100 mm petri dishes

Decoy oligonucleotides containing the palindromic p53 cis-element 5′-RRRCWWGYYY-3′ (R = A or G, Y = C or T, W = A or T), which allows self-hybridization and formation of a duplex hairpin that competes with p53 enhancers for binding of transcription factors, were used to inhibit p53-directed transcription in vivo, as previously described by us^{57 (link)}. The sequences of the p53 decoy and control phosphorothioate oligonucleotides (underscored) (Sigma-Aldrich) were as follows: p53 decoy, 5′-GGACATGCCCGGGCATGTCC-3′; control nonsense sequence, 5′-CTAGCTAGCTAGCTAGCTAGCTAG-3′. Cells were seeded at a density of 10⁶ cells onto in 100 mm Petri dishes (Sigma-Aldrich), differentiated as above. After that cells were collected and 800  μl of 2.5 × 10⁷ cell/ml, electroporated in presence of 500 μg sheared salmon sperm DNA (Sigma-Aldrich) and 1 μM of each phosphorothioate oligonucleotide at 330 V and 1000 uF using an Electroporator II (Invitrogen). Next, cells were plated onto 6-well plates (Sigma-Aldrich), grown in a complete medium overnight followed by its replacement with 1% FCIII containing 100 μM OT for 72 h and analysis using RT-PCR as said above.

Presumptive zygotes were denuded 18 h after IVF by vortexing for 120 s in BO-Wash medium, rinsed thrice in BO-Wash medium, and once in BO-IVC medium. Zygotes were cultured in 60 µL BO-IVC culture medium droplets (one zygote per droplet) overlaid with mineral oil in 100 mm Petri dishes (Sigma-Aldrich, St. Louis, MO, USA) at 38.5 °C under an atmosphere of 5% CO₂, 5% O₂, and 90% N₂ for up to 7 days. Controls were created by placing 60 µL of plain BO-IVC culture media droplets that were never in contact with an embryo under oil, and they were kept under the same conditions as zygotes (38.5 °C under an atmosphere of 5% CO₂, 5% O₂, 90% N₂). Fresh day 7 IETS Grade 1 blastocysts were transferred to the synchronized EHF recipient heifers using BO-Transfer media.