Supplementary Materials for E-00049-2025R1 in American Journal of Physiology-Endocrinology and Metabolism

The pituitary gland plays a central role in the regulation of development, metabolism, stress, reproduction, and homeostasis in the mammalians. All the endocrine cells secreting distinct pituitary hormones are derived from Prop1-expressing progenitors. Prop1 promoter-driven Cre transgenic mice are useful in the field of pituitary biology based on the Cre/LoxP system, however their ectopic activity complexes data interpretation. Here we demonstrate the robust and specific recombination activity of a Prop1-iCre knockin line in pituitary endocrine cells as well as in ovarian granulosa cells. Prop1-iCre mice were generated by introducing IRES-Cre cassette immediately downstream of the stop codon of the <i>Prop1 </i>gene by CRISPR/Cas9 system, which allows for the bicistronic expression of Prop1 and Cre recombinase simutanously. The Cre insertion had no effect on the expression of the <i>Prop1</i> gene per se or the development of pituitary endocrine cells. When crossed onto a Rosa-tdTomato reporter line, Prop1-iCre mice exhibited a robust recombination activity in all the pituitary endocrine cells, with no activity in hypothalamus, thyroid, adrenal gland, testis, heart, lung, liver, or kidney. Of note, the recombination activity was also detected in some of ovarian granulosa cells in female Prop1-iCre mice, which is consistent with their <i>Prop1</i> mRNA expression based on single-cell RNA sequencing analysis. Our findings support that the Prop1-iCre mouse line serves as a valuable tool for gene manipulation in pituitary endocrine cells, and also raise a caution regarding to its activity in the ovary.