The care and experimental use of pigs and mice were approved by the Institute of Laboratory Animal Resources, Seoul National University (SNU-140328-2). Unless otherwise stated, all chemicals were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA).
In vitro embryo production
The ovaries of the prepubertal gilts were obtained from a local slaughterhouse (Anyang-si, Gyeonggi-do, Korea) and transferred to the laboratory in warm saline. Cumulus-oocyte complexes (COCs) were collected by aspirating 3- to 7-mm follicles of the prepubertal gilts with a 10-mL syringe and an 18-gauge needle. Sediments were washed with TL–HEPES–PVA medium, and oocytes with compact cumulus cells and granulated cytoplasm were selected for in vitro maturation. The washed COCs were cultured in tissue culture medium (TCM-199; Life Technologies, Carlsbad, CA, USA) containing 10 ng/mL epidermal growth factor, 1 mg/mL insulin, and 10% porcine follicular fluid for 44 hours at 39°C at 5% CO2 and 100% humidity. The COCs were matured with 10 IU/mL gonadotropin hormone, pregnant mare serum gonadotropin (Lee Biosolutions, Maryland Heights, MO, USA), and human chorionic gonadotropin for the first 22 hours. The COCs were then matured under hormone-free conditions. To generate parthenotes, cumulus-free oocytes were activated with an electric pulse (1.0 kV/cm for 60 ms) in activation medium (280 mM mannitol, 0.01 mM CaCl2, 0.05 mM MgCl2) using a BTX Electrocell Manipulator (BTX, CA, USA), followed by 4 hours of incubation in PZM3 medium containing 2 mmol/L 6-dimethylaminopurine.
Cytoplasmic injection of the DNA-lipofectamine complex
For the microinjection assay, 10 μl of 90 ng/μl of DNA in combination with 1 μl of Lipofectamine (Stem
reagent; Thermo Fisher Scientific) was incubated for 5 min in Media-199 (Gibco), and the final DNA concentration was 15 ng/μl. One day after PA, the embryos at the 2-cell stage were injected with 2 pl of plasmid-lipofectamine solution in manipulation media. The microinjection procedure was conducted with a micromanipulator (Eclipse TE2000, Nikon, Tokyo, Japan) with holding and injection pipettes. We used a Femtotip Ⅱ (Eppendorf, Hamburg, Germany) as an injection pipette.
Immunocytochemistry
Each stage of embryos without zona pellucida was fixed in 4% paraformaldehyde for 15 min at room temperature. Fixed samples were permeabilized using 1% Triton X-100 for 1 hour at room temperature and washed three times with phosphate-buffered saline (PBS). The embryos were blocked using 10% goat serum or donkey serum in PBS for 1 hour at room temperature. Samples were stained with anti-SOX2 (5 μg/ml), NANOG (1 μg/ml), OCT4 (1 μg/ml), and SOX17 (1 μg/ml) in PBS containing 10% donkey serum at 4°C overnight. After washing 3 times in washing solution (PBS with 0.2% Tween-20 and 1% BSA for 10 min), embryos were incubated with donkey anti-rabbit Alexa594 (Invitrogen, Carlsbad, California, USA) in PBS with 10% donkey serum at RT for 1 hour. All samples were washed 3 times with washing solution after secondary antibody treatment. Immunostained embryos were mounted on a slide glass with Prolong gold with DAPI (Invitrogen) and cured for more than 24 hours. We described the list of antibodies in Table S2. The imaging tool of the micromanipulator was used to take fluorescence images. We used the ImageJ program to obtain images.
Quantitative RT-PCR
Pooled embryos at each stage of in vitro-produced embryos (2–3-cell, n=20; 4-cell, n=20; 6–8-cell, n=20; morula, n=10; and BL, n=5) were processed with an Arcturus® PicoPure® RNA Isolation Kit (Applied Biosystems, Foster City, California, USA) following the manufacturer’s instructions. cDNA was synthesized using a High-Capacity RNA-to-cDNA Kit (Applied Biosystems, USA). Extracted cDNA samples were amplified using Power SYBR Green Master Mix (Applied Biosystems, USA) containing 1 pmol of each primer set listed in Table S1 in a 10 μl reaction volume. Amplification and detection were conducted using the ABI 7300 Real-Time PCR System (Applied Biosystems, USA) under the following conditions: one cycle of 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension for 1 min (annealing/extension temperatures dependent on each primer set). The dissociation curves were analyzed, and the amplified products were loaded onto gels to confirm the specificity of the PCR products. The relative expression level was calculated by normalizing the threshold cycle (Ct) values of each gene to that of the reference gene beta-actin (ACTB) via the delta-delta Ct method.
Production of CRISPR Cas9 Vectors and SOX2 Gene Expression Vector
The selected guide sequence was inserted into the pX330 and pX458 vectors. The CRISPR-target site is depicted in Figure 2. The porcine SOX2 coding region sequence was synthesized and replaced in the human SOX2 site of the pCXLE-hS + Egfp vector, Addgene #74945. All vectors were verified by nucleotide sequencing.
Culture of porcine embryonic fibroblasts and plasmid transfection
Basic cell culture and lipofection were carried out following procedures in our previous report (1). Briefly, pEF cells were plated in 6-well plates and transfected with 300 ng of pX330 constructs and 300 ng of pCAG-EGxxFP constructs using Lipofectamine 3000 Reagent (Thermo Fisher Scientific, Waltham, MA, USA). We replaced culture media with fresh modified Dulbecco’s modified Eagle’s medium (DMEM) 24 hours after lipofection, followed by 2 days of culture. For genotyping of transfected pEF, genomic DNA was extracted from pEF using the G-spin Total DNA Extraction Kit (iNtRON Biotechnology, Korea). Genomic DNA samples were amplified using 10 pmol of porcine SOX2-specific primers and 2 X PCR master mix solution (iNtRON Biotechnology).
Statistical analysis
Statistical analysis of data was performed using GraphPad Prism Software (version 5.01; San Diego, CA, USA). Significant differences among experimental groups were determined by one-way analysis of variance followed by Tukey’s multiple comparison test. A p-value <0.05 was considered significant. Data are presented as the mean ± standard error.