Molecular Human Reproduction, Vol. 6, No. 9, 801-809,
September 2000
© 2000 European Society of Human Reproduction and Embryology
Embryo development |
Detection of human novel developmental genes in cDNA derived from replicate individual preimplantation embryos
1 Molecular Embryology Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, and 2 Assisted Conception Unit, Academic Department of Obstetrics and Gynaecology, Kings College School of Medicine and Dentistry, Denmark Hill, London, SE5 9RS, UK
Abstract
We have constructed amplified cDNA preparations from replicate samples of human oocytes and individual preimplantation embryos. Differential display of the cDNA preparations shows disparate patterns of gene expression in the individual embryos at all stages of preimplantation development. The variation in patterns of genes expressed is in part due to the low starting cell number undergoing the reverse transcription–polymerase chain reaction (RT–PCR) step in the preparation of the amplified cDNAs. Despite this variability, the use of replicate embryo samples makes it possible to identify and isolate human genes specifically expressed at the different stages of human preimplantation development from the unfertilized oocyte to the blastocyst stage.
cDNA libraries/differential display/developmental genes/human/preimplantation embryo
Introduction
The molecular programming of human early development has been difficult to study due to the rare availability of the embryo samples and the associated ethical considerations. These limitations to studies on gene expression in human embryos would be overcome to some extent if sensitive procedures could be developed to create amplified cDNA preparations, and cDNA libraries, representing the genes expressed at different stages of early development. Such a resource would enable extensive studies of gene expression in development, comparative studies within a single embryo, and the isolation of novel human developmental genes, without the continual requirement for additional human embryos.
Previously, we have reported procedures for the creation of cDNA libraries from single preimplantation embryos (Adjaye et al., 1997
, 1999). However, these initial libraries were limited to one embryo from each of the 2-, 4-, 8-cell and blastocyst stages so that reproducibility of results could not be confirmed. In addition, the cleavage embryos available at the time were those that failed to show ongoing development and thus may have been atretic and not representative of their stage of morphological development.
In this paper, we report the preparation of a new series of amplified cDNA preparations, from morphologically good quality (grade 1) embryos at the correct developmental stage for age, using four replicate single embryos at each of the 4-, 8-cell and blastocyst stages. We have analysed the overall patterns of gene expression in these embryo cDNAs by differential display to check the representation of mRNAs present and to identify stage specific gene expression. We show highly variable patterns of gene expression from embryo to embryo. In reconstruction experiments, using samples of four and 400 fibroblasts, we demonstrate that some of the variability is due to the low number of starting cells. Thus, although amplified cDNA preparations prepared by these methods may appear to be representative of all expressed embryonic sequences as judged by screening for the presence of specific genes by polymerase chain reaction (PCR), and their frequency by library hybridization (Adjaye et al., 1997, 1999), the differential display screen shows that a variable subset of genes are represented for each single embryo.
Nevertheless, despite the variability introduced by the low cell number in the patterns for the individual embryos, the use of replicate embryos in our work makes it possible to identify and isolate genes specifically expressed in human early development.
Materials and methods
Embryo samples
Human embryos, which were surplus to requirement for infertility treatment, were obtained with the couples' consent at King's College Hospital Assisted Conception Unit (KCH ACU). They were all healthy embryos at the right developmental stage for age and time in culture (Bolton et al., 1989). They were lysed on site at KCH ACU. Individual oocytes and preimplantation embryos were denuded of their zona pellucidae using acid Tyrode's solution and rinsed in PBS. Each was placed in 3 µl of ice-cold lysis buffer [0.8% Igepal (Sigma, UK), 1 IU/µl of RNase inhibitor (Gibco BRL, UK), 5 mmol/l DTT (Gibco BRL, UK)] and snap-frozen in liquid nitrogen and stored at –70°C until RNA extraction. The frozen lysed samples were transferred to the Institute of Child Health (ICH) for mRNA isolation and RT–PCR to create the amplified cDNA preparations. The research at KCH was licensed by the Human Fertilization and Embryo Authority (HFEA; licence number R0062).
Amplified cDNA preparations from fetal primordial germ cells included in some of the differential display experiments were kindly provided by Dr Tetsuya Goto. The germ cells were isolated from human fetuses at 10 weeks gestation, a time when the female germ cells are entering meiosis and the male germ cells are entering mitotic arrest. Differential display of the patterns of gene expression in these male and female primordial germ cells, and the isolation and characterization of genes specifically expressed in the germ cells (male, female or both) and not in the somatic cells have been previously described (Goto et al., 1999).
Fibroblast cells were obtained from the Biochemistry, Endocrinology and Metabolism Unit at the Institute of Child Health. They were washed in phosphate buffered saline and separated into groups of four fibroblasts, and ~400 fibroblasts, and transferred to Eppendorf tubes in 3 µl of lysis buffer exactly as described for embryos and oocytes (see above).
Preparation of polyadenylated RNA
Polyadenylated RNA [hereafter referred to as messenger RNA (mRNA)] molecules were isolated from embryos or fibroblast cells using an oligo(dT)25 nucleotide attached to magnetic beads (Dynabeads mRNA purification kit, Dynal, UK) as follows. Samples in 3 µl of lysis buffer were incubated for 5 min at 65°C to lyse the cells and release RNA. To this, 10 µl of the oligo(dT)25-magnetic beads in suspension were added, the sample was left at room temperature for 30 min and then the beads were collected with the magnetic station (provided with the beads). The beads were washed once in 10 µl of 1x washing buffer (supplied with the beads) and three times with 10 µl of 1x reverse transcription (RT) buffer (Gibco BRL, UK). The isolated mRNA attached to the beads was resuspended in 3 µl of double-distilled (dd) H2O. Total RNA from samples of fetal muscle, gut and brain (mesoderm, endoderm and ectoderm tissues respectively), extracted as described previously (Chirgwin et al., 1979), were subjected to the same bead isolation of mRNA and processed in the same way as the embryos.
cDNA synthesis and PCR amplification of cDNA
The cDNA synthesis was performed in solid phase, i.e. the mRNA was still attached to the beads during the RT procedure. The cDNA synthesis, and PCR amplification of cDNA molecules, were carried out by using SMARTTM PCR cDNA Library Construction Kit (initially the SMART2 primer and, when it became available, the SMART3 primer was used; Clontech, USA) essentially according to the manufacturer's instructions. The Clontech kit has previously been shown (Sasaki et al., 1998) to produce a high proportion of longer cDNAs.
For the reverse transcription step, the whole 3 µl of the resuspended mRNA were incubated for 1 h at 42°C in 10 µl of RT reaction mixture (Clontech, USA) with a 3' oligo(dT) primer (CDS/3' primer, Table I) and Moloney murine leukaemia virus (MMLV) reverse transcriptase (SuperScript II RNaseH- reverse transcriptase, Gibco BRL). The reaction mixture contains the SMART oligo that anneals with the three to five cytosine residues characteristically added by the reverse transcriptase at the 3' end of first strand cDNA molecules synthesized and serves as a further template for the extension of the cDNA by the reverse transcriptase (see Figure 1a). The SMART sequence thus tags the 3' ends of the first strand cDNA molecules synthesized by RT and makes it possible to amplify these cDNA molecules by PCR using the CDS/3' primer and the SMART primer. Tagging with the SMART3 primer allows extension of a 3' cDNA sequence, identified by differential display as showing stage-specific expression (see below), towards its 5' end by 5' rapid amplification of cDNA ends (RACE).
|
|
PCR amplification of the cDNA was carried out using 5 µl from the above RT reaction mixture, in 50 µl of PCR mixture, with initial denaturation for 1 min at 95°C, followed by 24 cycles of 15 s at 95°C and 5 min at 68°C, and final extension for 5 min at 72°C, using the CDS/3' and SMART primers (see Table I
for primer sequences) and Advantage 2 polymerase mix (provided in the Clontech kit) designed to produce longer length sequences (Barnes, 1994).
An aliquot (5 µl) of the amplified cDNA was electrophoresed on a 1% agarose gel to check the distribution of sizes of the cDNA molecules synthesized and amplified, and 1 µl of either a 1 in 5 or a 1 in 10 dilution of the amplified cDNA was used for genespecific PCR for two house-keeping genes, ß-actin and hypoxanthine phosphoribosyl transferase (HPRT) to verify successful reverse transcription and subsequent PCR amplification of cDNA molecules generated. Published sequences of the PCR primers used for the ß-actin and HPRT genes are shown in Table I. The PCR cycling parameters are 35 cycles of 1 min at 95°C, 1 min at 62°C and 1 min at 72°C for ß-actin (Heikinheimo et al., 1995) and 35 cycles of 1 min at 95°C, 1 min at 60°C and 1 min at 72°C for HPRT (modified from Daniels et al., 1997).
Differential display
Differential display was performed essentially as previously described (Liang and Pardee, 1997). The volume of PCR mixture was 20 µl, comprising 1 µl of the cDNA samples (concentrations adjusted for comparison), 2 µmol/l each of dNTPs, 0.2 µl of [
-33P]-dATP (10 mCi/ml, Amersham, UK), 0.2 pmol each of primers and 1.25 IU of Taq polymerase (Perkin Elmer, UK) in the supplied PCR buffer. Sequences of primers are shown in Table I. The random primers (H-AP1 and H-AP2, Table I) will define the size of fragment for each particular gene sequence, depending on the position [from the poly(A) tail] of homology to the random primer sequence. Primers, TG, TA and TC, are oligo(dT) primers for annealing with the poly(A) tail. One nucleotide, G, A or C, is attached to the 3' end of these oligo(dT) primers to ensure that the primer anneals at the beginning of poly(A) tail sequence. Therefore, PCR amplification with a primer set of one of the three 3' oligo(dT) primers (TG, TA or TC) and either H-AP1 or H-AP2 random primer produces a 3' end cDNA fragment of specific length for each gene cDNA in the differential display gel (Figure 1b).
The PCR cycle parameters were 40 cycles of 30 s at 94°C, 2 min at 40°C and 30 s at 72°C, followed by 10 min extension at 72°C. The annealing at 40°C allows 3–4 nucleotide mismatches between the random primers and cDNA (T.Goto, unpublished data) and benefits the amplification of a larger number of cDNA molecules with the limited number of primer sets.
Two µl of the PCR products were electrophoresed on a 6% denaturing polyacrylamide gel in 1x TBE at 1700 V until the xylene cyanol FF dye reached the bottom of the gel. The gel was transferred onto a 3MM paper (Whattman, UK), dried and exposed to Hyperfilm (Amersham) overnight at –70°C.
Re-amplification and cloning of differentially expressed cDNA fragments
The autoradiogram was superimposed on the dried gel to locate the cDNA fragments of interest. These were excised with a sterile blade, the DNA eluted from the gel by boiling in 100 µl of dd H2O for 15 min and precipitated with 100% ethanol in the presence of 50 µg of glycogen as a carrier. The DNA was washed with ice-cold 85% ethanol and resuspended in 10 µl of dd H2O.
To obtain a sufficient amount of the fragments for ligation to the cloning vector, pGem-T Easy (Promega, UK), 4µl of the resuspended DNA were re-amplified with the same set of primers as used for differential display and with the same PCR conditions except that the concentration of dNTPs was 20 µmol/l each, instead of 2 µmol/l, and [-33P]-dATP was omitted.
DNA sequencing
Re-amplified cDNA fragments were gel-purified by QIAEXII kit (Qiagen, UK) and cloned into the pGEM-T Easy vector according to the maufacturers instructions. DNA sequencing was carried out using the Thermo Sequenase radiolabelled terminator cycle sequencing kit (Amersham) with M13 forward primer, according to the manufacturer's instructions. All fragments were sequenced on both strands. Sequences were read manually.
Database analysis
Sequences were analysed with the NIX programme at the Human Genome Mapping Project (HGMP) web site that subjects a sequence to multiple analysis for, e.g., BLAST, GRAIL and Repeatmasker, in both directions.
Results
Our previous human embryo cDNA libraries, constructed in the process of refining procedures to the sensitivity of a few embryonic cells, were limited to a single embryo sample representative of each stage and, moreover, the only human cleavage embryos available at the time were developmentally delayed. Reproducibility could not be demonstrated and the representative nature of the libraries could not be confirmed. Also, it should be noted that further testing of the single blastocyst library prepared by Adjaye et al. (1999) showed a high proportion of concatamers of the SMART primer sequence in the clones (Chris Mundy, HGMP, personal communication). Nevertheless, the original libraries have proved valuable in the isolation of new embryo-specific members of the HOX gene family (Adjaye and Monk, 2000). The concatamers of SMART primer sequences have not been encountered in libraries prepared from the replicate oocyte and embryo cDNAs reported in this paper (HGMP, personal communication).
Here we report the construction of new preparations of amplified cDNA from human embryos using only grade 1 quality single embryos, at the correct developmental stage for their age, with replicate samples at each developmental stage. We considered that amplified cDNA preparations and libraries from replicate single embryos would give more significant results than preparations from batches of embryos at each stage. A batch of embryos may contain one or more degenerate or abnormal embryos that would interfere with the detection of normal patterns of gene expression in early development. Replicate amplified cDNA preparations from a number of individual embryos at each stage allows the confirmation of expressed genes, i.e. those occurring in the majority of replicate embryos. Four good quality embryos were used at each of the 4-cell, 8-cell and blastocyst stages and subjected to RT–PCR as described in the text.
As in previous work (Goto et al., 1999), we first showed that the embryonic cDNAs contained sequences corresponding to expressed genes, HPRT and ß-actin. Figure 2a shows the distribution of sizes of cDNA molecules from each of the replicate samples and Figures 2b and 2c show PCR amplification of these gene sequences in the replicate cDNAs for the oocytes, 4-cell, 8-cell and blastocyst stages and the somatic control cDNAs. Note that the expression of HPRT and ß-actin is not observed in all oocyte or embryo samples, nor to the same intensity in replicate samples. This is partly due to the fact that the amplified cDNA preparations are not fully qualitatively and quantitatively representative of the genes expressed at a particular stage due to the very low starting cell number in the single embryos.
|
To isolate and characterize embryo-specific and germ- cell-specific genes (i.e. genes not expressed in somatic cells), and identify which of these might be stage-specific, we carried out differential display on the amplified cDNAs to show the array of genes expressed in the cDNAs at the different stages. Figure 3
shows the differential display patterns of the amplified cDNA preparations from the replicate embryos. The most striking result is the disparate nature of the patterns of genes expressed. Each embryo shows a very different pattern even at the same stage of development. Despite the variability, there are clearly sequences that appear to be specific to particular stages. There are also sequences specific to muscle, gut or brain in the somatic cell patterns (m, g and b in Figure 3a).
|
In Figure 3
, it is clear that patterns of genes expressed in samples of higher cell number, the PGC samples (200–300 cells, Figure 3b) and the somatic cells (Figure 3a), are more reproducible and do not show the highly variable patterns seen for the oocytes and embryos. We have attempted to determine whether the strikingly disparate patterns of gene expression in the embryos, as revealed by differential display, are a real property of human early embryonic development, or whether the variability is a technical artefact, in the differential display procedures or the RT–PCR procedures, introduced by the low cell number in the individual embryos.
A comparison of Figures 3a and 3b shows that repeated differential display procedures from the same amplified cDNA preparations are reproducible. The reproducibility of the different bands in each sample confirms the oocyte- and embryo-specific bands for expressed genes (bands not appearing in the somatic samples). These are numbered in the figure and listed in Table II.
|
In Figure 3b
, we also show differential display of amplified cDNA preparations from replicate samples of four fibroblasts and 400 fibroblasts. A comparison of patterns of gene expression in these lanes indicates that the patterns of expressed genes in samples of four fibroblasts are more variable than those for 400 fibroblasts. Thus, the variability in the differential display between replicates is most probably due to low cell number and is introduced at the initial RT–PCR amplification of the mRNA populations in the embryos.
Figure 4 confirms the effect of low cell number on creating the variable patterns of gene expression. Highly variable differential display patterns of replicate samples of four fibroblasts may be compared with the more reproducible patterns for the replicate samples of 400 fibroblasts especially in the lower half of this gel. Therefore, in our hands, the patterns of gene expression in amplified cDNAs from individual human embryos are not reproducible and represent a variable subset of mRNAs present in these embryos. Nevertheless, despite the variability, the use of replicate individual embryos does allow the identification of individual bands within the patterns that are clearly specific to development, i.e. not appearing in the somatic controls, and some that are specific to a particular stage of development, i.e. to the oocyte, or appearing at the 8-cell stage.
|
To identify the oocyte- and embryo-specific genes, in these and further differential display experiments with different sets of primers, bands differentially expressed in oocytes or embryos (Table II
) were excised from the gels, re-amplified using the random primer and the polyT primer, gel-purified, and cloned into the pGEM T-easy vector, and sequenced (see Figure 1b). The embryo-specific bands 1, 2, 3, 9 and 11 were excised from the gel in Figure 3a and sequenced. Bands 1, 2 and 3 were the same sequence but truncated by one or two bases. Primers were designed within the sequences obtained and used to carry out one round of PCR of these sequences in the series of amplified oocyte, embryo and PGC cDNAs. For all bands, a product was obtained in oocytes, embryos and PGCs, with no product or very faint product (band 9) in somatic cell cDNAs and fibroblast cDNAs (data not shown). Thus the specificity of these developmental genes has been confirmed. In continuing work, we are obtaining the full length sequences of developmental genes identified in this way and characterizing the genes by database analysis before more extensive functional analysis. Discussion
Over the past few decades, we have designed a variety of single cell molecular procedures specifically for the study of the mutation, methylation and expression of specific genes in single embryonic cells of mouse and human. These highly sensitive micro methods have been successfully applied to a number of different studies including X-chromosome inactivation in mouse (Monk and Harper, 1979, for review, see Monk, 1992) and human (Daniels et al., 1997; Goto et al., 1997; for review, see Goto and Monk, 1998), the role of methylation in genetic programming in the embryo (Monk et al., 1987a; for review, see Monk, 1995), the regulation of X-linked (Grant et al., 1992) and imprinted gene expression (Zuccotti and Monk, 1995; Goto et al., 1998), and the first models of preimplantation diagnosis of genetic disease in mouse and human (Monk et al., 1987b; Holding and Monk, 1989; Monk and Holding, 1990). In this paper, we add to this repertoire of highly sensitive molecular technologies, procedures for the preparation of amplified cDNA complementary to expressed genes in human individual embryos and the use of differential display of the patterns of gene expression at different stages of preimplantation development to isolate human developmental genes.
Preparations of amplified cDNA from single embryos show a great deal of variability in gene expression patterns as demonstrated by differential display. There are valid reasons to expect some variability. The maternally-inherited mRNAs are degraded in cleavage stages, and the embryonic genes are being turned on one by one, starting as early as the fertilized one cell embryo (Daniels et al., 1995). It seems unlikely that silencing and activation of specific genes would be a highly synchronized process so variability from embryo-to-embryo, and even cell-to-cell, must be expected.
However, we have shown that a main component of variability in patterns of expressed genes detected is brought about by the low cell number in the starting samples. The variability does not occur in the differential display PCR step since the cDNA concentrations available for differential display are not limiting. Repeated differential display procedures are clearly reproducible for both the embryos and the somatic cells. On the other hand, it is our experience that RT–PCR procedures directly on individual embryos do not always detect known genes with 100% reliability. Therefore, the finding that reconstruction experiments using four and 400 fibroblast samples show greater variability in patterns of genes expressed for the low cell number is not unexpected. Due to this variability introduced by low cell number, we can not envisage the use of differential display to characterize patterns of gene expression in biopsied blastomeres as an indicator of embryo health in clinical situations at the present time, although it is possible that more representative cDNA procedures for single embryos and single cells might be designed in the future.
Nevertheless, despite the variability, the use of replicate embryos leads to clear identification of developmental genes specific to preimplantation development (i.e. genes not expressed in somatic tissues). In addition, embryo-specific genes that are stage specific, i.e. expressed predominantly in the oocyte (maternally expressed genes), or appearing at the 4-, 8-cell or blastocyst stages, are clearly identified and confirmed, but only by the use of replicate single embryo cDNAs. A similar approach to analyse embryonic gene expression in the mouse has been reported earlier by a number of authors (Rothstein et al., 1993; Zimmerman and Schultz, 1994; Corrick et al., 1995) using batches of 50 or more embryos at specific developmental stages. Sensitive RT–PCR procedures (e.g. Brady et al., 1990; Brady and Iscove, 1993), or the Clontech kit (Sasaki et al., 1998) have been used by these authors to prepare amplified cDNA for analysis of specific genes by PCR, or overall gene expression by random sequencing or differential display (see also Candeliere et al., 1999). Clearly, human embryos are not available for research in these numbers and, in addition, for the reasons outlined above and shown in this work, we consider that a better approach is to analyse replicate individual embryos rather than batch samples, despite the sensitivity required.
The characterization of oocyte- and embryo-specific genes (and PGC-specific genes, Goto et al., 1999) by this approach will be invaluable in the elucidation of maternal and embryonic contributions to development. The genes isolated will provide a profile of the genetic programme of normal development leading to a greater understanding of embryogenesis. This in turn will allow comparison with, and assessment of, gene expression in human embryonal stem cells (ES cells), leading to advances in the manipulation of these cells (e.g. using developmental gene regulatory sequences) and their use in tissue transplantation therapy.
The immortality, pluripotency and motility of early embryonic and germ cells suggests that the human developmental genes identified may be candidate genes for a role in tumourigenesis. A panel of CDNAs prepared from cancer cell lines and tumours will be screened for their expression with a view to the identification of embryo/cancer genes that could be targetted in cancer therapy and the preparation of DNA vaccines.
Notes
3 To whom correspondence should be addressed at: Molecular Embryology Unit, 30 Guilford Street, London WC1 1EH, UK. E-mail: mmonk{at}ich.ucl.ac.uk 
References
Adjaye, J. and Monk, M. (2000) Transcription of homeobox-containing genes detected in cDNA libraries derived from human unfertilized oocytes and preimplantation embryos. Mol. Hum. Reprod., 6, 707–711.
Adjaye, J., Daniels, R., Bolton, V. et al. (1997) cDNA libraries from single human preimplantation embryos. Genomics, 46, 337–344.[ISI][Medline]
Adjaye, J., Bolton, V. and Monk, M. (1999) Developmental expression of specific genes detected in high quality cDNA libraries from single human preimplantation embryos. Gene, 237, 373–383.[ISI][Medline]
Barnes, W.M. (1994) PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc. Natl Acad. Sci. USA, 91, 2216–2220.
Bolton, V.N., Hawes, S.M., Taylor, C.T., et al. (1989) Development of spare human embryos in vitro: An analysis of the correlations among gross morphology, cleavage rates and development to blastocyst. J In Vitro Fertil. Embryo Transfer, 6, 30–35.[ISI][Medline]
Brady, G., Barbara, M. and Iscove, N.N. (1990) Representative in vitro cDNA amplification from individual haemopoetic cells and colonies. Methods Mol. Cell. Biol., 2, 17–25.
Brady, G. and Iscove, N.N. (1993) Construction of cDNA libraries from single cells. Meth. Enzymol., 225, 611–623.[ISI][Medline]
Candeliere, G.A., Rao, Y., Floh, A. et al. (1999) cDNA fingerprinting of osteoprogenitor cells to isolate differentiation stage-specific genes. Nucleic Acids Res., 27, 1079–1083.
Chirgwin, J.M., Przybyla, A.E., McDonald, R.J. and Rutter, W.J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry, 18, 5294–5299.[Medline]
Corrick, C.M., Silvestro, M.J., Lahoud, M.H. et al. (1995) Construction of mouse blastocyst cDNA library by PCR amplification from total RNA. Mol. Reprod. Dev., 43, 7–16.
Daniels, R.D., Kinis, T., Serhal, P. et al. (1995) Expression of the myotin protein kinase gene in preimplantation human embryos. Hum. Mol. Genet., 4, 389–393.
Daniels, R., Zuccotti, M., Kinis, T. et al. (1997) XIST expression in human oocytes and preimplantation embryos. Am. J. Hum. Genet., 61, 33–39.[ISI][Medline]
Gibbs, R.A., Nguyen, P.-N., McBride, L.J. et al. (1989) Identification of mutations leading to the Lesch-Nyhan syndrome by automated direct DNA sequencing of in vitro amplified cDNA. Proc. Natl Acad. Sci. USA, 86, 1919–1923.
Goto, T., Adjaye, J., Rodeck, C. et al. (1999) Identification of genes expressed in human primordial germ cells at the time of entry of the female germ line into meiosis. Mol. Hum. Reprod., 5, 851–860.
Goto, T., and Monk, M. (1998) Regulation of X-chromosome inactivation in development in mice and humans. Microbiol. Mol. Biol. Rev., 62, 362–378.
Goto, T., Christians, E. and Monk, M. (1998) Expression of an Xist promoter-luciferase construct during spermatogenesis and preimplantation embryos: regulation by DNA methylation. Mol. Reprod. Dev., 49, 356–367.[ISI][Medline]
Goto, T., Wright E., and Monk, M. (1997) Paternal X-chromosome inactivation in human trophoblastic cells. Mol. Hum. Reprod., 3, 77–80.
Grant, M., Zuccotti, M. and Monk, M. (1992) Methylation of CpG sites of two X-linked genes coincides with X-inactivation in the female mouse embryo but not in the germ line. Nature Genet., 2, 161–166.[ISI][Medline]
Heikinheimo, O., Lanzendorf, S.E., Baka, S.G. et al. (1995) Cell cycle genes c-mos and cyclin-B1 are expressed in a specific pattern in human oocytes and preimplantation embryos. Mol. Hum. Reprod., 1, see Hum. Reprod., 10, 699–707.
Holding, C. and Monk, M. (1989) Diagnosis of beta-thalassaemia by DNA amplification in single blastomeres from mouse preimplantation embryos. Lancet, ii, 532–535.
Jolly, D.J., Okayama, H., Berg, P. et al. (1983) Isolation and characterization of a full-length expressible cDNA for human hypoxanthine phospho- ribosyltransferase. Proc. Natl Acad. Sci. USA, 80, 477–481.
Liang, P. and Pardee, A.B. (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science, 257, 967–971.
Liang, P. and Pardee, A.B. (1997) (eds) Differential Display Methods and Protocols. Humana Press Inc, New Jersey, USA, 3 pp.
Monk, M. (1992) The X chromosome in development in mouse and man. J. Inher. Metab. Dis., 15, 499–513.[ISI][Medline]
Monk, M. (1995) Epigenetic programming of differential gene expression in development and evolution. Dev. Genet., 17, 188–197.[ISI][Medline]
Monk, M., Boubelik, M. and Lehnert, S. (1987a) Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development, 99, 371–382.[Abstract]
Monk, M., Handyside, A., Hardy, K. et al. (1987b) Preimplantation diagnosis of deficiency of hypoxanthine phosphoribosyl transferase in a mouse model for Lesch–Nyhan syndrome. Lancet, ii, 423–426.
Monk, M. and Harper, M. (1979) Sequential X chromosome inactivation coupled with cellular differentiation in early mouse embryos. Nature, 281, 311–313.[Medline]
Monk, M. and Holding, C. (1990) Amplification of a beta-haemoglobin sequence in individual human oocytes and polar bodies. Lancet, 335, 985–988.[ISI][Medline]
Ponte, P., Ng, S.Y., Engel, J. et al. (1984) Evolutionary conservation in the untranslated region of actin mRNAs: DNA sequence of a human beta-actin cDNA. Nucleic Acids Res., 12, 1687–1696.
Rothstein, J.L., Johnson, D., Jessee, J. et al. (1993) Construction of primary and subtracted cDNA libraries from early embryos. In Methods in Enzymology. Vol. 225. Academic Press, New York, USA, 587 pp.
Sasaki, N., Nagaoka, S., Itoh, M. et al. (1998) Characterisation of gene expression in mouse blastocyst using single-pass sequencing of 3995 clones. Genomics, 49, 167–179.[ISI][Medline]
Zimmerman, I.W. and Schultz. R.M. (1994) Analysis of gene expression in the preimplantation embryo: Use of mRNA differential display. Proc. Natl Acad. Sci. USA, 91, 5456–5460.
Zuccotti, M. and Monk, M. (1995) Methylation of the mouse Xist gene in sperm and eggs correlates with imprinted Xist expression and paternal X-inactivation. Nature Genet., 9, 316–320.[ISI][Medline]
Submitted on January 24, 2000; accepted on June 21, 2000.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  G. M. Jones, D. S. Cram, B. Song, G. Kokkali, K. Pantos, and A. O. Trounson Novel strategy with potential to identify developmentally competent IVF blastocysts Hum. Reprod., August 1, 2008; 23(8): 1748 - 1759. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Monk, M. Hitchins, and S. Hawes Differential expression of the embryo/cancer gene ECSA(DPPA2), the cancer/testis gene BORIS and the pluripotency structural gene OCT4, in human preimplantation development Mol. Hum. Reprod., June 1, 2008; 14(6): 347 - 355. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M Hinkins, J Huntriss, D Miller, and H M Picton Expression of Polycomb-group genes in human ovarian follicles, oocytes and preimplantation embryos Reproduction, December 1, 2005; 130(6): 883 - 888. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Lefievre, S.J. Conner, A. Salpekar, O. Olufowobi, P. Ashton, B. Pavlovic, W. Lenton, M. Afnan, I.A. Brewis, M. Monk, et al. Four zona pellucida glycoproteins are expressed in the human Hum. Reprod., July 1, 2004; 19(7): 1580 - 1586. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Ghassemifar, J. J. Eckert, F. D. Houghton, H. M. Picton, H. J. Leese, and T. P. Fleming Gene expression regulating epithelial intercellular junction biogenesis during human blastocyst development in vitro Mol. Hum. Reprod., May 1, 2003; 9(5): 245 - 252. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Jurisicova, M. Antenos, S. Varmuza, J. L. Tilly, and R. F. Casper Expression of apoptosis-related genes during human preimplantation embryo development: potential roles for the Harakiri gene product and Caspase-3 in blastomere fragmentation Mol. Hum. Reprod., March 1, 2003; 9(3): 133 - 141. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Huntriss, R. Gosden, M. Hinkins, B. Oliver, D. Miller, A.J. Rutherford, and H.M. Picton Isolation, characterization and expression of the human Factor In the Germline alpha (FIGLA) gene in ovarian follicles and oocytes Mol. Hum. Reprod., December 1, 2002; 8(12): 1087 - 1095. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D.J. Bloor, A.D. Metcalfe, A. Rutherford, D.R. Brison, and S.J. Kimber Expression of cell adhesion molecules during human preimplantation embryo development Mol. Hum. Reprod., March 1, 2002; 8(3): 237 - 245. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. R. Ghassemifar, B. Sheth, T. Papenbrock, H. J. Leese, F. D. Houghton, and T. P. Fleming Occludin TM4-: an isoform of the tight junction protein present in primates lacking the fourth transmembrane domain J. Cell Sci., January 8, 2002; 115(15): 3171 - 3180. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Salpekar, J. Huntriss, V. Bolton, and M. Monk The use of amplified cDNA to investigate the expression of seven imprinted genes in human oocytes and preimplantation embryos Mol. Hum. Reprod., September 1, 2001; 7(9): 839 - 844. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Goto, A. Salpekar, and M. Monk Expression of a testis-specific member of the olfactory receptor gene family in human primordial germ cells Mol. Hum. Reprod., June 1, 2001; 7(6): 553 - 558. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||