111 CLONING AND CHARACTERIZATION OF PIG VASA HOMOLOG GENE AND ITS SPECIFIC EXPRESSION IN GERM CELL LINEAGE

Abstract

All of the vasa homologue genes in C. elegans (Caenorhabditis elegens, a free-living soil nematode), xenopus, zebrafish, mouse, human, chicken, trout, and rat exhibited a germ line-specific expression and are used as specific molecular probes to distinguish the developmental profile of germ cells. In order to determine a useful marker for the research of germ cell commitment and development in pigs, we investigated the cloning and expression profile of porcine vasa homolog gene (Pvh). A Pvh cDNA gene of size 2172 bps (submitted to NCBI gene Bank No. AY626785) was cloned from pig ovary by reverse transcription-polymerase chain reaction (RT-PCR) amplification. The amplification was repeated three times and each RT-PCR product was sequenced. The isolated cDNA had 724 deduced amino acids with significant homology to mouse (85%) or human (91%) vasa. The Pvh sequence presents five copies of the RGG motifs and the DEAD box. By RT-PCR amplification, the expression of Pvh mRNA was restricted to the ovary and testis and was undetectable in somatic tissues including brain, whole blood, heart, lung, kidney, spleen, intestine, and liver. When analyzed by RT-PCR amplification, during pre-implantation embryo development, Pvh was transcribed in oocytes and fertilized 2-cell embryos (no difference in the expression levels between oocytes and fertilized 2-cell embryos), but not in 4-cell, 8-cell, morula and blastocyst stages. Using mouse vasa antibody (kindly donated from Dr. Noce, Japan; tested in porcine cells with porcine oocytes and mouse oocytes as positive control and with porcine brain cells as negative control), immunohistochemical analysis of fetal (Day 100) and adult gonad sections revealed that Pvh protein was specifically expressed in proliferating primordial germ cells (PGC), oocytes and spermatocytes. Interestingly, Pvh protein was not expressed in embryonic germ cells, but it was strongly expressed in freshly isolated PGC. Our results indicate that Pvh gene is specifically transcribed in pig germ cells. This study was supported by grants from the Korean Ministry of Science and Technology (Biodiscovery) and the Biogreen 21-1000520030100000.