cDNA cloning of PRD-like homeobox genes expressed in bovine oocytes and early IVF embryos
We performed molecular cloning of PRD-like homeobox genes derived from MII oocyte, 8-cell, or 16-cell stage preimplantation bovine IVF embryos. The amplification of ARGFX, DUXA, LEUTX, NOBOX, TPRX1, TPRX2, and TPRX3 was carried out by PCR using 1 µl of cDNA library derived from different developmental stages of bovine oocytes or embryos. Specifically, cDNA from MII oocyte stage was used to amplify NOBOX, whereas cDNA from 8-cell stage embryos was employed to amplify LEUTX, and TPRX3. cDNA of the 16-cell stage was used for amplification of TPRX1, TPRX2, ARGFX, and DUXA. The PCR reaction was performed using Phusion™ High–Fidelity DNA Polymerase (ThermoFisher Scientific) in accordance with the manufacturer's protocol. The PCR cycling conditions were as follows: an initial denaturation at 98°C for 30 s, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 64°C for 30 s, and extension at 72°C for 80 s, with a final extension at 72°C for 10 min. The primer sequences used for amplification are listed in Table 1. The PCR products were extracted from 2% agarose gel using GeneJet Gel Extraction Kit (ThermoFisher Scientific) and were then cloned into the pCR4Blunt-TOPO vector using the Zero Blunt TOPO PCR Cloning kit (Invitrogen). Plasmid extraction was performed using the NucleoSpin Plasmid DNA Purification kit (Macherey Nagel) after inoculating individual colonies in LB medium with ampicillin (100 µg/ml) and incubating them for 12-16 hours at 37°C. The presence of inserts in the clones was verified by EcoRI digestion, and subsequently by Sanger sequencing using T7 and T3 primers (Core Facility of Genomics, Institute of Genomics, University of Tartu).