THE CONSTRUCTION OF CDNA LIBRARIES FROM HUMAN SINGLE PREIMPLANTATION EMBRYOS AND THEIR USE IN THE STUDY OF GENE-EXPRESSION DURING DEVELOPMENT

Purpose: The construction and application of polymerase chain reaction (PCR)-based cDNA libraries from unfertilized human oocytes and single preimplantation-stage embryos are described. The purpose of these stu dies is to provide a readily available resource for the study of gene expression during human preimplantation development. Methods: Rapid, r eproducible, and efficient procedures for the construction of PCR-base d cDNA libraries from fewer than 10 cells were first developed in smal l populations of fibroblast cells. We then constructed cDNA libraries from eight unfertilized oocytes and single two-cell, -4-cell, -7-cell, and blastocyst-stage embryos. Differential display PCR using the libr aries as template allows the analysis of stage-specific expression of embryonic Re,les. Genomic libraries are also prepared from parental sa mples (cumulus cells and sperm) corresponding to the individual embryo generating the cDNA library. Results: The complexities (between 10(5) and 10(6) clones) of the human embryo libraries indicate that they ma y represent the entire active gene population at these early stages of human development. Nucleotide sequence analyses of random clones show ed the presence of a variety of transcripts, such as the human transpo sable element, LINE-1. Also repent sequences, housekeeping genes, and tissue-specific genes, (e.g., alpha-globin, FMR-1, and interleukin-10) . Also present at the expected frequency are the ubiquitous cytoskelet al elements. beta-actin, keratin-18, and alpha-tubulin. In addition to cDNAs corresponding to known expressed sequence tags (ESTs) in the Ge nBank and dbEST databases, a high proportion of nov el sequences rt as also detected Several cDNAs were detected only at specific stages of preimplantation development by the differential display analysis. Conc lusions: PCR-based cDNA libraries from single human preimplantation em bryos provide a new and important resource for the identification and study of novel genes or gene families. As such, they will increase our basic understanding of the molecular control of human development. In the clinical context, the libraries identify the time of onset of spe cific genes, and hence the diseases resulting from mutation of these g enes, and provide information about new methods of preimplantation dia gnosis. The molecular analysis of early gene transcription in human em bryogenesis may be expected to lead to advances in contraception, assi sted reproduction, and preimplantation genetic diagnosis.