Tabriz University of Medical Sciences BioImpacts 2228-5652 16 1 2026 01 04 Generation and validation of ROSA26mT-mG knock-in mouse with CRISPR/Cas9 as a universal lineage tracing model 33474 33474 10.34172/bi.33474 EN Behrouz Asgari Abibeiglou https://orcid.org/0009-0001-1293-6071 Magid Fallahi Mahdi Hesaraki Meghdad Yeganeh https://orcid.org/0009-0005-4738-7761 Mohammad Rezaei Hossein Baharvand https://orcid.org/0000-0001-6528-3687 Mohsen Basiri https://orcid.org/0000-0003-2512-7641 Yaser Tahamtani https://orcid.org/0000-0003-2360-4985 Journal Article 10.34172/bi.33474 2026 01 29 2026 08 01 Introduction: Transgenic ROSA26mT-mG knock-in mice serve as an invaluable tool in developmental and differentiation studies, particularly for tracing and analyzing cell lineages throughout various stages of growth. Methods: In this study, we employed the CRISPR/Cas9 system to precisely introduce desired genetic material ("ROSA26mT-mG" vector) into the Rosa26 locus of the mouse genome. Results: Under fluorescent light, ROSA26mT-mG knock-in mice exhibit a red phenotype in the absence of Cre recombinase. However, when crossed with the "UBC-Cre/ER" transgenic mouse, the membrane-targeted tdTomato(mT) gene is excised at the LoxP site, leading to the expression of the membrane-targeted green fluorescent protein (mGFP) gene, thereby changing the mouse's appearance from red to green under fluorescent light. Conclusion: ROSA26mT-mG knock-in mice can serve as universal lineage tracing models in a diverse array of developmental biology and regenerative medicine studies, enabling researchers to investigate differentiation pathways, and tissue regeneration mechanisms in a clear and visually interpretable manner.