Refinement of inducible gene deletion in embryos of pregnant mice

Abstract

CreERT2‐mediated gene recombination is widely applied in developmental biology research. Activation of CreERT2 is typically achieved by injection of tamoxifen in an oily vehicle into the peritoneal cavity of mid‐gestation pregnant mice. This can be technically challenging and adversely impacts welfare. Here we characterize three refinements to this technique: Pipette feeding (not gavage) of tamoxifen, ex vivo CreERT2 activation in whole embryo culture and injection of cell‐permeable TAT‐Cre into Cre‐negative cultured embryos. We demonstrate that pipette feeding of tamoxifen solution to the mother on various days of gestation reliably activates embryonic CreERT2, illustrated here using β‐Actin CreERT2, Sox2 CreERT2, T CreERT2, and Nkx1.2 CreERT2. Pipette feeding of tamoxifen induces dose‐dependent recombination of Rosa26 mTmG reporters when administered at E8.5. Activation of two neuromesodermal progenitor‐targeting Cre drivers, T CreERT2, and Nkx1.2 CreERT2, produces comparable neuroepithelial lineage tracing. Dose‐dependent CreERT2 activation can also be achieved by brief exposure to 4OH‐tamoxifen in whole embryo culture, allowing temporal control of gene deletion and eliminating the need to treat pregnant mice. Rosa26 mTmG reporter recombination can also be achieved regionally by injecting TAT‐Cre into embryonic tissues at the start of culture. This allows greater spatial control over Cre activation than can typically be achieved with endogenous CreERT2, for example by injecting TAT‐Cre on one side of the midline. We hope that our description and application of these techniques will stimulate refinement of experimental methods involving CreERT2 activation for gene deletion and lineage tracing studies. Improved temporal (ex vivo treatment) and spatial (TAT‐Cre injection) control of recombination will also allow previously intractable questions to be addressed.

Cell non-autonomy amplifies disruption of neurulation by mosaic Vangl2 deletion in mice

“…Tamoxifen-inducible CreERT2 drivers are limited because early tamoxifen administration by intraperitoneal injection prevents embryo implantation and is teratogenic 46 . We recently validated an oral tamoxifen administration protocol which robustly activates embryonic CreERT2 without impeding NT closure 47 . Using this method to induce Sox2 CreERT2 led to extensive lineage tracing of neuroepithelial cells in the closed NT, but only to a very limited extent in the open PNP given the temporally-restricted window of CreERT2 activation (Supplementary Fig.…”

“…Cre-negative littermates generated in the course of colony maintenance were used as WT controls for embryo culture. Tamoxifen (10 mg/mouse) was administered at 8:00 a.m. on E8 as previously validated 47 . This dose of tamoxifen was halved when collecting foetuses later than E11 as the high dose produced foetotoxic effects in wild-types.…”

Cell non-autonomy amplifies disruption of neurulation by mosaic Vangl2 deletion

“…Tamoxifen-inducible CreERT2 drivers are limited because early tamoxifen administration by intra-peritoneal injection prevents embryo implantation and is teratogenic 45 . We recently validated an oral tamoxifen administration protocol which robustly activates embryonic CreERT2 without impeding NT closure 46 . Using this method to induce Figure 1B) 47 .…”

“…From 8 weeks of age, Nkx1.2 CreERT2/+ Vangl2 Fl/studs were bred with Vangl2 Fl/Fl Rosa26 mTmG/mTmG females to generate embryos with the desired genotypes. Tamoxifen (10 mg/mouse) was administered at 8:00 a.m. on E8 as previously validated 46 .…”

Rapid and efficient adaptation of the dTAG system in mammalian development reveals stage specific requirements of NELF

“…During pre-implantation embryo development, maternally deposited transcripts can mask early loss of function (knockout) phenotypes, often necessitating generation of maternal/zygotic mutants through complex and imperfect Cre/loxP genetic systems that knockout both maternal and zygotic genes (Sun et al, 2008). Early post-implantation, Cre-estrogen receptor (Cre-ER) systems can offer some spatio-temporal specificity, but can result in mosaic recombination across and within embryos, while tamoxifen injections can have teratogenic effects on embryos, as well as leading to implantation failure during these stages (Pimeisl et al, 2013; Savery et al, 2020; Ved et al, 2019). Thus, there is a need for a safe, efficient, rapid, inducible, and reversable perturbation system to probe gene functions in complex mammalian systems.…”