Generation of <i>Tpbpα‐iCre‐EGFP</i> Knock‐In Mice for Trophoblast Subtype‐Specific Gene Manipulation

The placenta plays an essential role during pregnancy in mammals, with the placental endocrine trophoblast subtypes secreting many growth factors and cytokines to promote fetal growth and maternal adaptation. These endocrine cells, including trophoblast giant cells (TGCs), glycogen trophoblast cells (GlyTs) and spongiotrophoblast cells (SpTs), are mainly derived from Tpbpα-positive (Tpbpα⁺) trophoblast cells primarily located in the ectoplacental cone (EPC) and later junctional zone (JZ) of the mouse placenta. However, the mechanism driving Tpbpα⁺ trophoblast cell differentiation and the functions of the factors secreted by these endocrine cells remain largely unknown. In the present study, we generated the Tpbpα-iCre-EGFP knock-in mice with codon-improved Cre recombinase (iCre) inserted into the endogenous locus of the Tpbpα gene. To examine the specificity and efficiency of iCre recombinase, we crossed the Tpbpα-iCre-EGFP mice with ROSA26Sor^{tm9(CAG-tdTomato)Hze} reporter mice. The co-expression of EGFP and Td-tomato was detected obviously in the EPC at E8.5 and E9.5, and in the JZ at E13.5. Meanwhile, employing lineage tracing and in situ hybridization, we demonstrated that Tpbpα⁺ trophoblast cells could differentiate into SpTs, GlyTs, maternal blood canal (C)-TGCs, parietal (P)-TGCs, and spiral artery-associated (Spa)-TGCs. In addition, no Tpbpα expression or iCre recombinase activity was detected in other organs examined, indicating the specificity of the iCre activity in placental trophoblast cells. In summary, we successfully generated the Tpbpα-iCre-EGFP knock-in mice with enhanced Cre recombinase for modulating specific genes and investigating their functions during pregnancy.