Demecolcine

Construction of semi-cloned embryos was performed following a published protocol [16 (link)] with a few modifications. DKO-phaESCs were maintained without MEFs in 2i plus LIF medium [32 (link), 33 (link)]. M-phase arrest was performed for DKO-phaESCs by culturing in the medium supplemented with 0.05 mg/ml demecolcine (Merck) for 8 hours. After staining with Hoechst, DKO-phaESCs with a 2n DNA content were sorted by flow cytometer. Sorted DKO-phaESCs were maintained in 2i plus LIF medium supplemented with 20 mM HEPES (Invitrogen) and the tube containing cell suspension was kept on ice until the use for injection. In parallel, MII oocytes were harvested from superovulated B6D2F1 females. To construct semi-cloned embryos, sorted single DKO-phaESCs were injected into MII oocytes using a piezo-driven micromanipulator (Eclipse Ti, Nikon; PiezoXpert, Eppendorf). After injection embryos were cultured in KSOM medium for 1 hour and subsequently activated for 6 hours in KSOM medium containing 5 mM strontium chloride and 2 mM EGTA. After activation, embryos were washed and cultured in KSOM medium at 37°C under 5% CO₂ in air.

To visualize SCE, 1 μM BrdU (Sigma, B5002) was added to medium for 20–40 h, depending on cell cycle length. Cells were arrested in metaphase by a 1-h treatment with 0.1 μg/ml demecolcine (Sigma, D1925), treated with 0.075 M KCl, fixed in methanol:acetic acid (3:1), spread onto glass slides and air-dried.

Cells were treated with 0.2μg/mL of colcemid (Demecolcine, SIGMA-Aldrich, Italy) for two hours, trypsinized and harvested by centrifugation at 1000 rpm for 10 min. Cells were swollen by adding 75mM KCl dropwise and incubated at 37°C for 10 min, then centrifugated at 800 rpm for 10 min. The pellet was resuspended adding dropwise 5 mL of cold Carnoy's fixative [methanol/acetic acid (3:1 v/v)] and incubated for 15 min on ice. After repeating the last step twice, cells were dropped onto iced slides. Chromosomes were stained either with Giemsa or with 1μg/mL of DAPI (4′,6-diamidino-2-phenylindole, SIGMA-Aldrich, Italy) and examined on a Zeiss Axioskop microscope equipped for fluorescence, images were captured with a CCD digital camera (AxioCam, Zeiss) and then transferred to Adobe PhotoShop. We evaluated at least 100 mitoses for each sample. The experiment was repeated twice.

For karyotype analysis, NP-derived iPSCs (passage 16) were treated with Demecolcine (0.07 mg/mL final concentration, Sigma-Aldrich) for 4 h. The cells were subsequently centrifuged and resuspended in 10 mL KCl solution (75 mM), followed by three rounds of fixation using an ice-cold methanol: glacial acetic acid (3:1) solution. The fixed cells were washed at least twice with 10 mL fixative solution before applying to chilled slides. Slides prepared for chromosome analysis were dried and treated with 0.0025% trypsin for 5 min and stained with Giemsa (1:10, Sigma-Aldrich,) for 5–10 min and finally, a karyogram was produced for two cells per clonal line.

For metaphase spreads, cells were incubated with demecolcine (Sigma) at 0.2 µg/ml for 4 h and then harvested. Cells were collected using standard cytogenetic techniques and fixed in 3 : 1 methanol:acetic acid. Fixed cells were dropped onto acetic acid-humidified slides.