201 IDENTIFICATION OF CANDIDATE X (INACTIVE)-SPECIFIC TRANSCRIPT (XIST) CODING REGION BY HOMOLOGY ANALYSIS OF SEQUENCE AND RELATIVE EXPRESSION ANALYSIS ON FERTILIZED EMBRYO AND PARTHENOTES IN PIG

Abstract

X (inactive)-specific transcript (XIST) has been known as long noncoding RNA, which regulates X-chromosome inactivation in female mammals to provide dosage equivalence between male and females. Its important roles in early embryo and stem cells development have been suggested in humans and mice. However, its coding region has been unclear in pigs. To determine the coding region of XIST in pigs, we examined candidate XIST coding region using BLAST, PCR, and sequencing techniques. By comparing pig whole genome sequence (Sus scrofa 10.2) with human, murine, and bovine XIST transcript sequence using BLAST, we selected candidate coding regions of XIST in pig. The result showed that XIST is coded on the minus strand of NW_003612825 contig, and its length was nearly 32 kb, which was similar to the length of human and bovine XIST genes. In order to identify the coding region of XIST in this candidate sequence, we designed 24 primer pairs and RT-PCR was performed using porcine embryonic fibroblast cells (PEF) established from porcine male and female lines.The XIST transcripts were expressed in female PEF but not in male PEF. Thus, by designing candidate intron-spanning primers, we confirmed the existence of introns between the first and last exons. The amplicon sequence was analysed and 5 short exons (<400 bp) that have similarity to the XIST coding region of other characterised species were identified. Moreover, to examine the XIST coding strand in pig genome, we conducted strand-specific reverse transcription. With BLAST homology searches, we confirmed that XIST was coded on the negative strand of the contig on the X chromosome. To analyse stage and origin of XIST transcript in the pig, we subsequently performed real-time PCR using cDNA template synthesised from sperm, oocyte, 1, 2, 4, 8, morular, and blastocyst of fertilized embryo and parthenotes, and intron-spanning primer sets were used. The results revealed that XIST was expressed after maternal to zygote transition (4- to 8-cell stage in pig) and not expressed in mature germ cells (sperm and oocyte). Moreover, parthenotes showed a higher expression level compared with fertilized embryos after the 8-cell stage. These results verify the existence of XIST in pig early embryos and may contribute to the comparative analysis of XIST mechanisms in different species. Further study using FISH and methylation analysis may reveal its mechanisms of action.

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST; No. 2012006276) and the Biogreen 21 program (PJ0081382011) of the Rural Development Administration, Republic of Korea.