239 IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN PORCINE PARTHENOTES AT 2-CELL AND BLASTOCYST STAGES USING ANNEALING CONTROL PRIMER TECHNOLOGY

Abstract

Identification of embryo-specific genes will provide insight into early embryonic development. However, the current methods employed to identify genes expressed at specific stages are labor intensive and produce a high degree of false positives. In the present study we employed an accurate PCR technology controlled by an annealing control primer (ACP, SeeGene, Seoul, Korea) to identify differentially expressed genes in presumptive porcine parthenotes. In vitro-matured porcine oocytes were parthenogenetically activated by square electrical direct current pulses. After 3 h of culture in North Carolina State University (NCSU) 23 medium with 0.4% BSA containing 7.5 mg mL⁻¹ cytochalasin B, embryos were washed and cultured in NCSU 23 medium with 0.4% BSA. Total RNA was prepared from a pool of 200 2-cell stage and a pool of 100 blastocyst-stage porcine parthenotes using a Dynabeads mRNA DIRECT Kit. First-strand cDNA synthesis was carried out using dT-ACP1, wherein the 3′-end core portion comprised a hybridizing sequence complementary to a poly (A) region of mRNA transcripts. Second-strand cDNAs were synthesized using PCR with an arbitrary ACP whereby the 3′-end core portion of the dT-ACP2 was prevented from annealing to the first-strand. The PCR products were electrophoresed on 2% agarose gel and stained with ethidium bromide. The differentially expressed bands were extracted and cloned into a TOPO TA cloning vector (Invitrogen, Seoul, Korea), sequenced and analyzed by BLAST search. To date we have identified 21 differentially expressed genes using 28 arbitrary ACPs. BLAST analysis revealed that most of these genes have strong homology (more than 95%) with known mouse and human genes. Differentially expressed genes in the 2-cell stage include GDP dissociation, putative translation initiation factor (SUI1) and succinate dehydrogenase iron-protein subunit. The blastocyst specific genes include rennin binding proteins, ATPase inhibitor, catenin, ribosomal protein S15a, ribosomal protein large P1, puromycin sensitive aminopeptidase, BMDP gene, arginine N-methyltrasferase p77 isoform, acylamino acid-releasing enzyme, ribosomal protein S14 and KIAA0630 protein. RT-PCR confirmed the expression of these genes in porcine parthenotes during preimplantation development. In conclusion, ACP technology is a specific and sensitive method to locate differentially expressed genes in preimplantation mammalian embryos.