fah KO mouse; In vivo gene silencing; Sleeping Beauty; Somatic transgenesis; Tumor model.

Fig. 1

In vivo transposon-based gene delivery into the liver of Fah^−/− and WT mice. a Schematic representation of the Sleeping Beauty (SB) transposon-based cloning platform and animal treatments. Black arrows, SB transposon inverted terminal repeats; red arrows, promoters. b Fah and EGFP immunostainings of liver sections from Fah^−/− mice 3 months after NTBC withdrawal. c Monitoring the amount of transcripts A and B following in vivo gene delivery. Liver RNA samples were collected from Fah^−/− mice at 3 months post-treatment. Samples were tested using Fah- and EGFP-specific RT-qPCR assays. Results were normalized to measurements of the ribosomal protein L27 (Rpl27) transcript as input control and data were presented as the mean ± standard deviation (SD) (n = 3) (see Additional file 2 for individual data values and statistics). d Live bioluminescence imaging of Fah^−/− and WT mice following in vivo gene delivery. Bioluminescence signals were obtained using an IVIS Lumina III imaging system at 3, 7, 14, 28, 56, and 84 days post-treatment. e Kinetics of bioluminescence changes during the first 3 months after gene delivery. For each experimental animal, the average radiance (photons/second/cm²/steradian (sr) [p/s/cm²/sr]) of circular regions of the same size covering the liver area was used for plotting. The numerical values were presented as box diagram from lowest to highest values with line at mean (n = 3) (see Additional file 2 for individual data values and statistics)

Fig. 2

In vivo amiR-based gene silencing in the mouse liver. a Brightfield and fluorescence stereomicroscopic images of the liver of Fah^−/− mice 5 months after the intrahepatic delivery of an amiR-free control and different amiR-expressing transposon vectors. b Monitoring the amount of the endogenous p53 mRNA and artificial transcripts A and B in the liver of Fah^−/− mice 5 months after intrahepatic delivery of an amiR-free control and different amiR-expressing transposon vectors. Liver RNA samples were collected from Fah^−/− mice at 5 months post-treatment. Samples were tested using Fah-, EGFP-, and p53 mRNA-specific RT-qPCR assays. Results were normalized to measurements of the ribosomal protein L27 (Rpl27) transcript as input control and data were presented as the mean ± SD (n = 3) (see Additional file 2 for individual data values and statistics). c Monitoring of endogenous p53 mRNA levels in NIH3T3 cells after stable transposon-based delivery of different amiR elements designed to silence Tp53 expression. RNA samples were collected from cultured cells after G418 selection and tested using a p53 mRNA-specific RT-qPCR assay. Results were normalized to measurements of the ribosomal protein L27 (Rpl27) transcript as input control. Data were presented as the mean ± SD as relative values compared to the value generated using an amiR-free control vector (n = 3) (see Additional file 2 for individual data values and statistics). d Copy numbers of the transgenes in the liver of Fah^−/− mice following intrahepatic delivery of different transposon vectors. Liver DNA samples were collected from Fah^−/− mice at 5 months post-treatment. Samples were tested using a Fah transgene-specific qPCR assay. Results were normalized to measurements of the olfactory receptor 16 (Olfr16) gene as an input control, and values were presented relative to one diploid genome (n = 3) (see Additional file 2 for individual data values and statistics)

Fig. 3

Induction of HCC using a predefined combination of drivers. a Immunohistochemical analysis of the Fah selection marker, Gpc3, and Afp HCC markers in liver sections from Fah^−/− mice treated with either control (no amiR, EGFP) or driver (amiR-mP53/1, hRas^G12V) transposon constructs at 5 weeks and 5 months post-treatment. For the analysis of tumors emerging 5 months after treatment with the driver construct, a vector mixture containing 1% driver transposon vector and 99% transposon vector expressing only the Fah selection marker protein was used. Scale bars, 100 μm. b Determination of the percentage of Fah-positive hepatocytes 5 weeks after treatment by machine learning-based measurement. Data were presented as the mean ± SD (n = 3) (see Additional file 2 for individual data values and statistics). c Monitoring of endogenous p53 mRNA levels in the liver of Fah^−/− mice treated with driver and control transposon constructs. Liver RNA samples were collected from Fah^−/− mice at 5 weeks post-treatment and tested using a p53 mRNA-specific RT-qPCR assay. Results were normalized to measurements of the ribosomal protein L27 (Rpl27) transcript as input control and data were presented as the mean ± SD (n = 3) (see Additional file 2 for individual data values and statistics). d Monitoring the amount of transcripts A and B in the liver of Fah^−/− mice treated with driver and control transposon constructs. Liver RNA samples were collected from Fah^−/− mice at 5 weeks post-treatment and tested using Fah-, EGFP-, and hRas^G12V-specific RT-qPCR assays. Results were normalized to measurements of the ribosomal protein L27 (Rpl27) transcript as input control and data were presented as the mean ± SD (n = 3) (see Additional file 2 for individual data values and statistics)