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. 2023 Jun 16;11:1208279. doi: 10.3389/fcell.2023.1208279

FIGURE 1.

FIGURE 1

Gsc:Cyp26A1-eGFP gene targeting design and mouse derivation. (A) The Gsc:Cyp26A1-eGFP cassette was targeted to exon 2 of the endogenous Gsc gene by homologous recombination. (B) Cyp26A1 was used to catabolize RA and all RA isoforms in cells expressing Gsc. eGFP is a co-expressed fluorescent marker used to identify these cells in in vitro and in vivo studies. The cassette was constructed with two cyclin T2A peptide-bond-skipping translation elements to translate both the Cyp26A1 and eGFP gene products as individual proteins when the Gsc promoter was activated. Neomycin is a mammalian selection marker for gene targeting, which was later removed in vivo by crossing with a Cre mouse. The G12 strain uses a T2A translational element for Cyp26A1. The H4 strain uses an IRES translational element for Cyp26A1. (C) All targeting steps in ES cells and C57BL/6N mice were sequence validated to ensure correct recombination events and intact functional elements. Gateway recombineering sites (green box) and primers (dark blue arrows), and loxP sites (red triangle) are indicated.