Expression profiles of the pluripotency marker gene POU5F1 and validation of reference genes in rabbit oocytes and preimplantation stage embryos

Abstract

Background: The surge in the number of gene expression studies and tendencies to increase the quality of analysis have necessitated the identification of stable reference genes. Although rabbits are classical experimental model animals, stable reference genes have not been identified for normalization. The aims of this study were to compare the expression profiles of the widely used reference genes in rabbit oocytes and preimplantation stage embryos, and to select and validate stable ones to use as reference.

Candidate gene expression patterns in rabbit preimplantation embryos developed in vivo and in vitro

“…The expression of candidate genes namely, OCT4, SOX2, NANOG, KLF4, HDAC1 and BCLXL were normalised with endogenous expression levels of reference genes namely, H2AFZ, HPRT1 and YWHAZ. These genes showed stable expression levels in all the developmental stages and thus agreeing with previous reports on rabbit preimplantation development [37].…”

“…In the current study, OCT4 transcripts were present in all stages of preimplantation development. The level of OCT4 declined in early stages of preimplantation development and increased post ZGA, which was in agreement with the earlier observations on OCT4 or POU5F1 expression in in vitro developed rabbit preimplantation embryos [37] and bovine preimplantation embryos [68]. But this observation was in discordance with what was observed in the porcine preimplantation development, where ZGA coincided with the peak expression of OCT4 [62].…”

“…Reference genes were selected based on reports demonstrating that H2A, HPRT1 and YWHAZ were expressed stably throughout the rabbit preimplantation embryo development [37]. Data analysis was done based on the geometric averaging and normalisation method described as in geNorm [44].…”

“…Earlier studies on rabbit embryo development were confined to the OCT4 expressions at transcriptional and translational levels [37][38][39][40][41] though it is very well established that BCLXL, HDAC1, SOX2, NANOG and KLF4 are also crucial for preimplantation embryo development. As more studies are needed in the context of preimplantation embryo development, this study was an attempt for the first time to monitor and compare expression of SOX2, NANOG, KLF4, HDAC1 and BCLXL simultaneously in addition to OCT4, both at the transcriptional and translational levels in rabbit preimplantation developmental stages (pronuclear, 2-cell, 4-cell, 8-cell, 16-cell, morula and blastocyst) between in vivo fertilized embryos, which were developed in vivo and in vitro.…”

Characterization of ovine herpesvirus 2-induced malignant catarrhal fever in rabbits

“…The specificity of the primers was confirmed by gel electrophoresis and sequencing of the PCR products. The set of primers used to amplify ORF73 and YWHAZ were previously described (Mamo et al, 2008;Meier-Trummer et al, 2009a). Nucleic acid amplification and detection of all genes were performed using a CFX96 TM real-time PCR Detection System (Bio-Rad).…”