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Expression of human CAR splicing variants in BAC-transgenic mice
Yu-Kun Jennifer Zhang1, Hong Lu, Curtis D Klaassen
1Department of Pharmacology, University of Kansas Medical Center, Kansas City, KS, USA.
Abstract:
The nuclear receptor constitutive androstane receptor (CAR) is a key regulator for drug metabolism in liver. Human CAR (hCAR) transcripts are subjected to alternative splicing. Some hCAR splicing variants (SVs) have been shown to encode functional proteins by reporter assays. However, in vivo research on the activity of these hCAR SVs has been impeded by the absence of a valid model. This study engineered an hCAR-BAC-transgenic (hCAR-TG) mouse model by integrating the 8.5-kbp hCAR gene as well as 73-kbp upstream and 91-kbp downstream human genomic DNA into the genome of CAR-null mice. A series of experiments demonstrate that (1) the expression of major hCAR mRNA SVs, SV0-4, in livers of hCAR-TG mice is comparable to that in human livers; (2) the hCAR SVs are predominantly expressed in liver, which resembles the tissue distribution of CAR in humans, but diverges from that in mice; and (3) major hCAR mRNA SVs increase markedly in postnatal livers of hCAR-TG mice, which mimics the ontogeny of CAR mRNA in humans. Thus, the transgene likely contains all the functional regulatory elements controlling proper spatial and temporal expression of the hCAR gene. Moreover, hCAR-TG mice respond to the hCAR-specific agonist 6-(4-chlorophenyl)imidazo[2,1-b] [1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime instead of the mouse CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, as well as the common CAR activator, phenobarbital, suggesting that hCAR is fully functional in livers of transgenic mice. In summary, the hCAR-TG mice developed by this study represent a valid model for studying in vivo function and regulation of hCAR and its splicing variants.
Insights
Researchers developed a new human constitutive androstane receptor (hCAR) transgenic mouse model. This model accurately mimics human hCAR gene expression and function in vivo, enabling better study of drug metabolism regulation.
Area of Science:
- Pharmacology
- Genetics
- Molecular Biology
Background:
- The constitutive androstane receptor (CAR) is crucial for regulating drug metabolism in the liver.
- Human CAR (hCAR) transcripts exhibit alternative splicing, producing functional variants.
- Previous in vivo studies of hCAR splicing variants (SVs) were limited by the lack of a suitable animal model.
Purpose of the Study:
- To engineer a humanized constitutive androstane receptor (hCAR) bacterial artificial chromosome (BAC)-transgenic (hCAR-TG) mouse model.
- To validate the in vivo expression pattern and functionality of hCAR and its splicing variants in the developed mouse model.
- To establish a reliable platform for studying the in vivo regulation and function of hCAR and its SVs.
Main Methods:
- Engineered hCAR-BAC-transgenic mice by integrating human hCAR gene and flanking genomic DNA into CAR-null mice.
- Analyzed the expression of major hCAR mRNA SVs (SV0-4) in mouse liver using quantitative methods.
- Assessed the tissue distribution and developmental expression of hCAR mRNA in hCAR-TG mice.
- Evaluated the in vivo functionality of hCAR in hCAR-TG mice by challenging with specific agonists and activators.
Main Results:
- hCAR-TG mice exhibited expression of major hCAR mRNA SVs comparable to human liver expression.
- hCAR SVs were predominantly expressed in the liver, mirroring human CAR distribution.
- Postnatal liver expression of hCAR mRNA in hCAR-TG mice mimicked human CAR ontogeny.
- hCAR-TG mice responded to hCAR-specific agonists and common CAR activators, indicating functional hCAR.
Conclusions:
- The developed hCAR-TG mouse model accurately recapitulates human hCAR gene expression, including alternative splicing, spatial, and temporal regulation.
- This model provides a valid platform for in vivo investigation of human CAR function and the biological roles of its splicing variants.
- The findings support the utility of this hCAR-TG mouse model for preclinical drug metabolism and toxicity studies.
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