The use of amplified cDNA to investigate the expression of seven imprinted genes in human oocytes and preimplantation embryos.
Imprinted genes are characterized by expression of only one of the two alleles according to its inheritance from the mother or the father. This mono-allelic expression must arise from primary differential epigenetic modification of the parental alleles of the imprinted gene in the spermatozoon and the oocyte. Most of the information on the onset of imprinted gene expression, and on the molecular mechanisms regulating mono-allelic expression, have been derived from studies in the mouse. In this paper, we investigate the expression of seven imprinted genes in human preimplantation development. Due to limitations imposed by the rarity of human embryos available for research, our approach has been to screen amplified cDNA preparations prepared from human unfertilized oocytes and individual embryos at each of the 4-cell, 8-cell and blastocyst stages. Gene-specific primers were used to investigate expression of the imprinted genes by polymerase chain reaction (PCR) analysis of these amplified cDNA. We found that expression is inherently variable in the amplified cDNA from embryo to embryo but the use of several samples at each stage showed that the SNRPN, UBE3A and PEG1 genes are expressed throughout human preimplantation development. This was confirmed by direct analysis by gene-specific reverse transcription-PCR on a limited number of lysed embryos (one gene analysed per embryo). Thus, the amplified cDNA may be used to rapidly identify those imprinted genes expressed in preimplantation development and, hence, those genes amenable to investigation of the epigenetic mechanisms regulating mono-allelic expression. Confirmation of preimplantation expression also identifies those imprinted diseases amenable to preimplantation diagnosis, and the imprinted genes which may be used in assessment of possible perturbations of imprinting following new procedures in assisted reproduction. Our series of single embryo amplified cDNA are established as a valuable resource for comparative studies of gene expression within one embryo and between embryos throughout early human development. The amplified cDNA thus circumvent the need for a continuous supply of human embryos for studies on embryonic gene expression.
Salpekar
A
A
Molecular Embryology Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK.
Huntriss
J
J
Bolton
V
V
Monk
M
M
D018076:DNA, Complementary / Q000235:genetics*; D047108:Embryonic Development / Q000235:genetics*; D005260:Female; D005784:Gene Amplification; D020869:Gene Expression Profiling / Q000379:methods; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D018392:Genomic Imprinting / Q000235:genetics*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D009865:Oocytes / Q000502:physiology*; D011247:Pregnancy
D018076:DNA, Complementary