Identification of differentially regulated genes in bovine blastocysts using an annealing control primer system

Abstract

The identification of embryo-specific genes would provide insights into early embryonic development. However, the current methods employed to identify the genes that are expressed at a specific developmental stage are labor intensive and suffer from high rates of false positives. Here we employed a new and accurate reverse transcription-polymerase chain reaction (RT-PCR) technology that involves annealing control primers (ACPs) to identify the genes that are specifically or prominently expressed in bovine early blastocysts and hatched blastocysts produced in vitro. Using these techniques, a total of nine expressed sequence tags (ESTs) of genes that were differentially expressed in hatched blastocysts, as compared to blastocyst embryos, were cloned and sequenced. The cloned genes or ESTs (C1-C9) all exhibited significant sequence similarity with known bovine genes (99-100%; FTL, RPS12, LAPTM4a, and RPL12) or ESTs (80-94%; AIBP, CULLIN-1, HDLP, COX5a, and RECS1) of other species. As revealed by real time RT-PCR, these genes were regulated upstream in the hatched blastocyst stage during early implantation. These results suggest that this new, PCR-based differential display RT-PCR technique is a very useful tool for the identification of stage-specific genes of preimplantation embryos.

Tenascin-X and leukemia inhibitory factor receptor are down-regulated in leiomyoma compared with normal myometrium

“…5). 15 However, there are insufficient number of studies which have used the ACP system, so methodologic role of ACP systems for translational research has not been yet proven clearly.…”

Reduced Mitochondrial Properties in Putative Progenitor/Stem Cells of Human Keratinocytes

“…2B). which contains distinct 3'-and 5'-end regions separated by a regulator, and the interaction of each portion of this primer during two-stage PCR 12,13 . This system facilitates the identification of DEGs from small samples without generating false positive results 13 .…”

“…The goal of this study was to use the adhesion assay and annealing control primer based differentially displayed polymerase chain reaction (PCR) to further characterize the KSCs. We evaluated whether the ad-hesion assay showed a marked difference in some of the important characteristics of rapid adhering cells compared to non adhering cells by differentially expressed genes (DEGs) 12,13 . Subsequent Western blot analysis was used to confirm the differences observed.…”

Characterization of Expressed Genes Under Ozone Stress in Soybean

“…To detect DEGs transcribed at low levels, highly specific polymerase chain reaction (PCR) amplification is required. Novel annealing control primer (ACP)-based DDRT-PCR technology has been used to identify differentially expressed genes in tissues Hwang et al (2004). This method specifically targets sequence hybridization to the template via a polydeoxyinosine linker Hwang et al (2003).…”