Elsevier

Gene

Volume 237, Issue 2, 17 September 1999, Pages 373-383
Gene

Developmental expression of specific genes detected in high-quality cDNA libraries from single human preimplantation embryos

https://doi.org/10.1016/S0378-1119(99)00329-7Get rights and content

Abstract

We describe an improved highly sensitive method for generating cDNA libraries containing a high proportion of cDNAs enriched with 5′-coding sequences from single human preimplantation embryos and a 10 week old whole foetus. The embryonic mRNA was isolated using oligo-(dT) linked to magnetic beads. First-strand cDNA synthesis was carried out directly on the bound mRNA, followed by PCR designed to amplify the cDNA molecules synthesized in their entirety. The complexities of the libraries are between 105 and 106 independent clones. The average cDNA size is 1.0 kb, and the size range is 0.5–3.0 kb. PCR analysis of the embryonic libraries for specific genes has revealed transcripts for genes known to be transcribed in preimplantation stages, such as the imprinted gene SNRPN, developmental genes WNT11, HOX, OCT-1 and the embryonic OCT-4, cytoskeletal genes keratin-18 and β-actin, the cell cycle gene C-MOS, and housekeeping genes GAPDH and HPRT. Sequencing of random clones showed the presence of a variety of sequences, such as human chorionic gonadotrophin, ubiquitin, TFIIA, guanine nucleotide-binding protein (β-subunit), annexin I, a gene encoding a kinesin-like protein, and TWIST, which encodes a basic helix–loop–helix (bHLH) transcription factor implicated in Saethre–Chotzen syndrome (characterized by craniofacial and limb anomalies). Approximately 40% of these randomly analysed clones were full length. In addition to cDNAs matching known ESTs (Expressed Sequence Tags) in the GenBank and dbEST databases, novel sequences were detected at a frequency of 16% of randomly picked clones. The libraries are a valuable resource, providing longer cDNAs representing genes expressed during human preimplantation development.

Introduction

The earliest stages of development in mammals are regulated by maternally inherited information. During the transition from maternal to embryonic control of development, which begins immediately after fertilization and is probably complete by the blastocyst stage in the human, maternal transcripts (common to both the unfertilized oocyte and early embryo) are depleted, and embryo-specific transcripts involved in early embryogenesis are generated. Key developmental events occur in the embryo before implantation, resulting in changes in cell shape at compaction of the eight-cell embryo and segregation of cells to the inner cell mass (ICM) and the trophectoderm (TE) of the blastocyst. These cell lineages differ in morphology, function, and developmental fate. The ICM is initially a non-differentiated, proliferating stem-cell population with the capacity to form all the tissues of the foetus. The trophectoderm cells are typically epithelial and, following proliferation, form extraembryonic structures such as the placenta. It is assumed that differential gene expression underlies the delineation of these two cell types. Despite the fundamental importance of this transition from maternal to embryo-coded gene transcription, and the observed morphological changes in the developmental program, very little is known about the molecular basis of embryonic gene activation in mammals (especially humans) and the repertoire of genes involved.

So far, detailed studies of gene expression in preimplantation human embryos have been limited to previously characterized genes using such methods as in-situ hybridization with labelled antisense probes and reverse transcription-polymerase chain reaction (RT-PCR) amplification with known primer sequences. Such analyses have been hampered by the scarcity and small size of human embryos, and there has been little opportunity to screen for the many human gene sequences that are rapidly becoming available through the various EST (Expressed Sequence Tags) databases. Another limitation to these approaches is that only known sequences can be analysed, and the available ESTs only comprise genes expressed during the later stages of foetal development and the adult. Thus, genes expressed specifically during human preimplantation development remain unidentified.

To overcome these limitations, we developed a reproducible technique for generating cDNA libraries from single embryos (Adjaye et al., 1997, Adjaye et al., 1998). These original libraries contained a high proportion of partial cDNAs restricted to the 3′-regions of genes. In this paper, we describe a new improved method for producing high-quality cDNA libraries enriched with longer cDNAs extending into the more 5′-coding sequences (Sasaki et al., 1998). Human oocyte and single embryo (two-cell to blastocyst) cDNAs are synthesized directly from purified mRNAs bound to oligo-(dT)-linked magnetic beads. This solid-phase cDNA synthesis is an established technique that has been used previously to obtain reusable pools of first-strand cDNA from a few cells (Roeder, 1998).

We show that the embryonic cDNA libraries show high complexities (105–106 cfu), are representative of embryonic cells and contain longer cDNA inserts than previously reported human cDNA libraries (Adjaye et al., 1997, Verlinsky et al., 1998). We demonstrate the expression of OCT4, a marker gene for pluripotent cells, thus confirming the embryonic origin of these libraries. A number of other developmental genes, the imprinted gene, SNRPN, cytoskeletal and cell cycle genes are also shown to be expressed in these preimplantation stages of development. The libraries are shown to be an excellent resource for carrying out research on gene expression during human embryogenesis.

Section snippets

Human oocytes and embryo samples

Human oocytes and embryos derived by in-vitro fertilization (IVF), and forming part of a research project on embryo culture conducted by V. Bolton, were donated for research with consent by patients attending the Assisted Conception Unit, King's College Hospital (KCH) London. Pituitary suppression, ovarian stimulation and oocyte retrievals were carried out as described previously (Waterstone and Parsons, 1992). IVF was performed as described previously (Bolton et al., 1989) except that the

Preparation of the cDNA libraries and screening for specific genes by PCR

The cDNA synthesis and long distance (LD) PCR amplification strategy (Barnes, 1994) were employed to prepare cDNA libraries from mRNA samples isolated from four unfertilized oocytes, single two-cell, four-cell, eight-cell and blastocyst stage embryos, and a 10 week old whole foetus. The procedure is shown in the diagram in Fig. 1. Important features of this method are: (1) all reactions are performed directly on the oligo-(dT) magnetic beads (solid phase) thus minimizing losses that might occur

Discussion

The construction of high-quality PCR-based cDNA libraries from human unfertilized oocytes and single two-cell, four-cell, eight-cell and blastocyst stage embryos is described. A library from a 10 week gestation whole foetus was also prepared to serve as a somatic control, representing all tissues, for comparisons of gene expression in embryonic and somatic cells. The complexities of the libraries are between 105 and 106 (cfu), and we may expect that they are fully representative of genes

Acknowledgments

The use of embryos is covered by HFEA licence number R0063 to Dr Virginia Bolton.

This work was supported by the Medical Research Council.

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