Abstract
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 studies is to provide a readily available resource for the study of gene expression during human preimplantation development.
Methods:Rapid, reproducible, and efficient procedures for the construction of PCR-based cDNA libraries from fewer than 10 cells were first developed in small 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 libraries as template allows the analysis of stage-specific expression of embryonic genes. Genomic libraries are also prepared from parental samples (cumulus cells and sperm) corresponding to the individual embryo generating the cDNA library.
Results:The complexities (between 105and 106clones) of the human embryo libraries indicate that they may represent the entire active gene population at these early stages of human development. Nucleotide sequence analyses of random clones showed the presence of a variety of transcripts, such as the human transposable element, LINE-1, Alu repeat sequences, housekeeping genes, and tissue-specific genes, (e.g., α-globin, FMR-1, and interleukin-10). Also present at the expected frequency are the ubiquitous cytoskeletal elements, β-actin. keratin-18, and α-tubulin. In addition to cDNAs corresponding to known expressed sequence tags (ESTs) in the GenBank and dbEST databases, a high proportion of novel sequences was also detected. Several cDNAs were detected only at specific stages of preimplantation development by the differential display analysis.
Conclusions:PCR-based cDNA libraries from single human preimplantation embryos 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 specific genes, and hence the diseases resulting from mutation of these genes, and provide information about new methods of preimplantation diagnosis. The molecular analysis of early gene transcription in human embryogenesis may be expected to lead to advances in contraception, assisted reproduction, and preimplantation genetic diagnosis.
Keywords: human preimplantation embryos, cDNA libraries, gene expression, genomic imprinting, zygotic gene activation
Full Text
The Full Text of this article is available as a PDF (552.2 KB).
REFERENCES
- 1.Adams MD, Kelley JM, Gocayne JD, Dubnick M, Polymeropoulos MH, Xiao H, Merril CR, Wu A, Olde B, Moreno RF, Kerlavage AR, McCombie WR, Venter JC. Complementary DNA sequencing: Expressed sequence tags and human genome project. Science. 1991;252:1651–1656. doi: 10.1126/science.2047873. [DOI] [PubMed] [Google Scholar]
- 2.Adjaye J, Daniels R, Bolton V, Monk M. cDNA libraries from single human preimplantation embryos. Genomics. 1997;46:337–344. doi: 10.1006/geno.1997.5117. [DOI] [PubMed] [Google Scholar]
- 3.Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988;239:487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
- 4.Smith DE, Gridley T. Differential screening of a PCR-generated mouse embryo cDNA library: Glucose transporters are differentially expressed in early postimplantation mouse embryos. Development. 1992;116:555–561. doi: 10.1242/dev.116.3.555. [DOI] [PubMed] [Google Scholar]
- 5.Buraczynska MJ, Van Keuren ML, Buraczynska KM, Chang YS, Crombez E, Kurnit DM. Construction of human embryonic cDNA libraries: HD, PKD1 and BRAC1 are transcribed widely during embryogenesis. Cytogenet Cell Genet. 1995;71:197–202. doi: 10.1159/000134106. [DOI] [PubMed] [Google Scholar]
- 6.Rothstein JL, Johnson D, DeLoia JA, Skowronski J, Solter D, Knowles B. Gene expression during preimplantation mouse development. Genes Dev. 1992;6:1190–1201. doi: 10.1101/gad.6.7.1190. [DOI] [PubMed] [Google Scholar]
- 7.Temeles GL, Ram PT, Rothstein JL, Schultz RM. Expression patterns of novel genes during mouse preimplantation embryogenesis. Mol Reprod Dev. 1994;37:121–129. doi: 10.1002/mrd.1080370202. [DOI] [PubMed] [Google Scholar]
- 8.Weng DE, Morgan RA, Gearhart JD. Estimates of mRNA abundance in the mouse blastocyst based on cDNA library analysis. Mol Reprod Dev. 1989;1:233–241. doi: 10.1002/mrd.1080010403. [DOI] [PubMed] [Google Scholar]
- 9.Bevan SG, Southey MC, Armes JE, Venter DJ, Newgreen DF. Spatiotemporally exact cDNA libraries from Quail embryos: A resource for studying neural crest development and neurocristopathies. Genomics. 1996;38:206–214. doi: 10.1006/geno.1996.0617. [DOI] [PubMed] [Google Scholar]
- 10.Beliavsky A, Vinogradova T, Rajewsky K. PCR-based cDNA library construction: General cDNA libraries at the level of a few cells. Nucleic Acids Res. 1989;17:2919–2932. doi: 10.1093/nar/17.8.2919. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratories Press; 1989. [Google Scholar]
- 12.Liang P, Pardee AB. Differential display of eucaryotic messenger RNA by means of the polymerase chain reaction. Science. 1992;257:967–971. doi: 10.1126/science.1354393. [DOI] [PubMed] [Google Scholar]
- 13.Daniels R, Lowell S, Bolton V, Monk M. Transcription of tissue-specific genes in human preimplantation embryos. Hum Reprod. 1997;12:2251–2256. doi: 10.1093/humrep/12.10.2251. [DOI] [PubMed] [Google Scholar]
- 14.Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
- 15.Zimmermann JW, Schultz RM. Analysis of gene expression in the preimplantation mouse embryo: Use of mRNA differential display. Proc Natl Acad Sci USA. 1994;91:5456–5460. doi: 10.1073/pnas.91.12.5456. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Nothias J-Y, Majumder S, Kaneko KJ, DePamphilis ML. Regulation of gene expression at the beginning of mammalian development. J Biol Chem. 1995;270:22077–22080. doi: 10.1074/jbc.270.38.22077. [DOI] [PubMed] [Google Scholar]
- 17.Schultz RM. Regulation of zygotic gene activation in the mouse. BioEssays. 1993;15:531–538. doi: 10.1002/bies.950150806. [DOI] [PubMed] [Google Scholar]
- 18.Diatchenko L, Lau Y. C., Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, Siebert PD. Suppression subtractive hybridisation: A method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA. 1996;93:6025–6030. doi: 10.1073/pnas.93.12.6025. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Daniels R, Kinis T, Serhal P, Monk M. Expression of the myotonin protein kinase gene in preimplantation human embryos. Hum Mol Genet. 1995;4:389–393. doi: 10.1093/hmg/4.3.389. [DOI] [PubMed] [Google Scholar]
- 20.Cross SH, Bird AP. CpG islands and genes. Curr Opin Genet Dev. 1995;5:309–314. doi: 10.1016/0959-437x(95)80044-1. [DOI] [PubMed] [Google Scholar]
- 21.Reed ML, Leff SE. Maternal imprinting of human SNRPN, a gene deleted in Prader-Willi syndrome. Nature Genet. 1994;6:163–167. doi: 10.1038/ng0294-163. [DOI] [PubMed] [Google Scholar]
- 22.Mc Allister G, Amara SG, Lerner MR. Tissue-specific expression and cDNA cloning of small nuclear ribonucleoprotein-associated polypeptide N. Proc Natl Acad Sci USA. 1988;85:5296–5300. doi: 10.1073/pnas.85.14.5296. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Barlow DP. Gametic imprinting in mammals. Science. 1995;270:1610–1613. doi: 10.1126/science.270.5242.1610. [DOI] [PubMed] [Google Scholar]