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Erschienen in:

10.04.2023 | Original Article

Generation of bicistronic Dmp1-Cre knock-in mice using a self-cleaving 2A peptide

verfasst von: Takashi Nakamura, Sayako Honda, Shinichirou Ito, Toshihide Mizoguchi, Takehiro Yamamoto, Masataka Kasahara, Yasuaki Kabe, Koichi Matsuo, Makoto Suematsu

Erschienen in: Journal of Bone and Mineral Metabolism | Ausgabe 4/2023

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Abstract

Introduction

The conditional manipulation of genes using the Cre recombinase-locus of crossover in P1 (Cre/loxP) system is an important tool for revealing gene functions and cell lineages in vivo. The outcome of this method is dependent on the performance of Cre-driver mouse strains. In most cases, Cre knock-in mice show better specificity than randomly inserted Cre transgenic mice. However, following knock-in, the expression of the original gene replaced by Cre is lost.

Materials and methods

We generated a new differentiated osteoblast- and osteocyte-specific Cre knock-in mouse line that carries the viral T2A sequence encoding a 2A self-cleaving peptide at the end of the coding region of the dentin matrix protein 1 (Dmp1) gene accompanied by the Cre gene.

Results

We confirmed that Dmp1-T2A-Cre mice showed high Cre expression in osteoblasts, osteocytes, odontoblasts, and periodontal ligament cells and that the 2A self-cleaving peptide efficiently produced both Dmp1 and Cre proteins. Furthermore, unlike the Dmp1 knockout mice, homozygous Dmp1-T2A-Cre mice showed no skeletal abnormalities. Analysis using the Cre reporter strain confirmed differentiated osteoblast- and osteocyte-specific Cre-mediated recombination in the skeleton. Furthermore, recombination was also detected in some nuclei of skeletal muscle cells, spermatocytes, and intestinal cells.

Conclusion

2A-Cre functions effectively in vivo, and Dmp1-T2A-Cre knock-in mice are a useful tool for studying the functioning of various genes in hard tissues.

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Titel: Generation of bicistronic Dmp1-Cre knock-in mice using a self-cleaving 2A peptide
verfasst von: Takashi Nakamura
Sayako Honda
Shinichirou Ito
Toshihide Mizoguchi
Takehiro Yamamoto
Masataka Kasahara
Yasuaki Kabe
Koichi Matsuo
Makoto Suematsu
Publikationsdatum: 10.04.2023
Verlag: Springer Nature Singapore
Erschienen in: Journal of Bone and Mineral Metabolism / Ausgabe 4/2023
Print ISSN: 0914-8779
Elektronische ISSN: 1435-5604
DOI: https://doi.org/10.1007/s00774-023-01425-y

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Generation of bicistronic Dmp1-Cre knock-in mice using a self-cleaving 2A peptide