WO2002077275A2

WO2002077275A2 - Diagnostic method for determining abnormality of cloned embryos

Info

Publication number: WO2002077275A2
Application number: PCT/GB2002/001398
Authority: WO
Inventors: Wendy Lee Dean; Wolf Reik
Original assignee: Babraham Institute
Current assignee: Babraham Institute
Priority date: 2001-03-23
Filing date: 2002-03-22
Publication date: 2002-10-03
Anticipated expiration: 2003-09-23
Also published as: GB0107267D0; AU2002244843A1; WO2002077275A3

Abstract

The present invention relates to a method for determining abnormal development of cloned embryos. The method provides for the detection and/or measurement of aberrant epigenetic reprogramming in cloned mammalian embryos. Aberrant reprogramming may lead to abnormality in cloned embryos. According to a first aspect of the invention there is provided a method for detecting and/or measuring aberrant epigenetic reprogramming in a cloned mammalian embryo, which includes the step of determining the methylation state of said embryo. According to a second aspect of the invention there is provided a method of diagnosis of aberrant epigenetic reprogramming in a cloned mammalian embryo, which includes the step of determining the methylation state of said embryo. Furthermore, the invention provides a method of diagnosis of the propensity of a cloned mammalian embryo for abnormal development, which method includes the step of determining the methylation state of said embryo.

Description

Diagnostic Method for Determining Abnormality of Cloned Embryos

Introduction

The present invention relates to a method for deterniining abnormal development of cloned embryos. The method provides for the detection and/or measurement of aberrant epigenetic reprogramming in cloned mammalian embryos. Aberrant reprogramming may lead to abnormality in cloned embryos.

Background

Cloning of mammals using somatic cell nuclei is now possible in several species but the success rate of development to term is very low^1"3. It is not known what the reasons are for this low rate of development and postnatal problems. For example, "Dolly" the sheep (Nature, 385, 810-813, 1997), the first large animal cloned from an adult cell, is now believed to suffer premature arthritis. Normal embryos in several mammalian species undergo 'epigenetic reprograniming' of gametic epigenetic information⁴. This has been shown for DNA methylation, which is generally high in mature gametes, and is lost first from the paternal genome in the zygote by an active (replication independent) demethylation mechanism, and from the maternal genome during preimplantation development by a passive (replication dependent) mechanism^5"12.

In vitro manipulation of murine embryonic stem(ES) cells has previously been shown ²⁰ to result in epigenetic drift from the normal patterns of establishment of a number of specific gene loci (Igf2, Igf2r, H19, and U2afl-rsl). These genes are known to have a specific pattern of gene expression such that only a single copy is expressed and the identity of the expressed copy is stipulated by parent-of-origin phenomena known as genomic imprinting. The two parental alleles are 'marked' for expression in the respective germlines and these 'marks' are frequently formalised by the establishment of differential methylation patterns such that the silent copy is usually methylated.

Pluripotent ES cells may drift from the normal epigenetic profile with prolonged life under culture conditions, and may result in inappropriate gene expression. Derivation of completely ES cell derived foetuses from ES cells carrying altered imprinted genotypes indicated that abnormal phenotypes often resulted from these aberrantly imprinted embryonic stem cells. These results highlight the important element that many epigenetically established states are influenced by environmental conditions and that aberrant patterns once formalised, often by methylation of specific genes, are stable and heritablely maintained thereafter. Despite differentiation these errors are perpetuated and result in phenotypic abnormalities. The previous work²⁰ indicates that errors are not corrected without reprogramming of the genome.

Summary of the Invention Reprogramming (on a genome-wide basis) is at the centre of the issue of abnormality of cloned embryos. Reprogramming is important for developmental totipotency of the genome. We examined the reprogramming of genomic DNA methylation patterns in cloned bovine¹³ preimplantation embryos. We found that demethylation and de novo methylation occurred aberrantly in cloned embryos. All nuclei in cloned morulae were highly methylated in contrast to normal morulae in which a substantial proportion of nuclei had low levels of methylation¹¹. Reorganisation of nuclear methylation patterns to resemble those of differentiated donor nuclei occurred precociously in cloned embryos. We believe that aberrant epigenetic reprogramming is responsible for the high rate of abnormal development of clones. We believe that abnormally high methylation levels in all cells of the morulae, and in trophectodermal cells of the blastocyst (which go on to form placental tissues) would impair placental gene expression and function, which is the most consistent abnormality in cloned embryos^1"3.

According to a first aspect of the invention there is provided a method for detecting and/or measuring aberrant epigenetic reprogramming in a cloned mammalian embryo, which includes the step of deteπnining the methylation state of said embryo.

According to a second aspect of the invention there is provided a method of diagnosis of aberrant epigenetic reprogramming in a cloned mammalian embryo, which includes the step of deterrriining the methylation state of said embryo.

Furthermore, the invention provides a method of diagnosis of the propensity of a cloned mammalian embryo for abnormal development, which method includes the step of deteπnining the methylation state of said embryo.

Additionally, the invention provides a method as described above, wherein the methylation state of an embryo is utilised in a step of selection, to select an embryo having correct epigenetic reprogramming and hence having a propensity for normal development.

Where legally permissible the present invention provides a direct diagnostic method for determining the presence or absence of abnormality in cloned mammalian embryos. This may constitute a method of diagnosis practised on the human or animal body. Such methods of diagnosis may be carried out in vivo on cloned embryos. However, it is generally expected that the invention will be used for in vitro testing in order to determine abnormality of cloned embryos and/or abnormality in nuclei for use in nuclear transfer to produce cloned embryos. The in vitro methods may be used to screen test samples of nuclei and/or embryos in order to determine the methylation state of the nuclei and/or embyros. Such methods of in vitro testing and diagnosis do not constitute direct methods of diagnosis practised on the human or animal body.

Our approach accepts that specific variability occurs on cloning, but that it is the incorrect genome-wide organisational aspect, reflected by the distribution of DNA methylation and reinforced by histone methylation, which is the most accurate and likely to be predictive for developmentally normal outcomes. That is, the whole genome must undergo reprogramming for cloning to succeed and that there is an inherent tolerance of specific gene expression permitted. Detailed Description of the Invention

The invention will now be described by way of example, with reference to Figure 1 which shows methylation patterns in normal and cloned bovine preimplantation embryos:

Fig 1 a-f, Anti-5 methylcytosine immunofluorescence of normal bovine embryos, b, 2 cell embryos stain intensely (> 10). c, 4 cell embryos (>a, Zygotes 12 h post fertilisation (inset panel shows staining of the DNA with YOYOl™) (25, number of embryos analysed). 15) and, d, 8 cell embryos show reduced staining (> 10). e, 10-16 cell embryos have a dramatic increase in methylation (> 10). f, morulae (>24 cells) maintain the high intensity signal (> 10). g-1, Cloned embryos, g, cloned 1 cell embryos stain faintly . Inset panel, DNA staining. (> 10). h, 2 cell cloned embryos (15). I, in 4 cell and, j, 8 cell clones there are two populations of embryos with high and low staining, respectively, (7/14) in each group, k, 10-16 cells (20). 1, morulae stage (> 10). Scale bar, (a-1 ;50 μm). m-o, Confocal projection of normal and cloned morula, and fibroblast donors, m, normal morula stains heterogeneously. Inset panel, nuclei (300%) indicate the two patterns of staining, n, cloned morula stains homogeneously (see inset panel, 300%). o, methylation organisation in fetal fibroblast donor cells. Inset (300%) indicates detail, p-u, Organisation of methylation patterns in normal and cloned bovine embryos, p-r, normal bovine embryos, p, normal 4, q, 8 and ,r, 16 cell embryos, s-u, cloned bovine embryos, s, 4 cell, t, 8 cell and, u, 16 cell cloned embryos. Note de novo methylation and fine granular staining in normal morulae (r) and the same pattern in cloned 4 cell embryos (s) followed by organisation into fewer and more intense foci in cloned 8 cell embryos (t). Scale bar, 20 μ (m-u).

We used immunostaining with a 5-methyl cytosine antibody to examine genome wide methylation patterns⁶'¹² of interphase nuclei in normal and cloned bovine preimplantation embryos (Fig. 1). Normal embryos showed demethylation of the larger (male) pronucleus in zygotes (Fig.l a) as in the mouse⁶'¹¹'¹². In cloned embryos the somatic donor nucleus was also demethylated in the zygote (Fig. lg, compare with fibroblast donor nucleus in Fig.lo). Nuclei in normal 2 cell to 8 cell embryos were further demethylated presumably by passive demethylation (Fig. 1 b-d,p,q), but no further demethylation occurred in cloned embryos (Fig. 1 h-j,s,t). Instead, a proportion of cloned 4 cell and 8 cell embryos underwent de novo methylation (Fig. 1 1, j,s,t) and all nuclei in morulae were highly methylated (Fig. In). By contrast, all normal embryos underwent de novo methylation from the 8-16 cell stage (Fig. 1 d,e,q,r) but a substantial proportion of nuclei remained undermethylated (Fig. 1 m). This happens earlier than in mouse embryos in where de novo methylation occurs after implantation. A similar pattern was found comparing normal and cloned blastocysts.

Earlier de novo methylation in cloned embryos preceded a characteristic change of methylation patterns from finely granular to fewer but larger and more intense foci from 4 to 8 cell stage (Fig. 1 s,t), which strikingly resembles that of the fibroblast donor nuclei (Fig. lo). The large intense foci have been attributed to centromeric heterochromatin in bovine somatic cells¹⁴. Our results show that bovine somatic nuclei undergo rapid demethylation in 1 cell embryos but are not further demethylated in subsequent cleavage divisions, perhaps because these nuclei possess the somatic form of Dnmtl¹⁰. Precocious de novo methylation in clones may be brought about by Dnmt3a,b¹⁵ and may lead to an earlier reorganisation of nuclear methylation patterns which resemble those of differentiated nuclei. This aberrant de novo methylation may restrict the potential for differentiation in cloned embryos. A substantial proportion of cloned embryos are lost during preimplantation development^1"3, consistent with the heterogeneity of methylation abnormalities observed in 4 and 8 cell clones (Fig. 1 i,j). Further substantial losses occur after implantation of cloned embryos^1"3, consistent with abnormally high levels of methylation in cloned as compared to normal morulae (Fig. 1 m,n). Cloned mouse fetuses had normal patterns of X chromosome inactivation suggesting that this aspect of epigenetic reprogramming was successful in embryos that survived¹⁶. Our results show that global epigenetic reprogramming of somatic nuclei is aberrant in most preimplantation cloned embryos. Future work will need to address the effects on expression of individual genes, both imprinted and nonimprinted ones. Our study indicates the view that correct reprograπiming of DNA methylation patterns is necessary for normal development of clones⁴.

We have further investigated the extent of epigenetic reprogramming in normal and cloned bovine and mouse embryos, extending our studies to histone methylation of H3 at K9 residues. Work in Neurospora suggests that DNA methylation patterns are dependent wholly or in part on methylation of histone H3 at K9 residues¹⁷. We have extended our studies using an antibody to methyl groups on histones, particularly histone H3 lysine 9 (K9), and using an antibody that recognises primarily branched rather than linear occurrence of this histone modification¹⁸. This occurs primarily in pericentromeric heterochromatin in which we also primarily detect meC in the previous work. Using a technique which enables us to detect the two methylation types, histone and DNA, on the same embryo has revealed that histone methylation very closely parallels DNA methylation, with the exception that there are some foci in the nucleus that stain positive for histone but not DNA methylation. These results show that:

(a) Histone methylation reprogramming occurs in the early embryo and parallels DNA methylation reprogramming. This suggests that H3 K9 is causative for DNA methylation but that this relationship can be regulated.

(b) Histone methylation reprogramming is aberrant in the majority of cloned embryos.

(c) There are some sites in the nucleus of clones that are histone methylated but not DNA methylated, suggesting that histone H3 K9 methylation may provide a more comprehensive marker for aberrant reprogramαiing in clones than DNA methylation.

Further investigation and consideration of the precise patterns of histone and DNA methylation in the morula and blastocyst stage embryo suggests that cells of the trophectoderm, rather than the inner cell mass, are affected by inappropriately high levels of DNA and histone methylation. The trophectoderm cells go on to form large parts of the placenta. The inner cell mass gives rise to all the tissues of the fetus. We propose that the aberrant epigenetic reprogramming we detect particularly affects gene expression and function of the placenta. This is supported by two observations. First, the placenta is the organ that is most consistently adversely affected in cloned animals in various species^1"3. Second, a recent analysis of gene expression in cloned mice revealed overall normal expression of a number of genes in the fetus, but reduced expression of a number of genes in the placenta¹⁹.

We propose that the placenta is the organ most affected in cloning. To show this, inner cell masses from cloned embryos can be introduced into tetraploid blastocysts²⁰. The tetraploid normal cells will form large parts of the placenta, whereas the cloned cells will form all the embryonic lineages. Therefore as placental function, and consequently fetal health, is adversely affected in clones, this procedure results in much better development and survival of clones.

Our results and their interpretation give the following insights into the cloning problem. In normal development epigenetic patterns including histone and DNA methylation are reprogrammed genome wide, giving rise to a totipotent genome. The great majority of cloned mouse and bovine preimplantation embryos show aberrant patterns of DNA methylation and histone H3 K9 methylation. Aberrant DNA methylation and histone methylation patterns can overlap but can also occur exclusively. We interpret these aberrant patterns as the inability of the normal reprograrmning machinery to remove comprehensively the epigenetic modifications of the donor nuclei at the time of nuclear transfer. Occasionally cloned embryos are found that are indistinguishable in their epigenetic patterns from normal controls. We propose that diagnosis of such patterns in preimplantation cloned embryos, by biopsy and immunofluorescence analysis of single blastomeres (leaving the remainder of the embryo viable), would be predictive for the developmental outcome of the clone. That is better development would occur with clones that had a more normal epigenetic pattern in biopsied blastomeres. We further propose that donor cells used for cloning might be epigenetically different or heterogeneous, so it will be possible by epigenetic analysis to identify and define more efficient donor cell nuclei in terms of cloning outcome. We further propose that it will be possible to manipulate the epigenetic state of donor cells prior to nuclear transfer by (a) drug treatments such as Azacytidine or TSA which interfere with epigenetic patterns or (b) transiently inducible knockouts of enzymes such as DNA methyltransferases or histone methyltransferases that are involved in controlling the epigenetic state of the donor nucleus. We further propose that, because our results highlight problems in the placenta, transfer of cloned inner cell mass cells from blastocysts into tetraploid normal (uncloned) blastocysts will improve development of cloned genotypes. Even simply aggregating cloned and normal tetraploid preimplantation embryos may result in a higher frequency of development to term. References

1. Wilmut, 1., Young,L. & Campbell, K.H. Embryonic and somatic cell cloning. Reprod. Fertil. Dev. 10, 639-643 (1998)

2. Solter, D. Mammalian cloning: advances and limitations. Nature Rev. Genet.l, 199-207 (2000).

3. Colman, A. Somatic cell nuclear transfer in mammals: progress and applications. Cloning 1, 185-200 (1999).

4. Reik, W., Dean, W. & Walter, J. Epigenetic reprogramming in mammalian development. Science 293, 1089-1093 (2001).

5. Rougier, N. et al . Chromosome methyiation patterns during mammalian preimplantation development. Genes Dev. 12, 2108-2113 (1998).

6. Mayer, W., Niveleau, A., Walter, J., Fundele, R. & Haaf, T. Demethylation of the zygotic paternal genome. Nature 403, 501-502 (2000).

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18. Peters AH, O'Carroll D, Scherthan H, Mechtler K, Sauer S, Schofer C, Weipoltshammer K, Pagani M, Lachner M, Kohlmaier A, Opravil S, Doyle M, Sibilia M, Jenuwein T. Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability. Cell 107, 323-337 (2001).

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Claims

1. A method for detecting and/or measuring aberrant epigenetic reprogramming in a cloned mammalian embryo, which includes the step of determining the methylation state of said embryo.

2. A method of diagnosis of aberrant epigenetic reprogramming in a cloned mammalian embryo, which includes the step of determining the methylation state of said embryo.

3. A method of diagnosis of the propensity of a cloned mammalian embryo for abnormal development, which method includes the step of determining the methylation state of said embryo.

4. A method according to any of claims 1 to 3 which includes the use of immunostaining with an antibody to examine genome- wide methylation in the nucleus of an embryo.

5. A method according to claim 4 which uses immunostaining with a 5-methyl cytosine antibody to examine the DNA methylation pattern.

6. A method according to claim 4 which uses an antibody to methyl groups on histones to examine the histone methylation pattern.

7. A method according to claim 6 which uses an antibody to histone H3 lysine 9 (k9).

8. A method according to any of claims 1 or 7 which examines the methylation pattern of an interphase nucleus of a cloned preimplantation embryo.

9. A method according to any of claims 1 to 7 which exainines the methylation pattern of a morula stage embryo.

10. A method according to any of claims 1 to 7 which examines the methylation pattern of a blastocyst stage embryo.

11. A method according to any of claims 1 to 10 which examines methylation patterns of normal and cloned embryos.

12. A method according to any preceding claim wherein the methylation state of an embryo is utilised in a step of selection, to select an embryo having correct epigenetic reprogramming and hence having a propensity for normal development.

13. A method according to claim 12, wherein the selection includes the step of examining the methylation pattern in a preimplantation cloned embryo, by biopsy and immunofluorescence analysis of a single blastomere (leaving the remainder of the embryo viable), in order to predict the developmental outcome of the clone.

14. A method for identifying an effective donor cell nucleus (in terms of successful cloning outcome) for use in cloning by nuclear transfer, which uses epigenetic analysis of the methylation state of the nucleus to identify a nucleus having a normal epigenetic methylation pattern.

15. A method for controlling the epigenetic methylation state of donor nucleus prior to nuclear transfer by (a) drug treatments such as Azacytidine or TSA which interfere with epigenetic patterns or (b) transiently inducible knockouts of enzymes such as DNA methyltransferases or histone methyltransferases that are involved in controlling the epigenetic state of the donor nucleus.

16. A method for improving development of cloned genotypes in a cloned embryo, which includes the step of transferring cloned inner cell mass cells from a blastocyst into a tetraploid normal (uncloned) blastocyst.

17. A method for increasing the frequency of development of cloned embryos successfully to term, which includes the step of aggregating cloned and normal tetraploid preimplantation embryos.

18. A method according to any preceding claim wherein a developmentally abnormal methylation state is indicative of aberrant epigenetic reprogramming.

19. A method according to claim 18 wherein the methylation state is abnormally high.