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Transgenic mouse model for conditional expression of influenza hemagglutinin-tagged human SLC20A1/PIT1.

ABSTRACT: To further investigate the role of the phosphate (Pi) transporter PIT1 in Pi homeostasis and tissue mineralization, we developed a transgenic mouse expressing the C-terminal influenza hemagglutinin (HA) epitope-tagged human PIT1 transporter under control of the cytomegalovirus/chicken beta actin/rabbit beta-globin gene (CAG) promotor and a loxP-stop-loxP (LSL) cassette permitting conditional activation of transgene expression (LSL-HA-hPITtg/+). For an initial characterization of this conditional mouse model, germline excision of the LSL cassette was performed to induce expression of the transgene in all mouse tissues (HA-hPIT1tg/+). Recombination was confirmed using genomic DNA obtained from blood samples of these mice. Furthermore, expression of HA-hPIT1 was found to be at least 10-fold above endogenous mouse Pit1 in total RNA isolated from multiple tissues and from cultured primary calvaria osteoblasts (PCOB) estimated by semi-quantitative RT-PCR. Robust expression of the HA-hPIT1 protein was also observed upon immunoblot analysis in most tissues and permits HA-mediated immunoprecipitation of the transporter. Characterization of the phenotype of HA-hPIT1tg/+ mice at 80 days of age when fed a standard chow (0.7% Pi and 1% calcium) showed elevated plasma Pi, but normal plasma iPTH, iFGF23, serum calcium, BUN, 1,25-dihydroxy vitamin D levels and urine Pi, calcium and protein excretion when compared to WT littermates. Likewise, no change in bone mineral density was observed upon uCT analysis of the distal femur obtained from these mice. In conclusion, heterozygous overexpression of HA-hPIT1 is compatible with life and causes hyperphosphatemia while bone and mineral metabolism of these mice are otherwise normal.

SUBMITTER: Chande S

PROVIDER: S-EPMC6793878 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background & aimsDevelopment of new and more effective therapies against hepatitis B virus (HBV) is limited by the lack of suitable small animal models. The HBV transgenic mouse model containing an integrated overlength 1.3-mer construct has yielded crucial insights, but this model unfortunately lacks covalently closed circular DNA (cccDNA), the episomal HBV transcriptional template, and cannot be cured given that HBV is integrated in every cell.MethodsTo solve these 2 problems, we generated a novel transgenic mouse (HBV1.1X), which generates an excisable circular HBV genome using Cre/LoxP technology. This model possesses a HBV1.1-mer cassette knocked into the ROSA26 locus and is designed for stable expression of viral proteins from birth, like the current HBV transgenic mouse model, before genomic excision with the introduction of Cre recombinase.ResultsWe demonstrated induction of recombinant cccDNA (rcccDNA) formation via viral or transgenic Cre expression in HBV1.1X mice, and the ability to regulate HBsAg and HBc expression with Cre in mice. Tamoxifen-inducible Cre could markedly downregulate baseline HBsAg levels from the integrated HBV genome. To demonstrate clearance of HBV from HBV1.1X mice, we administered adenovirus expressing Cre, which permanently and significantly reduced HBsAg and core antigen levels in the murine liver via rcccDNA excision and a subsequent immune response.ConclusionsThe HBV1.1X model is the first Cre-regulatable HBV transgenic mouse model and should be of value to mimic chronic HBV infection, with neonatal expression and tolerance of HBV antigens, and on-demand modulation of HBV expression.Lay summaryHepatitis B virus (HBV) can only naturally infect humans and chimpanzees. Mouse models have been developed with the HBV genome integrated into mouse chromosomes, but this prevents mice from being cured. We developed a new transgenic mouse model that allows for HBV to be excised from mouse chromosomes to form a recombinant circular DNA molecule resembling the natural circular HBV genome. HBV expression could be reduced in these mice, enabling curative therapies to be tested in this new mouse model.

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Transgenic mouse model for conditional expression of influenza hemagglutinin-tagged human SLC20A1/PIT1.

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