GoKawiilMaintenance of mouse lines
All animal procedures were approved by the Austrian Federal Ministry of Women, Science and Research in accordance with the Austrian and European Union animal law (license number: BMWF-66.018/0007-II/3b/2012; BMWFW-66.018/0006-WF/V/3b/2017; GZ: 2020-0.579.989 and GZ: 2025-0.597.515). Experimental mice were bred and maintained according to regulations approved by the institutional animal care and use committee and institutional ethics committee and the guidelines of the preclinical facility (PCF) at ISTA. Mice with specific pathogen-free status according to FELASA recommendation57 were bred and maintained in experimental rodent facilities (room temperature 21 ± 1 °C (mean ± s.e.m.); relative humidity 40–55%; photoperiod 12 h light:12 h dark). Food (V1126, Ssniff Spezialitaten) and tap water were available ad libitum. Mouse lines with MADM cassettes inserted on chromosome 1158, Emx1-cre59, Emx1-creER60 and mTmG reporter61 have been reported previously and were used to generate experimental mice. All mouse lines were kept in a mixed C57/Bl6 and CD1 genetic background. Mice were used at an age range of 2–8 months for general breeding, 2–4 months (females) for collecting blastocysts, and at P21 for clonal analysis experiments in vivo. All efforts were made to minimize the number of animals by following the 3R principles.
Timed breeding and superovulation for the generation of genetically defined blastocysts
MADM-11TG/TG and MADM-11GT/GT;Emx1cre/+ or MADM-11GT/GT;Emx1-creER+/− stock mice were crossed to generate MADM-11GT/TG;Emx1cre/+ or MADM-11GT/TG;Emx1-creER+/− blastocysts, respectively. For scRNA-seq experiments (see ‘scRNA-seq’), mTmG reporter mice were crossed to Emx1cre/+ mice to generate mTmG;Emx1cre/+ blastocysts. Superovulation was performed according to the ISTA Preclinical Facility protocol. In brief, to synchronize the oestrous cycle and induce superovulation, 0.1 ml (5 IU) pregnant mare serum gonadotropin (PMSG; Sigma) was administered by intraperitoneal injection during the afternoon (between 16:00 and 18:00) of day −3 before ovulation. On day −1 (46–48 h after PMSG), 0.1 ml (5 IU) human chorionic gonadotropin (Sigma) was administered by intraperitoneal injection and the female immediately added to the male cage. Hormones in lyophilized powder form were resuspended in Dulbecco’s PBS (Sigma) and stored in aliquots at −20 °C until use.
Derivation and culture of mESCs
Blastocyst collection at E3.5 and derivation of mESCs from individually cultured blastocysts were performed as described previously62. Blastocysts were flushed from the uterine horn with M2 medium (Sigma) using a syringe, collected with a micropipette and washed with 1 ml of M2 medium. Blastocysts were cultured for up to 24 h in KSOM medium (Sigma) until expansion of the blastocoel and/or hatching was observed. Each blastocyst was transferred to a single well in a 96-well plate that was prepared with mouse embryonic fibroblasts (MEFs; Thermo Fisher Scientific) the day before at a density of 1.5 × 104 cells per well. Blastocysts were cultured in KO-DMEM (Thermo Fisher Scientific) medium containing 15% knockout serum replacement (Thermo Fisher Scientific), 1 mM sodium pyruvate (Thermo Fisher Scientific), 0.1 mM non-essential animo acids (Thermo Fisher Scientific), 0.1 mM 2-mercaptoethanol (Sigma), 2 mM GlutaMAX (Thermo Fisher Scientific), 50 U ml−1 penicillin/streptomycin (Thermo Fisher Scientific), 2i (1 µM PD0325901 and 3 µM CHIR99021, Sigma) and LIF (1 2 ng ml−1, batch tested, Thermo Fisher Scientific). Once large outgrowth was observed (~7 days) the cells were passaged for the first time. mESCs were maintained on MEFs in medium containing ES-qualified FBS (Thermo Fisher Scientific)/LIF, with passaging every 3 days on average. mESCs were moved off MEFs 2 passages before generating organoids, and were plated on EmbryoMax 0.01% gelatin (Sigma) coated wells in medium containing FBS/LIF/2i63 for a maximum of 10 passages. mESCs were routinely tested for mycoplasma using the LookOut Mycoplasma PCR Detection Kit (Sigma). Standard cell culture conditions (37 °C with 5% CO 2 ) were used throughout all procedures. Early passage (P3–P8) cells were frozen in liquid nitrogen cryovials in large stocks, using ES-qualified FBS with 20% DMSO as the freezing medium. Early passage stocks were used for all experiments (final passage of cells used to make organoids between P8 and P15).
Methanol fixation for karyotyping of mESC lines
Methanol fixation was performed according to instructions provided by Cell Guidance Systems’ karyotyping service. In short, cell cultures in 6-well plates were incubated with medium supplemented with 10 µg µl−1 KaryoMAX colcemid solution (Thermo Fisher Scientific) for 30 min at 37 °C. Medium was removed and colonies were dissociated with 400 µl of pre-warmed 0.05% Trypsin EDTA (Thermo Fisher Scientific) for 5 min at 37 °C. Trypsin was deactivated with 800 µl of warm medium and cells were transferred to a 15 ml conical tube and centrifuged for 5 min at 200g. The supernatant was discarded and the cell pellet was broken by flicking the tube 20 times. Cells were treated with 2 ml of warm 0.075 M KCl drop by drop, followed by a further 2 ml dispensed slowly down the wall of the tube. The suspension was mixed by inversion and incubated for 15 min at 37 °C. Next, 10 drops of cold freshly prepared fixative (3 parts methanol (VWR) to 1 part acetic acid (VWR) by volume) were added, using a 1 ml Pasteur pipette. The suspension was mixed by gentle inversion. Samples were centrifuged for 5 min at 150g, the supernatant was discarded and the pellet was broken by flicking the tube 20 times. Cells were washed with 4 ml of cold fixative, added very slowly, and centrifuged for 5 min at 150g. The supernatant was discarded, pellet broken, and cells were finally resuspended in 1.5 ml fixative. Samples were shipped off to Cell Guidance Systems’ karyotyping service. Chromosome 8 and 11 abnormalities have been previously reported in mESC lines64,65.
Metaphase spread for chromosome counts
Cells for metaphase chromosome spreads were fixed as described above. In preparation, Superfrost glass slides (Thermo Fisher Scientific) were cooled at −20 °C for 5 min. 100 µl of the fixed cell suspension was dropped, as a single drop, from 10–15 cm above the slide. The slide was air dried, and a coverslip was placed with Mowiol 4-88 (Carl Roth) and 1,4-diazabicyclooctane (Carl Roth) with DAPI (4′,6-diamidino-2-phenylindole, Thermo Fisher Scientific, 1:5,000 dilution) to stain DNA. Slides were imaged with a Plan-Apochromat 40×/1.2 water immersion objective using an inverted LSM 800 series confocal microscope (Zeiss), and images were processed using Zeiss ZEN Blue 2.3 and 2.6 software (Zeiss). Chromosomes were counted manually in the Zeiss ZEN Blue 2.6 software (Zeiss) and plotted using Graphpad Prism 10.2.2 (Dotmatics) software.
... continue reading