Placental gene editing via trophectoderm-specific Tat-Cre/loxP recombination

Abstract

The ways in which placental defects affect embryonic development are largely overlooked because of the lack of a trophoblast-specific approach for conditional gene ablation. To tackle this, we have established a simple, fast and efficient method for trophectodermal Tat-Cre/loxP recombination. We used the natural permeability barrier in mouse blastocysts in combination with off-the-shelf Tat-Cre recombinase to achieve editing of conditional alleles in the trophoblast lineage. This direct approach enables gene function analysis during implantation and placentation in mice, thereby crucially helping to broaden our understanding of human reproduction and development.

Early developmental plasticity enables the induction of an intermediate extraembryonic cell state

“…As the blastocoel cavity is sealed by tight junctions ( 13 ), treatment with cell-permeant Cre protein (Tat-Cre) results in Tat-Cre uptake only by the directly exposed TE, whereas the ICM remains out of reach (Fig. 2A) ( 14 ). We removed zona pellucida of E3.5 embryos, and after 2 hours of incubation with Tat-Cre, the blastocysts were transferred into individual wells containing MEFs and iXTE medium.…”

“…This strain harbors membrane-targeted tandem dimer Tomato (mT) cassette flanked by loxP sites, followed by membrane-targeted GFP (mG), integrated in the Rosa26 locus. Z ona -free mT/mG blastocysts were incubated with 1.5 μM recombinant cell-permeant Cre protein (Tat-Cre, Millipore) in prewarmed KSOM medium (Millipore) for 2 hours at 37°C in 5% CO 2 atmosphere in air, according to a previously published protocol ( 14 ). After that, the embryos were washed and cultured in KSOM overnight at 37°C in 5% CO 2 .…”

Polarity inversion reorganizes the stem cell compartment of the trophoblast lineage

“…To examine the role of integrin beta-1 in vivo , we implemented the Tat-Cre/ loxP recombination approach for the deletion of conditional alleles in the trophoblast. 20 Tat-Cre treatment of E3.5 embryos harboring integrin beta-1 floxed alleles 29 resulted in a TE-specific depletion of the integrin beta-1 receptor by E4.5 ( Figure 6 E). After embryo transfer into surrogate mothers, both the non-treated (control) and the Tat-Cre-treated embryos established epithelial polarity in the ExE ( Figure 6 E), in accord with the results of the integrin beta-1 blocking in TSCs, in vitro .…”

“…This approach results in Tat-Cre uptake only by the directly exposed TE cells, whereas the ICM remains out of reach. 20 , 21 Thus, the nTom cassette is excised only in the trophoblast, which gains a constitutive nGFP expression ( Figure 3 F). We performed time-lapse microscopy on nTom/nGFP embryos cultured in vitro using our recently established 3D biomimetic culture system 9 ( Figures 3 G and 3H, Video S8 ).…”

Conditionally mutant animal model for investigating the invasive trophoblast cell lineage

“…Conditional mutagenesis has also become a mainstay for mouse placental research ( Woods et al, 2018 ). Several trophoblast cell-specific regulatory sequences have been used to direct Cre recombinase to an assortment of different mouse trophoblast cell lineages ( Wenzel and Leone, 2007 ; Hu and Cross, 2011 ; Mould et al, 2012 ; Ouseph et al, 2012 ; Zhou et al, 2012 ; Crish et al, 2013 ; Pimeisl et al, 2013 ; Nadeau and Charron, 2014 ; Outhwaite et al, 2015 ; Kong et al, 2018 ; Wattez et al, 2019 ; Ozguldez et al, 2020 ). Research in rat has lagged, and model systems for the generation of conditional mutations within the rat trophoblast cell lineage are yet to be reported.…”