214 DIFFERENT GENE EXPRESSION OF INDIVIDUAL BLASTOMERES IN EARLY MOUSE EMBRYO DETECTED BY REAL TIME PCR

Abstract

The objective of this study was to detect gene expression differences among blastomeres from 4- and 8-cell stage mouse embryos by a sensitive method. In earlier studies gene expression was determined in pooled or single embryos. With the higher levels of precision afforded by our method, we could quantify mRNA in single blastomeres. For comparison of gene expression, different inner cell mass specific (Oct-4, Nanog) and trophectoderm-specific (Glut-3) genes and the housekeeping beta-actin gene (for reference) were chosen. Late 4- and early 8-cell stage embryos were obtained from oviducts 58 and 66 h post-hCG, respectively. The embryos were collected in CZB-HEPES and the zonae pellucidae were dissolved by acid Tyrode solution. Blastomeres were separated mechanically from each other by gentle pipetting with a fire-polished glass capillary. Messenger RNA was isolated from the frozen blastomeres using the Dynabeads® mRNA DIRECTTM Micro Kit (Kvalitex Kft., 1136 Budapest, Hungary) following the manufacturer's protocol. The reverse transcription reactions were performed at 25°C for 10 min, and then at 42°C for 1 h followed by 99°C for 5 min. Real-time PCR was performed in an iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories, 1094 Budapest, Hungary). Reaction mixtures, which were separated for each gene, consisted of QuantiTect iQ SYBR Green Supermix (Bio-Rad Laboratories), 500 nm of both primers, and a 1/8 aliquot of the blastomere cDNA. The cDNA template was denatured by heating to 95°C for 2 min and amplified by 50 cycles of 95°C for 20 s, 60°C for 20 s, and 72°C for 20 s, with a single fluorescence measurement at each cycle. When the reaction was finished, melting curves were plotted to confirm product purity. By this procedure we could measure the expression level of four genes in six 4- and 8-cell-stage embryos. Glut-3 expression was not detectable in the blastomeres of 4-cell stage embryos. In spite of the 1.3–1.5-fold technical error, Oct-4 and Nanog showed 10-fold and 9-fold differences, respectively. Among the blastomeres of the same embryo, one of the 4-cell-stage showed notably high expression of Oct-4. In the 8-cell stage blastomeres Oct-4 expression showed slight differences (2–8-fold), and Nanog and Glut-3 showed 28-fold and 12-fold differences, respectively. Two of the 8-cell blastomeres showed notably high expression of Nanog. In conclusion, this method was accurate and sensitive enough to measure the expression of different genes in a single blastomere. We found that the mRNA levels of Oct-4 and Nanog have remarkably discrete differences within the blastomeres of a 4-cell-stage embryo. Expression of Nanog gene showed notable differences within blastomeres of an 8-cell-stage embryo. These differences may show a very early sign of the cell fate determination. This work was funded by the Wellcome Trust International Senior Fellowship and Bio-Rad Laboratories.