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Stage‐specific expressed sequence tags obtained during preimplantation bovine development by differential display RT‐PCR and suppression subtractive hybridization
Author(s) -
Ponsuksili Siriluck,
Tesfaye David,
ElHalawany Nermin,
Schellander Karl,
Wimmers Klaus
Publication year - 2002
Publication title -
prenatal diagnosis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.956
H-Index - 97
eISSN - 1097-0223
pISSN - 0197-3851
DOI - 10.1002/pd.501
Subject(s) - suppression subtractive hybridization , biology , differential display , blastocyst , inner cell mass , expressed sequence tag , embryo , microbiology and biotechnology , complementary dna , cdna library , gene , genetics , embryogenesis
Differential display RT‐PCR (DDRT‐PCR) and suppression subtractive hybridization (SSH) were applied in order to detect preimplantation stage‐specific expressed sequence tags (ESTs) of bovine embryos. Seventeen ESTs were detected from the differential display RT‐PCR approach. All clones but two showed homology to genes or ESTs known in human, cattle or other species. One of the clones similar to H. sapiens mRNA for KIAA1764 protein was used exemplarily to quantify the transcripts by real‐time PCR. The result of quantitative differential screening was found to be in agreement with DDRT‐PCR banding patterns. In the second approach, a blastocyst‐stage enriched cDNA library was constructed using SSH of blastocyst versus morula transcripts. The 71 clones that were analysed represent 33 distinct loci including candidate genes for the regulative processes during differentiation of inner cell mass (ICM) and trophoblast cells and the initial phase of embryo implantation, such as galectin‐3 and fibronectin. As revealed by real‐time PCR, the mRNA level of galectin‐3 was three times higher in the blastocyst stage than in the morula stage. DDRT‐PCR and SSH are both powerful tools for the identification of stage‐specific expressed gene in preimplantation bovine embryos. Real‐time PCR allows to test and confirm the outcome and to add quantitative data of selected transcripts of interest. Copyright © 2002 John Wiley & Sons, Ltd.