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This Article Full Text Full Text (PDF) [Supplemental Tables] All Versions of this Article: 80/1/194    most recent biolreprod.107.064691v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal My Folders Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by May, A. Articles by Haaf, T. Search for Related Content PubMed PubMed Citation Articles by May, A. Articles by Haaf, T. Agricola Articles by May, A. Articles by Haaf, T.

Multiplex RT-PCR Expression Analysis of Developmentally Important Genes in Individual Mouse Preimplantation Embryos and Blastomeres¹

Institute for Human Genetics,³ Johannes Gutenberg University Mainz, 55101 Mainz, Germany Advalytix AG,⁴ 81377 Munich, Germany Institute of Medical Biostatistics, Epidemiology and Informatics,⁵ Johannes Gutenberg University Mainz, 55101 Mainz, Germany

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 (1-cell, 2-cell, 4-cell, and morula stage) embryos and in all analyzed blastomeres from 16-cell embryos, indicating a 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 the following 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 most or all nuclei of 40–60-cell embryos is a good indicator of functional activity of genes that are activated by the 16-cell stage.

blastomere, developmental biology, DNA methyltransferase, early development, embryo, gene regulation, genome reprogramming, in vitro fertilization, methyl-CpG-binding domain protein, multiplex RT-PCR, preimplantation development, single-cell analysis

¹Supported by research grant HA 1374/9-1 from the German Research Foundation.

Correspondence: ²FAX: 49 6131 175690; e-mail: haaf{at}humgen.klinik.uni-mainz.de