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BAC transgenic mice to study the expression of P2X2 and P2Y₁ receptors.

ABSTRACT: Extracellular purines are important signaling molecules involved in numerous physiological and pathological processes via the activation of P2 receptors. Information about the spatial and temporal P2 receptor (P2R) expression and its regulation remains crucial for the understanding of the role of P2Rs in health and disease. To identify cells carrying P2X2Rs in situ, we have generated BAC transgenic mice that express the P2X2R subunits as fluorescent fusion protein (P2X2-TagRFP). In addition, we generated a BAC P2Y₁R TagRFP reporter mouse expressing a TagRFP reporter for the P2RY1 gene expression. We demonstrate expression of the P2X2R in a subset of DRG neurons, the brain stem, the hippocampus, as well as on Purkinje neurons of the cerebellum. However, the weak fluorescence intensity in our P2X2R-TagRFP mouse precluded tracking of living cells. Our P2Y₁R reporter mice confirmed the widespread expression of the P2RY1 gene in the CNS and indicate for the first time P2RY1 gene expression in mouse Purkinje cells, which so far has only been described in rats and humans. Our P2R transgenic models have advanced the understanding of purinergic transmission, but BAC transgenic models appeared not always to be straightforward and permanent reliable. We noticed a loss of fluorescence intensity, which depended on the number of progeny generations. These problems are discussed and may help to provide more successful animal models, even if in future more versatile and adaptable nuclease-mediated genome-editing techniques will be the methods of choice.

SUBMITTER: Grohmann M

PROVIDER: S-EPMC8410928 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:p53 is an important tumor suppressor and stress response mediator. Proper control of p53 level and activity is tightly associated with its function. Posttranslational modifications and the interactions with Mdm2 and Mdm4 are major mechanisms controlling p53 activity and stability. As p53 protein is short-lived and hardly detectable in unstressed situations, less is known on its basal level expression and the corresponding controlling mechanisms in vivo. In addition, it also remains obscure how p53 expression might contribute to its functional regulation. In this study, we established bacterial artificial chromosome transgenic E.coli ?-galactosidase Z gene reporter mice to monitor p53 expression in mouse tissues and identify important regulatory elements critical for the expression in vivo. We revealed preferentially high level of p53 reporter expressions in the proliferating, but not the differentiated compartments of the majority of tissues during development and tissue homeostasis. In addition, tumors as well as regenerating tissues in the p53 reporter mice also expressed high level of ?-gal. Furthermore, both the enhancer box sequence (CANNTG) in the p53 promoter and the 3' terminal untranslated region element were critical in mediating the high-level expression of the reporter. We also provided evidence that cellular myelocytomatosis oncogene was a critical player regulating p53 mRNA expression in proliferating cells and tissues. Finally, we found robust p53 activation preferentially in the proliferating compartment of mouse tissues upon DNA damage and the proliferating cells exhibited an enhanced p53 response as compared with cells in a quiescent state. Together, these results suggested a highly regulated expression pattern of p53 in the proliferating compartment controlled by both transcriptional and posttranscriptional mechanisms, and such regulated p53 expression may impose functional significance upon stress by setting up a precautionary mode in defense of cellular transformation and tumorigenesis.

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BAC transgenic mice to study the expression of P2X2 and P2Y₁ receptors.

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BAC transgenic mice to study the expression of P2X2 and P2Y1 receptors.

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