Embryo

Multiplex RT-PCR Expression Analysis of Developmentally Important Genes in Individual Mouse Preimplantation Embryos and Blastomeres

Abstract
We have developed a microfluidic chip-based qualitative assay for sensitive (10 RNA copies) detection of multiple transcripts in single cells. We determined the expression patterns of 17 developmentally important genes and isoforms in individual mouse preimplantation embryos from superovulated matings and blastomeres. The ubiquitously expressed histone variant H3f3a and the transcription factor Pou5f1 generated mRNA-derived products in all analyzed (one-cell, two-cell, four-cell and morula-stage) embryos and in all analyzed blastomeres from 16-cell embryos, indicating a rather uniform reactivation of pluripotency gene expression during mouse preimplantation development. In contrast, mRNA expression of different methyltransferases for DNA methylation, methylcytosine-binding proteins for chromatin modification, and base excision repair enzymes, which may provide a mechanism for active demethylation, varied considerably between individual cells from the same embryo and even more dramatically between cells from different embryos. We conclude that at a given point in time the transcriptome encoding the reprogramming machinery and, by extrapolation, genome reprogramming differs between blastomeres. By studying the cell-to-cell variability in gene expression, we can distinguish two classes: mouse 16-cell embryos in which most cells express the reprogramming machinery and embryos in which most cells do not contain detectable mRNA levels of DNA and chromatin modification genes. Immunolocalization of DNMT3A, MBD3, APEX1, and LIG3 in the majority or all nuclei of 40-60-cell embryos is a good indicator of functional activity of genes that are turned on by the 16-cell stage.

Key words: Embryo • Developmental biology • Early development • Gene regulation • In vitro fertilization