The multiple roles of Ggt1-Cre in the generation of transgenic mice

The Cre/loxP system continues to serve as a well-established and widely adopted strategy for generating conditional gene knockout or knock-in mouse models, facilitating precise genetic manipulations. The Ggt1 gene, which exhibits specific expression in proximal tubular epithelial cells (TECs) of the kidney, has been extensively employed as a Cre driver for tissue-specific gene targeting within these cells. In this study, to achieve conditional Fam134b knockout in proximal TECs, we generated Fam134b floxed mice and crossed them with Ggt1-Cre transgenic mice. After several generations of selective breeding, we successfully obtained conditional Fam134b knockout mice, which displayed specific deletion of the target gene in proximal TECs. This was confirmed by western blot analysis, which demonstrated a marked deficiency of the FAM134B protein in the renal cortex of these mice. During the mating experiments, we unexpectedly found that we could obtain systematic Fam134b knockout mice, suggesting that Ggt1-Cre might be expressed and functional in germ cells. Genomic and transcriptomic sequencing analysis unequivocally confirmed the deletion of exon 4, while western blot analysis revealed complete absence of FAM134B protein in both heart and kidney tissues of these knockout mice. Through the implementation of different mating strategies, we determined that Ggt1-Cre mediated gene knockout occurs in germ cells that have completed the first meiotic division, rather than in germ cells prior to this developmental stage. Furthermore, qPCR and western blot analyses demonstrated the expression of Cre driven by the Ggt1 promoter in both testes and ovaries, providing additional evidence for its germline activity. Lineage tracing experiments revealed that Ggt1-Cre is expressed in both the kidneys and testes of B6-G/R f/+; Ggt1-Cre transgenic mice, where it effectively catalyzes Cre recombinase activity, leading to the conversion of green fluorescent protein-expressing cells to red fluorescent protein-expressing cells. These findings collectively highlight that Ggt1-Cre is not only a reliable proximal TEC-specific Cre driver but also an effective germline-specific Cre driver. Consequently, it can be utilized to achieve gene knockout or overexpression in both proximal TECs and post-first meiotic division germ cells, thereby enabling in-depth in vivo functional studies of genes in these distinct cell types.