WO2012029957A1

WO2012029957A1 - Method for creating cloned animal

Info

Publication number: WO2012029957A1
Application number: PCT/JP2011/070062
Authority: WO
Inventors: 淳郎小倉; 貴美子井上; 史敏石野; 尚幸田; 澤井　健
Original assignee: 独立行政法人理化学研究所; 国立大学法人東京医科歯科大学; 国立大学法人岩手大学
Priority date: 2010-09-03
Filing date: 2011-09-02
Publication date: 2012-03-08

Abstract

It was discovered that the gene expression pattern in a reconstructed embryo obtained by means of somatic cell nuclear transfer normalized by deleting the Xist function on the active X chromosome of the reconstructed embryo, thereby improving cloning efficiency by 7 to 8 times.

Description

How to make cloned animals

The present invention relates to a method for producing a cloned animal by somatic cell nuclear transfer.

The somatic cell cloning technique is a technique for creating a new individual by removing a nucleus from a somatic cell of a mature individual, transplanting it to an enucleated unfertilized egg, and returning it to the maternal fallopian tube or uterus. This technique makes it possible to produce an individual having the same gene as the parent individual, and is expected to be applied to biological pharmaceutical production, regenerative medicine, and agriculture in the future.

Somatic cell nuclear transfer (SCNT) has been successfully applied to more than 16 animal species. However, the efficiency in terms of normal birth has always remained low despite many efforts to improve this technology (Non-Patent Document 1). For example, since there is no correlation between cloning efficiency and the differentiation state of donor cells, screening of effective donor cells has had limited success (Non-Patent Documents 2 to 4).

With increasing evidence for epigenetic changes in somatic cell nuclear transfer embryos, drugs used to modify chromatin structure and methylation status in donor cells and reconstructed embryos are promising for overcoming developmental deficits. It has been attracting attention as a process. One of the most successful means is that histone deacetylation inhibitors such as trichostatin A (TSA) (Non-patent Documents 5 and 6) and scriptoids (Non-patent Document 7) are used in recipient mouse oocytes. (HDACi). This treatment led to a significant increase in cloning efficiency. Since histone acetylation is a central factor for controlling chromatin accessibility (Non-patent Document 8), histone deacetylation inhibitors are presumed egg cytoplasmic factors (ooplasmic factors) It is believed to facilitate access to the transplanted nuclear genome. Therefore, the effects of histone deacetylation inhibitors are global rather than specific and vary depending on the epigenetic state of the donor genome.

In order to increase the success rate of cloning, many efforts have been made to improve somatic cell nuclear transfer techniques, including the above-mentioned drug treatment. Unfortunately, however, the success rate for cloning is only 1-5% in all cases.

The present invention has been made in view of the above-described problems of the prior art, and its object is to make it possible to dramatically increase the success rate in a method for producing a cloned animal by somatic cell cloning technology. Is to provide.

Many somatic cell nuclear transfer-specific phenotypes such as placental abnormalities (S. Tanaka, M. Oda et al., Biol Reprod 65, 1813-1821 (2001)), obesity (K. L. Tamashiro, T. Wakayama et al., Nat Med 8, 262-267 (2002)) and immunodeficiency (N. Ogonuki, K. Inoue et al., Nat Genet 30, 253-254 (2002)) are inevitable for somatic cell nuclear transfer. Suggesting that certain epigenetic errors are induced in the donor genome. These are probably non-random and limiting features caused by the fundamental epigenetic differences that exist between the somatic and germ cell genomes. Therefore, the present inventors thought that if such an error could be improved, this would be a technical breakthrough in mammalian cloning by somatic cell nuclear transfer. However, no evidence of this presumption has been presented, perhaps because the epigenetic state of individual cloned genomes is highly diverse.

Therefore, the present inventors conducted earnest research to elucidate epigenetic errors in the donor genome induced by somatic cell nuclear transfer. As a result, the inventor found that Xist, which is normally inactivated on one of the two X chromosomes in females, is ectopically expressed from active X (Xa) chromosomes in both male and female mouse embryos. I found out. Deletion of Xist in the Xa chromosome normalized the overall gene expression pattern and improved cloning efficiency by 7 to 8 times. Furthermore, the present inventor has also identified a non-random mechanism that is independent of Xist and suppresses a series of X-linked genes via somatic-type repressive histone control.

In addition, the present inventors produced siRNA for Xist and introduced it into a cloned embryo. As a result, we found that the introduction of siRNA improved the birth rate of clones by more than 10 times. Furthermore, combining trichostatin A treatment with the introduction of siRNA resulted in a synergistic effect on the birth rate of the clones.

Thus, the inventor has identified the world's first non-random reprogramming error induced by somatic cell nuclear transfer, and by normalizing this error, the success rate of mammalian somatic cell nuclear transfer As a result, the present invention has been completed.

More specifically, the present invention provides the following inventions.

(1) A method for producing a cloned animal by somatic cell nuclear transfer, which suppresses abnormal gene expression of the active X chromosome in a reconstructed embryo formed by transplanting the nucleus of a somatic cell into an enucleated oocyte A method characterized by.

(2) The method according to (1), wherein the gene expression abnormality of the active X chromosome is increased expression of the Xist gene.

(3) The method according to (2), wherein somatic cells in which the expression of the Xist gene on the active X chromosome is artificially suppressed are used.

(4) The method according to (2), wherein suppression of increase in the expression of the Xist gene is performed using siRNA against the Xist gene.

(5) The method according to any one of (1) to (4), wherein trichostatin A treatment is further performed on somatic cells or reconstructed embryos.

(6) A kit for carrying out the method according to (1) to (5), wherein an active X chromosome in a reconstructed embryo formed by transplanting a somatic nucleus into an enucleated oocyte A kit comprising a molecule having an activity of suppressing abnormal gene expression.

(7) A cloned animal produced by the method according to any one of (1) to (5).

For the first time, the present invention has identified two types of somatic cell nuclear transfer-related errors (SCNT-associated errors) that specifically affect the X chromosome. The first is the ectopic expression of Xist from the Xa chromosome, and the second is the persistence of inhibitory histone modifications (H3K9me2) in the Magea and Xlr regions. Furthermore, in the present invention, it has been clarified that correction of the Xist expression pattern in the cloned embryo can improve the cloning efficiency at a level superior to any cloning technique (Non-patent Document 1) applied so far. . In the present invention, it was also found that the expression of Xist was increased in both male and female bovine somatic cell nuclear transfer embryos, and therefore, the present invention can be widely applied to animals. According to the present invention, by using gene targeting, RNA knockdown, and other techniques to suppress (normalize) gene expression abnormality in the Xa chromosome, the efficiency of producing cloned animals by somatic cell nuclear transfer can be improved in a wide range of animals. It can be improved dramatically.

It is a figure which shows the large-scale expression fall of the X linkage gene in a somatic cell nuclear transfer (SCNT) embryo. A plots a representative pattern of relative gene expression levels of B6D2F1 in vitro fertilized embryos (IVF; left) and cumulus cell cloned embryos (right) at the genomic location from chromosome 1 to X (except Y). FIG. The expression level was normalized with the expression level of the IVF embryo. Red bars indicate X-linked genes with reduced expression in cloned embryos. B is a graph showing the expression level ratio of the X-linked gene to the autosomal gene. Data were expressed as mean ± sem ^{a, a ′} P <0.05, ^{b, b ′} P <0.0001, ^{c, c ′} P <0.05 (one-way analysis of variance and Student's t test). C is a graph plotting relative gene expression levels of cumulus cell cloned embryos (red), trichostatin A-treated cumulus cell embryos (blue), and IVF embryos (gray) at the position of the X chromosome. . The dotted line represents one embryo and the solid line shows their average value. It is a figure which shows that Xist is ectopically expressed on an active X chromosome in a male and female clone embryo. A is a graph showing the expression of Xist in male and female embryos. B is a photomicrograph of an embryo stained with Xist RNA and nuclei. C is a graph showing the ratio (0-2) of blastomeres classified by the number of Xist RNA domains in one embryo. Each bar represents one embryo. It is a figure which shows that the deletion of Xist on the active X chromosome (Xa) in a somatic cell nuclear transfer embryo improves the gene expression pattern and developmental ability in vivo. A is a graph showing the number of genes with reduced expression in somatic cell nuclear transfer embryos compared to the corresponding IVF embryos. B and C are graphs in which the relative expression level of the X-linked gene is plotted at the X chromosome position in female and male clones, respectively. Xlr clusters are indicated by triangles with a number of 1, and Magea clusters are indicated by triangles with a number of 2. D is a graph showing the birth rate in the transplanted embryo. E is a photograph of a fetus born after nuclear transfer using Sertoli cells with (left) or without (right) the Xist gene. It is a graph which shows the expression increase of Xist gene in the somatic cell nuclear transfer embryo of a bovine male and female. It is a graph which shows the effect of specific siRNA with respect to the Xist RNA level of the parthenogenetic activation embryo which introduce | transduced siRNA at different time. The horizontal line of each group shows an average value. SiRNA was introduced before activation (mid-phase II; MII) and 6 hours after activation (pronuclear phase; PN). It is a figure which shows the result of having detected the transient suppression of ectopic Xist expression in the embryo | germ generated by somatic cell nuclear transfer by Xist-siRNA introduction | transduction. (A) Quantitative RT-PCR of Xist in cloned embryos introduced with control or Xist-siRNA and cultured for 48 hours (4 cell stage), 72 hours (morula stage) or 96 hours (blastocyst stage) It is a graph which shows a result. The horizontal line in each group shows the average value of each expression level. (B) It is a photograph which shows the result of the FISH analysis of Xist in the cloned embryo which introduce | transduced siRNA. Arrows indicate “pinpoint” signals of Xist-siRNA-treated cloned embryos. The scale bar is 50 μm. (C) It is a graph which shows the result of having analyzed the ratio of the blastomere classified by the cloud or pinpoint expression pattern of Xist by RNA FISH. Each column represents a single embryo. It is a figure which shows the effect of Xist-siRNA in the development after implantation of a clone embryo. (A) It is a graph which shows the development rate of the embryo evaluated in embryonic period (E) 5.5 and the last stage of pregnancy. In some experiments, trichostatin A was added to the medium at 5 or 50 nM. The numbers above the bar graph indicate the rate of normal shaped embryos (E5.5) and full-term births relative to the transferred embryos. * P <0.05, ** P <0.001 (Fisher exact test). (B) Representative photomicrographs of siRNA-treated cloned embryos recovered at E5.5. In the figure, “Epi” indicates the upper definitive endoderm region, “EXE” indicates the extraembryonic ectoderm, and “EPC” indicates the trophoblast cone. (C) A photograph showing a litter of pups generated by somatic cell nuclear transfer from Sertoli cells obtained after treatment with Xist-siRNA and 50 nM trichostatin A. (D) Results of immunostaining of H3K27me3 (green) and Oct4 (red) in control or Xist-siRNA-treated cloned embryos at 96 hours (blastocyst) in vitro and E5.5 in utero It is a photograph which shows. “Epi” indicates the upper definitive endoderm region, and “EXE” indicates the extraembryonic ectoderm. The arrow is just one H3K27me3-positive cell found in the sample. The scale bar is 50 μm. It is the schematic which shows the influence on the survival of the male embryo | germ generated by Xist suppression via siRNA and somatic cell nuclear transfer. In the figure, (upper) shows the Xist expression level, and (lower) shows the survival rate of the cloned embryo.

The present invention provides a method for producing a cloned animal by somatic cell nuclear transfer. The method of the present invention is characterized by suppressing abnormal gene expression of active X chromosomes in embryos formed by transplanting somatic cell nuclei into enucleated oocytes.

The animal for producing a clone in the present invention is not particularly limited as long as it is a non-human animal capable of producing a clone by somatic cell nuclear transfer. For example, mouse, rat, cow, horse, pig, sheep, goat, monkey , Rabbits, dogs, cats, ferrets, camels, deer and the like.

The recipient oocyte used in the method of the present invention can be collected from, for example, an animal in which superovulation is induced by administration of a hormone such as gonadotropin or a slaughtered animal ovary. When collected as a cumulus / oocyte complex, it is preferable to remove the cumulus cells by hyaluronidase treatment. Enucleation in an oocyte can be performed, for example, by treating the oocyte with cytochalasin B, which is a cytoskeleton formation inhibitor, and then removing the nucleus from the oocyte using a micropipette.

Examples of donor somatic cells used in the method of the present invention include cumulus cells, Sertoli cells, fibroblasts, nerve cells, mammary cells, blood cells, and lymphocytes, but are not limited thereto. These somatic cells may be derived from stem cells such as embryonic stem cells, tissue stem cells, and cells to which pluripotency is imparted by genetic manipulation (for example, iPS cells).

In the present invention, in order to suppress abnormal gene expression of the active X chromosome in a reconstructed embryo formed by transplanting a somatic nucleus into an enucleated oocyte, the above-mentioned somatic cell, oocyte, or reconstructed embryo is used. Then, a process for suppressing the gene expression abnormality is performed. In this example, the active X chromosome in the reconstructed embryo was found to increase the expression of the Xist gene, but by suppressing the expression of the Xist gene, the overall gene expression pattern was normalized, Cloning efficiency was significantly improved. Therefore, the treatment for suppressing abnormal gene expression of the active X chromosome is preferably a treatment for suppressing the expression or function of the Xist gene.

The treatment for suppressing the expression or function of the Xist gene includes, for example, destroying the Xist gene in somatic cells, or a molecule having an activity of suppressing the expression of the Xist gene (for example, siRNA, antisense described later) Introducing RNA, RNA having ribozyme activity, and vectors expressing them) into somatic cells, oocytes, or reconstructed embryos. The gene sequence of the Xist gene is known (Borsani, G., Tonlorenzi, R. Nature 351 325-329 (1991), J. Kawai, A. Shinagawa, Nature 409, 685-690 (2001). NCBI Reference ID: AF138745, AK051106, NR_001464.2, U50910.1, GU372693.1, EF619477.1).

Other treatments for suppressing the expression or function of the Xist gene include, for example, BRCA1 binding to Xist (Ganesan et al., Cell 111, 393-405 (2002), Sirchia, et al., PLoS One 4 ( 5), PRC1,2 related genes (Plath et al, Science 300, 131-135 (2003), which is a polycomb protein that suppresses the expression or function of the gene encoding e5559 (2009)) and maintains X chromosome inactivation. Plath et al, JCB 167 (6), 1025-1035 (2004), Hernandez-Munoz et al., PNAS 102, 7635-7640 (2005)) expression or function suppression, macro-H2A related genes (Costanzi et al ., Development 127, 2283-2289 (2000), Hernandez-Munoz et al., PNAS 102, 7635-7640 (2005)), or suppression of function. A body that destroys these genes in somatic cells and has an activity that suppresses the expression of these genes (for example, siRNA, antisense RNA, RNA having ribozyme activity and a vector that expresses these RNAs described later) By introducing into a cell, oocyte, or reconstructed embryo, it is possible to suppress the expression or function of the Xist gene.

As other processing to suppress the expression or function of Xist gene, SATB1,2 (Agrelo, et al., Developmental Cell 16, 507-516 (2009)), which is a factor involved in gene silencing by Xist, is encoded. Tsix (Lee et al., Cell 99, 47-57 (1999), Lee et al., Cell 103, 17-27 (2000), Sado, which is a regulator that suppresses the increase in gene expression or function, and Xist et al., Development 128, 1275-1286 (2001), Ogawa et al, Science 320. 1336-1341 (2008)) may also include treatment for increasing the expression or enhancing the function of the gene. In order to increase the expression of the gene or enhance the function, for example, a vector expressing the gene or a synthetic RNA may be introduced into a somatic cell, an oocyte, or a reconstructed embryo.

In the present invention, H3K27me3 (Plath et al, Science 300, 131-135 (2003), Rougeulle et al., Mol Cell Biol. 24, 5475-5484 (2004), which is a histone modification that maintains X chromosome inactivation. )) To suppress the expression or function of the Xist gene.

On the other hand, in this Example, it was also found that the expression of the genes described in Table 1 below was decreased in the active X chromosome in the reconstructed embryo, so that the abnormal gene expression of the active X chromosome was suppressed. In order to achieve this, it may be possible to enhance the expression or function of the genes listed in Table 1.

The above-described treatment method for suppressing abnormal gene expression of the active X chromosome can be applied alone, but a plurality of methods can be applied in combination as appropriate.

Furthermore, it can be applied in combination with other treatments that can increase the efficiency of production of cloned animals by somatic cell nuclear transfer. Such other treatments include trichostatin A treatment of somatic cells or reconstructed embryos. Trichostatin A is a potent histone deacetylation inhibitor that mitigates histone-related repression of donor chromatin during genome reprogramming in vitro and in vivo, It is known to promote the development of cloned embryos (Kishigami, S. et al., Biochem. Biophys. Res. Commun. 340, 183-189 (2006)). In fact, in this example, when the introduction of siRNA that suppresses expression of Xist and trichostatin A treatment were combined, a synergistic effect was shown in the development of cloned embryos (FIG. 7A).

In the present invention, “suppression of gene expression” means both complete suppression and partial suppression of gene expression. It also includes both transcriptional and translational repression. Further, in the present invention, “suppression of gene function” means both suppression of the function of a gene transcription product and suppression of the function of a translation product. As a method for suppressing the expression or function of a gene in the present invention, methods well known to those skilled in the art, such as a method for destroying a target gene and a method using an RNA interference technique, can be used.

In the method of destroying the target gene, for example, a knockout technique using homologous recombination can be used. In the knockout technique, a targeting DNA construct having a sequence homologous to at least a part of the target gene region is introduced into a cell so that homologous recombination occurs with the target gene region. The targeting DNA construct typically has a structure in which DNA consisting of a sequence homologous to the sequence of the target DNA site is adjacent to each end of the DNA for destroying the target gene. That is, the homologous DNA is in the left and right arms of the targeting DNA construct, and the DNA for destroying the target gene is located between the two arms. Here, the term “homologous” includes not only the case where sequences are completely identical (ie, 100%) but also the case where some sequences are different as long as homologous recombination occurs. Usually, at least 95% or more, preferably 97% or more, more preferably 99% or more of the sequences are identical. The target gene region on the chromosome and the homologous sequence of the targeting DNA construct interact to exchange a specific sequence of the target gene region with the DNA on the targeting DNA construct, thereby knocking out the target gene. it can. In the preparation of the targeting DNA construct, for example, DNA in which a marker gene is inserted into the cloned target gene can be used.

In the method using RNA interference, a dsRNA (double stranded RNA) complementary to a transcription product of a target gene or a DNA encoding the dsRNA is used. DNA encoding dsRNA includes antisense DNA encoding antisense RNA for any region of the transcript (mRNA) of the target gene, and sense DNA encoding sense RNA for any region of the mRNA, Antisense RNA and sense RNA can be expressed from the antisense DNA and the sense DNA, respectively. Moreover, dsRNA can be produced from these antisense RNA and sense RNA.

The configuration in which the dsRNA expression system is held in a vector or the like includes a case where antisense RNA and sense RNA are expressed from the same vector, and a case where antisense RNA and sense RNA are expressed from different vectors, respectively. The antisense RNA and sense RNA are expressed from the same vector. For example, an antisense RNA expression cassette in which a promoter capable of expressing a short RNA such as polIII is linked upstream of the antisense DNA and the sense DNA. And sense RNA expression cassettes are constructed, and these cassettes are inserted into the vector in the same direction or in the opposite direction.

It is also possible to construct an expression system in which antisense DNA and sense DNA are arranged in opposite directions so as to face each other on different strands. In this configuration, one double-stranded DNA (siRNA-encoding DNA) in which an antisense RNA coding strand and a sense RNA coding strand are paired is provided, and antisense RNA and sense RNA are separated from each strand on both sides. A promoter is provided oppositely so that it can be expressed. In this case, in order to avoid adding extra sequences downstream of the sense RNA and antisense RNA, a terminator is added to the 3 'end of each strand (antisense RNA coding strand, sense RNA coding strand). It is preferable to provide. As this terminator, a sequence in which four or more A (adenine) bases are continued can be used. Further, in this palindromic style expression system, the two promoter types are preferably different.

In addition, as a configuration for expressing antisense RNA and sense RNA from different vectors, for example, antisense RNA expression in which a promoter capable of expressing a short RNA such as polIII is linked upstream of antisense DNA and sense DNA, respectively. A cassette and a sense RNA expression cassette are constructed, and these cassettes are held in different vectors.

The above dsRNA can be prepared by those skilled in the art by chemically synthesizing each strand.

The dsRNA used in the present invention is preferably siRNA. siRNA means double-stranded RNA consisting of short strands in a range that does not show toxicity in cells. The chain length is not particularly limited as long as the expression of the target gene can be suppressed and it does not show toxicity. The dsRNA chain length is, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs, and more preferably 21 to 30 base pairs.

In the present invention, siRNA composed of the RNA described in SEQ ID NO: 5 and the RNA described in embryonic row number: 6 can be suitably used.

The DNA encoding dsRNA need not be completely identical to the base sequence of the target gene, but is at least 70% or more, preferably 80% or more, more preferably 90% or more (eg, 95%, 96%, 97 %, 98%, 99% or more). Sequence identity can be determined by the BLAST program.

Other methods for suppressing gene expression include antisense technology using DNA encoding antisense RNA complementary to the target gene transcript (antisense DNA) and cleaving target gene transcripts specifically. It is also conceivable to use a ribozyme technique using DNA encoding RNA having ribozyme activity.

The above nucleic acid molecule for suppressing abnormal gene expression of the active X chromosome can be introduced into host animal cells by a known transformation technique such as lipofection, electroporation, or viral vector method. it can.

In a preferred embodiment of the method of the present invention, in order to suppress abnormal gene expression of the active X chromosome in the reconstructed embryo, the donor somatic cell has an activity of suppressing the expression of the somatic cell in which the Xist gene is disrupted or the Xist gene. Somatic cells into which siRNA having the same is introduced are used. By transferring the nuclei of these donor somatic cells to the enucleated oocytes that are recipients, reconstructed embryos can be obtained. In another preferred embodiment of the method of the present invention, an enucleated oocyte into which siRNA having an activity to suppress the expression of the Xist gene is introduced as the recipient enucleated oocyte. A reconstructed embryo can be obtained by transplanting the nucleus of a donor somatic cell into this enucleated oocyte. In another preferred embodiment of the method of the present invention, after obtaining a reconstructed embryo by transplanting the nucleus of a donor somatic cell into an enucleated oocyte, the expression of the Xist gene is suppressed in the reconstructed embryo. SiRNA having the activity to be introduced is introduced.

Transplantation of a somatic cell nucleus into an oocyte can be performed by a known method such as a method of directly injecting a somatic cell nucleus into an enucleated oocyte or a method of fusing a somatic cell and an enucleated oocyte. it can. Oocytes are activated before or after nuclear transfer. The activation of the oocyte can be carried out, for example, by treatment with strontium or ethanol, calcium ionophore treatment or electrical stimulation.

The reconstructed embryo thus obtained is cultured for a certain period of time (for example, until reaching the 2-cell stage, 4-cell stage, morula stage, or blastocyst stage) and then transplanted into the oviduct or uterus of the animal. And thus a cloned animal can be obtained.

The present invention also provides a kit for carrying out the above-described method of the present invention. The kit of the present invention includes at least one of the above-described molecules having an activity of suppressing abnormal gene expression of the active X chromosome in the reconstructed embryo, and may further include instructions for use. In addition, a reagent capable of increasing the efficiency of production of a cloned animal by somatic cell nuclear transfer, for example, trichostatin A may be included.

Hereinafter, the present invention will be described more specifically based on examples and comparative examples, but the present invention is not limited to the following examples.

[Materials and methods (Examples 1 to 3)]

(1) Animals 8-10 weeks old (C57BL / 6´DBA / 2) F1 (BDF1) and 8-12 weeks old ICR female mice were collected from recipient oocytes and embryo transfer recipients, respectively. Used to do. Male and female BDF1 and male (C57BL / 6′129 / Sv-ter) F1 mice were used as nuclear donors. In some experiments, Xist-deficient (X ^DXist ) C57BL / 6 females (T. Sado, Y. Hoki, H. Sasaki, Dev Cell 9, 159-165 (2005)) were mated with DBA / 2 males. BDF1 donor mice were prepared. Animals were given free access to water and diet for experimental mice and were kept under controlled lighting conditions (7: 00-21: 00 sunshine). Animals were maintained under specific pathogen removal conditions. All animal experiments were performed according to the RIKEN BioResource Center guidelines.

(2) Preparation of donor cells Immature Sertoli cells were collected from 3-7 day old male newborns. Cells are described in the literature (K. Inoue, N. Ogonuki et al., Biol Reprod 69, 1394-1400 (2003), A. Ogura, K. Inoue et al., Biol Reprod 62, 1579-1584 (2000)). Prepared as described. That is, the collected testis cells were treated with 0.1 mg / mL collagenase (Sigma-Aldrich) and 0.01 mg / mL deoxyribonuclease (Sigma-Aldrich) for 30 minutes at 37 ° C., followed by 0.2 mg / mL trypsin (Sigma- Aldrich) for 5 minutes at 37 ° C. The testicular cell suspension was washed with PBS containing 4 mg / mL bovine serum albumin without addition of Ca ²⁺ / Mg ²⁺ and used for injection. Cumulus cells were collected from cumulus / oocyte complexes after treatment with KSMO medium containing 0.1% “bovine testicular hyaluronidase” (Calbiochem).

(3) Nuclear transplantation Nuclear transplantation is performed in the literature (K. Inoue, N. Ogonuki et al., Biol Reprod 69, 1394-1400. (2003), Y. Seki, K. Hayashi et al., Dev Biol 278, 440. -458 (2005), T. Wakayama, AC Perry et al., Nature 394, 369-374 (1998)). That is, superovulation by injecting 7.5 IU “pregnant mare serum gonadotropin” (Sankyo-yell) and 7.5 IU “human chorionic gonadotropin” (hCG; Aska-Pharmaceutical) into BDF1 female mice at 48-hour intervals. Induced. At 15 hours after hCG injection, cumulus-oocyte complexes were collected from the fallopian tube and cumulus cells were seeded in KSOM medium containing 0.1% bovine testicular hyaluronidase. Oocytes were enucleated in hepes buffered KSOM containing 7.5 μg / mL cytochalasin B. The donor nucleus was introduced into the enucleated oocyte using “Piezo-driven micromanipulator” (PMM-150FU, Primetech). After culturing with KSOM for 1 hour, the oocytes were activated for 1 hour in KSOM containing 2.5 mM SrCl ₂ without addition of Ca ²⁺ . The reconstructed embryo was cultured in KSOM containing 5 μg / mL cytochalasin B for 5 hours.

(4) Embryo transfer The reconstructed embryo that reached the 4-cell stage after 48 hours in culture was transplanted into the oviduct of a pseudopregnant ICR female mouse on the 0.5th day (the day after mating with the vas deferens male). On day 19.5, pregnant females were born by caesarean section and their offspring were bred in lactating ICR female mice.

(5) Preparation and amplification of RNA Cloned blastocysts cultured for 96 hours were used for total RNA extraction with TRIzol reagent (Invitrogen). RNA from one blastocyst was subjected to two rounds of linear amplification using “TargetAmp Two-Round Aminoallyl-aRNA Amplification Kits” (Epicentre) according to the manufacturer's instructions. For control embryos, one IVF blastocyst was subjected to total RNA extraction and two rounds of RNA amplification. Amplified RNA is labeled with Cy3 dye (GE Healthcare) and hybridized to “whole mouse genome oligo DNA microarray” (4′44K, Agilent Technologies) for 16 hours at 65 ° C. according to the manufacturer's instructions. I let you.

(6) Microarray analysis Scanning of microarray slides was performed at a resolution of 5 μm using a DNA microarray scanner (Agilent Technologies). The scanned image file was detected for signal intensity using “Feature Extraction software” (Agilent Technologies). For comparison of gene expression levels in each chromosome, quantile normalization was performed to adjust for differences in signal intensity between different chips. Signal intensities with average values greater than 50 in IVF embryos were selected and plotted according to their location on the chromosome. In order to extract genes with different expression, all raw data were loaded into “Gene Spring GX 7.3 software” (Agilent Technologies) and quantile normalization was performed. In all wild type IVF and cloned embryos, probes with a value lower than 9 were excluded from further analysis. Normalized values were used to extract genes that expressed differently between IVF and cloned embryos. This extraction utilized the Student's t-test with a significance level of 5% using the procedure of Benjamin and Hochberg. The X: A expression ratio was calculated using the average of the raw intensity data.

(7) RNA fluorescence in situ hybridization (RNA FISH)
RNA FISH was performed with slight modifications to the description in the literature (M. Sugimoto, K. Abe, PLoS Genet 3, e116 (2007)). A probe for detecting Xist RNA was prepared from a Xist genomic clone containing a 7.5 kb fragment of exon 1 by nick translation using Cy3-dCTP (GE Healthcare). Embryos were incubated in PBS containing 0.1% Triton X-100 for 10 seconds and fixed in PBS containing 4% paraformaldehyde for 10 minutes at room temperature. Hybridization was performed overnight at 37 ° C. Following stringent washing, embryonic nuclei were stained with TO-PRO-3 (Invitrogen). Embryos were sampled in 90% glycerol, 0.1'PBS, and 1% Dabco (Sigma-Aldrich) and fluorescent images were obtained using an "LSM510 meta confocal laser scanning microscope" (Zeiss).

(8) Gene expression analysis of bovine embryos Regarding bovine IVF, nuclear transfer and in vitro culture of embryos are basically performed in the literature (K. Sawai, S. Kageyama et al., Cloning Stem Cells 7, 189-198 (2005 ), K. Sawai, S. Kageyama et al., J Reprod Dev 53, 77-86 (2007)). Bovine blastocyst stage embryos or nuclear transfer embryos were treated with 0.5% protease for 5 minutes and washed 3 times with PBS containing 1% polyvinylpyrrolidone. One embryo was added to an aliquot of 5 μl lysate and stored at −80 ° C. Embryo samples obtained from IVF or nuclear transfer embryos were heated at 80 ° C. for 5 minutes and 0.8 μl of solution was used for sex determination. PCR for the sexing of embryos was performed as described in the literature (M. Sugimoto, K. Abe, PLoS Genet 3, e116 (2007)). The protocol used to determine the relative amount of bovine Xist transcript is as described in the literature (A. Ogura, K. Inoue et al., Biol Reprod 62, 1579-1584. (2000)). For normalization of the relative expression of Xist, the relative amount of transcript relative to GAPDH in the same sample was determined. PCR was performed using StepOne unit (Applied Biosystems), and cDNA was synthesized with SYBR Green (Qiagen) and a primer for bovine XIST (SEQ ID NO: 1 / 5′-AATAATGCGACAGGCAAAGG-3 ′ and SEQ ID NO: 2/5). Amplified in a 20 μl reaction mixture containing '-TCCCGCTCATTTTCCATTAG-3') or primers for GAPDH (SEQ ID NO: 3 / 5'-CCAGAAGACTGTGGATGGCC-3 'and SEQ ID NO: 4 / 5'-CTGACGCCTGCTTCACCACC-3').

(9) Analysis of public database Normalized values obtained from public GEO database (GSE13445) (B. Wen, H. Wu et al., Nat Genet 41, 246-250 (2009)) are in the 1000 bp range. The average value was calculated. The degree of H3K9me2 was analyzed using Subio platform (Subio).

[Materials and Methods (Examples 4 to 6)]

(1) Animals 8-12 week old (C57BL / 6′DBA / 2) F1 (BDF1) and ICR female mice were used for oocyte collection and embryo transfer recipients, respectively. 1-9 day old BDF1 male mice are used for collection of Sertoli cells as described in the literature (Ogura, A. et al., Biol. Reprod. 62, 1579-1584 (2000)). It was. To prepare in vitro fertilized (IVF) control embryos, oocytes collected from C57BL / 6 female mice were fertilized in vitro using DBA / 2 sperm. All mice were purchased from SLC Japan (Hamamatsu, Japan) and maintained under specific pathogen removal conditions and controlled lighting conditions (sunshine from 7: 00-21: 00). All animal experiments were performed according to the RIKEN BioResource Center guidelines.

(2) siRNA design Synthetic siRNA duplexes were designed by “Stealth ^™ designer” (Invitrogen Life Technologies Japan, Tokyo, Japan). The siRNAs used are as follows.

For Xist (SEQ ID NO: 5 / 5'-AUAACAGUAAGUCUGAUAGAGGACA-3 'and SEQ ID NO: 6 / 5'-UGUCCUCUAUCAGACUUACUGUUAU-3')
For negative control (SEQ ID NO: 7 / 5'-UUACUCAUGUGUCAUAACACAGGUG-3 'and SEQ ID NO: 8 / 5'-CACCUGUGUUAUGACACAUGAGUAA-3')
MSS201293 and MSS293979 (Invitrogen Life Technologies Japan) were used for G9a (Ehmt2) and Glp (Ehmt1), respectively. siRNA duplex mixtures were prepared as 200 μM stock solutions in nuclease-free water and stored at −80 ° C. until use.

(3) Preparation of mRNA mRNAs of Jhdm2a were synthesized with “T7 mMESSAGE mMACHINE kit” (Ambion, Austin, TX, USA) according to the manufacturer's instructions. The synthesized mRNA was purified by phenol / chloroform extraction and precipitated with ethanol. mRNA was dissolved in nuclease-free water to a final concentration of 100 pg / ml and stored at −80 ° C. until use.

(4) Preparation of Donor Cells Immature Sertoli cells were collected from 1-9 day old BDF1 strain male mice. After removal of the white membrane, the seminiferous tubules were treated with 0.1 mg / mL collagenase (Sigma-Aldrich) and 0.01 mg / mL deoxyribonuclease (Sigma-Aldrich) for 30 minutes at 37 ° C., followed by 0.2 mg / mL trypsin ( (Sigma-Aldrich) for 5 minutes at 37 ° C. The testicular cell suspension was washed 4 times with PBS containing 4 mg / mL bovine serum albumin and used for nuclear transfer.

(5) Nuclear transplantation Nuclear transplantation is described in the literature (Wakayama, T., Perry, AC, Zuccotti, M., Johnson, KR & Yanagimachi, R., Nature 394, 369-374 (1998), Ogura, A. et al. , Biol. Reprod. 62, 1579-1584 (2000)). That is, the recipient oocytes were 7.5 IU equine serum gonadotropin and 7.5 IU human chorionic gonadotropin (hCG; Aska-Pharmaceutical) at 48 hour intervals. BDF1 female mice were harvested by inducing superovulation by injection. At 15-17 hours after hCG injection, cumulus-oocyte complexes were collected from the fallopian tube and cumulus cells were plated in KSOM medium containing 0.1% bovine testicular hyaluronidase. Oocytes were enucleated in hepes buffered KSOM containing 7.5 μg / mL cytochalasin B. Using “Piezo-driven micromanipulator” (PMM-150FU, Primetech), the nuclei of donor Sertoli cells were introduced into the enucleated oocytes. After culturing with KSOM for 1 hour, somatic cell nuclear transfer oocytes were activated for 1 hour in KSOM containing 2.5 mM SrCl ₂ without addition of Ca ²⁺ . The reconstructed embryo was cultured in KSOM containing 5 μg / mL cytochalasin B for 5 hours, and further cultured in KSOM not containing cytochalasin B. In some experiments, trichostatin A (Sigma-Aldrich) (final concentrations of 5 and 50 nM) was added to each medium for 6 and 8 hours (total), respectively, from the beginning of oocyte activation.

(6) Development of polyploid parthenogenetic embryos Mature oocytes collected from BDF1 females were activated with SrCl _{2 in} the presence of cytochalasin B. After washing, they were incubated with KSOM for 96 hours.

(7) Introduction of siRNA or mRNA siRNA or mRNA microinjection was performed using "Piezo-driven micropipette" (Prime Tech). To investigate the specificity and time schedule of siRNA introduction, siRNA (final concentration 5 mM in nuclease-free water) was introduced into oocytes before and after parthenogenetic activation (FIG. 5). SiRNA was introduced into embryos generated by somatic cell nuclear transfer 6-7 hours after activation corresponding to the pronuclear (1 cell) stage (FIG. 8). After introduction, embryos were washed and cultured in KSOM prior to analysis or embryo transfer. In some experiments, mRNA (1-100 pg / mL) was introduced into oocytes just prior to somatic cell nuclear transfer.

(8) Embryo Transfer and Recovery Reconstructed embryos that reached the 2-cell stage after 24 hours of culture in KSOM were transferred to the oviduct of ICR recipient female mice one day after pseudopregnancy induction. On the 20th day, the recipient females investigated for full-term fetuses and raised live births in lactating ICR foster parents. Some recipient females were sacrificed on day 6 corresponding to E5.5 and the implanted embryos were carefully removed from the uterus using a dissecting microscope.

(9) RNA amplification and microarray analysis The total RNA content of a single blastocyst was amplified by a two-round amplification method. Extract total RNA from 96-hour cultured blastocysts using TRIzol reagent (Invitrogen) and use `` TargetAmp Two-Round Aminoallyl-aRNA Amplification Kits '' (Epicentre) according to the manufacturer's instructions for 2 rounds Was subjected to linear amplification. For control embryos, one IVF blastocyst was subjected to total RNA extraction and two rounds of RNA amplification.

The amplified RNA was labeled with Cy3 dye (GE Healthcare) and purified with RNeasy Mini kit (Qiagen, Tokyo, Japan). The labeled RNA was hybridized to “whole mouse genome oligo DNA microarray” (4′44K, Agilent Technologies) for 17 hours at 65 ° C. After washing, the image of the microarray slide was scanned using a DNA microarray scanner (Agilent Technologies), and the signal intensity was detected using “Feature Extraction software” (Agilent Technologies). In order to compare gene expression levels in each experiment, all raw data was loaded into “Gene Spring GX 11 software” (Agilent Technologies) and quantile normalization was performed to adjust for differences in signal intensity between different chips did. Normalized values of genes that expressed differently between each group of embryos were compared by one-way analysis of variance (ANOVA) using Tukey's post-hoc test (P <0.05 is considered significant).

(10) Quantitative RT-PCR
Single embryo cDNA was synthesized by “Cell to cDNAII kits” (Ambion Japan, Tokyo, Japan). Quantitative PCR was performed using “QuantiTect SYBR Green PCR kit” (Qiagen) and “ABI Prism 7900HT system” (Applied Biosystems). All PCRs were performed in duplicate at 60 ° C annealing temperature and 50 amplification cycles. Primer sequences are as follows.

For Xist (SEQ ID NO: 9 / 5'-GTCAGCAAGAGCCTTGAATTG-3 'and SEQ ID NO: 10 / 5'-TTTGCTGAGTCTTGAGGAGAATC-3')
For Gapdh (SEQ ID NO: 11 / 5'-CAACAGCAACTCCCACTCTTC-3 'and SEQ ID NO: 12 / 5'-CCTGTTGCTGTAGCCGTATTC-3')
Melting curve analysis was used to confirm amplification specificity.

(11) Immunofluorescence The embryos were fixed with 4% paraformaldehyde at 4 ° C. overnight. After washing 3 times with PBS, it was dialyzed against PBST-BSA (0.5% Triton X-100, 0.1% BSA in PBS) for 1 hour at room temperature. The embryos were then incubated overnight at 4 ° C. with `` rabbit anti-H3K27me3 antibody '' (1: 100 dilution; Millipore, Billerica, MA, USA) and `` goat anti-Oct4 antibody '' (1: 100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing 3 times with PBST-BSA, embryos were mixed in a mixture of secondary antibodies conjugated with `` Alexa Fluor 488 '' (anti-rabbit, Invitrogen) or `` Alexa Fluor 546 '' (anti-sheep, Invitrogen) for 1 hour at room temperature. Incubated with. After washing, specimens were made on glass slides using Vectashield (Vector Laboratories, Burlingame, CA, USA). The fluorescence signal was observed using a “confocal scanning laser microscope” (Digital Eclipse C1; Nikon, Tokyo, Japan).

(12) RNA fluorescence in situ hybridization (RNA FISH)
A probe for detecting Xist RNA was prepared from a Xist genomic clone containing a 7.5 kb fragment of exon 1 by nick translation using Cy3-dCTP (GE Healthcare). Embryos were incubated on ice for 10 seconds in PBS containing 0.1% Triton X-100 and fixed in PBS containing 4% paraformaldehyde for 10 minutes at room temperature. Hybridization was performed overnight at 37 ° C. Following stringent washing, embryonic nuclei were stained with TO-PRO-3 (Invitrogen). Embryos were sampled in 90% glycerol, 0.1'PBS, and 1% Dabco (Sigma-Aldrich) and fluorescent images were obtained using an "LSM510 meta confocal laser scanning microscope" (Zeiss).

(13) Statistical analysis In vitro and in vivo embryo development rates were compared using Fisher's exact test. Relative transcript levels in embryos determined by quantitative RT-PCR were analyzed with Student's t test to compare group means. Microarray data sets were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test, where P <0.05 was statistically significant.

[Example 1] Expression of X-linked genes in somatic cell nuclear transfer (SCNT) embryos In order to elucidate epigenetic features specific to somatic cell nuclear transfer, the present inventor has strictly standardized conditions in his laboratory. Below, mouse embryos were reconstructed from different donor cells (K. Inoue, N. Ogonuki et al., Biol Reprod 69, 1394-1400 (2003)). The overall gene expression pattern of one clonal blastocyst was analyzed by comparing it to a genotype matched control by in vitro fertilization (IVF) in the same environment.

First, when the relative expression levels of cloned embryo genes selected with a 44k oligo DNA microarray were plotted on chromosome 20 (excluding Y), the expression on the X chromosome was specifically decreased. (FIG. 1A). This phenomenon was independent of sex and genotype, as the average X: autosomal (X: A) expression ratio in the three types of cloned embryos was always lower compared to the corresponding control embryo ( FIG. 1B). From the detailed observation of the entire X chromosome, it seems that there are some gene-specific variations, but the expression level of the X-linked gene was greatly reduced in most regions (FIG. 1C).

The inventor then performed a statistical analysis using a t-test to identify the number of affected genes in the cloned embryo. In each clonal group, 2341 to 7865 of the 39301 gene probes had different expression compared to the matched IVF control. However, only 157 genes (164 probes) were affected in common to all clone groups, 128 had increased expression, and 29 had decreased expression. Thus, somatic cell nuclear transfer caused dysregulation of a large subset of genes, but most were specific for each donor cell type. Interestingly, 62% (18/29) of commonly decreased genes (hereinafter referred to as “CDGs”) are located on the X chromosome compared to an unbiased group of genes with increased expression. (Table 1). In Table 1, the X-linked genes are colored. The dark color showed what is contained in the Xlr cluster or Magea cluster (XqA7.2-7.3 and XqF3).

Next, the present inventor examined whether or not the reduced expression of the X-linked gene in the cloned embryo could be improved by treating the reconstructed oocyte with trichostatin A. Regarding the effect of trichostatin A treatment in cloned mice, the inventor's laboratory has confirmed that the birth rate is increased 2 or 3 times. However, no significant improvement was observed in X: A expression ratio (FIG. 1B) or X-linked gene expression levels (FIG. 1C) compared to untreated cloned embryos.

[Example 2] Expression of Xist in male and female cloned embryos Extensive reduction of expression on the X chromosome in cloned embryos is reminiscent of X inactivation (XCI). This process usually results in the inactivation of one of the two X chromosomes in the female embryo so that it can be balanced with the male in gene dosage (KD Huynh, JT Lee, Nat Rev Genet 6, 410- 418 (2005)). Since X-chromosome inactivation is established by Xist RNA coating in cis, the present inventor next investigated whether Xist is overexpressed in cloned embryos. As reported (S. Bao, N. Miyoshi et al., EMBO Rep 6, 748-754 (2005), LD Nolen, S. Gao et al., Dev Biol 279, 525-540 (2005)) Xist expression levels were significantly higher in female blastocysts derived from cumulus cells than in female IVF embryos (FIG. 2A). In male blastocysts derived from Sertoli cells, Xist was expressed at high levels, in contrast to male IVF embryos that expressed little or no Xist (FIG. 2A). Quantitative real-time PCR (RT-PCR) experiments confirmed the overexpression of Xist in male and female cloned embryos. From this discovery, the present inventor assumed that Xist was ectopically expressed from the activated X chromosome (Xa) in male and female cloned embryos.

The inventor then observed the number of 'Xist domains' in the nucleus of each blastomere by RNA fluorescence in situ hybridization (RNA FISH). As expected, about half of the IVF embryos always showed one domain in each blastomere and the other half did not show any domain. This is thought to represent female and male embryos, respectively (FIGS. 2B, 2C). In female clones, all four embryos contained blastomeres with an unusual biallelic Xist domain. However, the frequency varied from 20.0% to 51.7% for individual embryos (FIGS. 2B, 2C). This is considered to represent that the Xist expression level is relatively easily changed in the female cloned embryo (FIG. 2A). In male clones, all seven embryos contained one strong Xist RNA domain in the majority of blastomeres (FIGS. 2B, 2C). These results clearly support our hypothesis that Xist is ectopically activated on Xa in male and female clones. Interestingly, the present inventor has also found that expression of XIST is elevated in bovine amphoteric somatic cell nuclear transfer blastocysts (FIG. 4). This indicates that Xist (XIST) expression from ectopic Xa is specific to somatic cell nuclear transfer and is a common phenomenon in the mouse and bovine genomes. It has been reported that XIST is overexpressed in cow somatic cell nuclear transfer embryos (C. Wrenzycki, A. Lucas-Hahn et al., Biol Reprod 66, 127-134 (2002)). With respect to Xi (or conversely Xa), the major mechanisms of genetic memory (imprinting) may differ between embryos and somatic cells before transplantation. The former memory is borne during gametogenesis and is independent of the Xist promoter (L.E.McDonald, C.A. Paterson, G.F.Kay, Genomics 54, 379-386 ( 1998), P. Navarro, I. Chambers et al., Science 321, 1693-1695 (2008)), the latter memory was borne by the somatic genome during random X chromosome inactivation after transplantation. It greatly depends on the methylation state of the cage Xist promoter (K. D. Huynh, J. T. Lee, Nat Rev Genet 6, 410-418 (2005)). Reconstruction of Xi (or Xa) memory in the somatic cell-derived genome in cloned embryos appears to be incomplete.

[Example 3] Effect of deletion of Xist on active X chromosome (Xa) in somatic cell nuclear transfer embryos on gene expression pattern and developmental ability Xist has an overall suppressive effect on the X-linked gene with cis Therefore, the present inventor next examined to what extent the ectopic expression contributed to abnormal gene expression in the cloned embryo. For this study, the present inventor determined that Xa is Xist deficient (X ^DXist ) (T. Sado, Y. Hoki, H. Sasaki, Dev Cell 9, 159-165 (2005)). Cloned and analyzed the gene expression pattern of the embryo. Xist-deficient donor cells have the same B6D2F1 genotype as wild-type donor cells. In both the female (cumulus cells) and male ( ^Certoli cells) X ^DXist clones, the number of X-linked genes whose expression was reduced was significantly reduced compared to the wild-type clone (FIG. 3A). That is, 11% (119 → 13) for females and 13% (135 → 17) for males. This effect is clearly shown by the upward shift in gene expression levels plotted on the X chromosome (FIGS. 3B, 3C) and A: X ratio (FIG. 1B). Interestingly, this effect is genome-wide, and the number of autosomal genes that are down-regulated is also 6% (1298 → 74) and 25% (327 → 82) in females and males, respectively. It decreased (FIG. 3A). These results indicate that ectopic Xist expression in clones can adversely affect gene expression throughout the genome of cloned embryos.

Next, the present inventor hopes that the development after transplantation will be better than that of wild-type somatic cell nuclear transfer embryos, so that the somatic cell nuclear transfer embryos containing the Xist-deficient Xa chromosome were transferred to a pseudopregnant female recipe. Transplanted to the ent. As a result, embryo development was significantly improved in both cumulus cells and clones derived from Sertoli cells. The respective birth rates reached 12.1% and 13.0% of the transferred embryos, which were 7-8 times higher than the wild type control (FIGS. 3D, 3E). Cloned mice derived from a pedigree with a standard genetic background (B6D2F1) did not reach such high efficiencies even when treated with trichostatin A or scriptaid (up to about 3-5%) (Non-Patent Documents 5 to 7).

As mentioned above, the majority of X-linked genes whose expression was reduced in the clones were corrected for their expression due to the loss of Xist on the Xa chromosome. However, there were two distinct gene groups that were still repressed (FIGS. 3B, 3C). These were the Magea cluster located in XqF3 and the Xlr gene cluster located in XqA 7.2-7.3 (FIG. 1C). Interestingly, 11 of the 18 X-linked CDGs were classified into one of these clusters (Table 1). This fact indicates that these X-linked clusters are always repressed in clones by some mechanism different from Xist-dependent inactivation. The Magea family gene encodes a “melanoma antigen A” (MAGE-A) protein that is expressed only in germ cells, placenta and cancer cells (melanoma) (E. De Plaen, K. Arden et al., Immunogenetics 40, 360-369 (1994), E. De Plaen, O. De Backer et al., Genomics 55, 176-184 (1999)). They are ectopically expressed in embryonic stem cells (ES cells) lacking G9a (histone methyltransferase that dimethylates histone H3 at lysine 9) (M. Tachibana, K Sugimoto et al., Genes Dev 16, 1779-1791 (2002)). H3K9me2 is responsible for gene silencing with a constitutive heterochromatin state, and this histone-modified region is a tissue-specific block called LOCKs (large organized chromatin K9 modifications) (B. Wen, H. Wu et al., Nat Genet 41, 246-250 (2009)).

The present inventor then determined whether or not the two identified regions with decreased expression are related to LOCKs, such as the public database of Wen et al. (B. Wen, H. Wu et al., Nat Genet 41, 246-250 (2009)). Interestingly, not only the Magea family gene but also the Xlr family gene was present in LOCKs common to all undifferentiated and differentiated ES cells (B. Wen, H. Wu et al., Nat Genet 41, 246. -250 (2009)). In contrast, F8a in the Xlr gene was outside of these regions and expression levels were restored in Xist knockout cloned embryos. The present inventor has shown that the expression of Magea family gene and Xlr family gene is very low in undifferentiated ES cells by microarray analysis, whereas these genes are actively expressed in their blastocyst counterparts. Showed that. Therefore, the inventor has shown that the suppressive state of the Magea region and the Xlr region via somatic cell type LOCKs in donor cells is resistant to reprogramming by putative egg cytoplasmic factors. Think of it as being transmitted to the cloned embryo. The expression level of the G9a gene is variable at the blastocyst stage, but in the 2-cell cloned embryo, the Glp (G9a-like-protein) gene that forms a heterocomplex with G9a to methylate H3K9 Similar to (M. Tachibana, J. Ueda et al., Genes Dev 19, 815-826 (2005)), the expression level was high. Since the primary embryonic gene activation (EGA) phase begins in the 2-cell stage in mice, this finding suggests that cloned embryos activate G9a and Glp as early as EGA, and somatic LOCKs in their genomes. It is shown that it is maintained. BIX, a G9a inhibitor, can improve the production efficiency of differentiated somatic cell-derived induced pluripotent stem cells (Y. Shi, C. Desponts et al., Cell Stem Cell 3, 568-574 (2008 )). Therefore, erasure of H3K9me2 memory appears to be an essential step of effective dedifferentiation. In fact, global demethylation of H3K9me2 is known to occur in the early germ cell genome, and this hypomethylation is maintained throughout the germ cell development process (Y. Seki, K. Hayashi et al., Dev Biol 278, 440-458 (2005)).

The reprogramming mechanism is initially present in the egg cytoplasm to change the epigenetic state of the gamete to the epigenetic state of the zygote. Somatic cell nuclear transfer technology takes advantage of this mechanism in some way to reprogram the donor's somatic genome, which is epigeneticly different from the gamete. Therefore, various epigenetic errors are considered to occur in cloned embryos (J. Fulka, Jr., N. Miyashita, T. Nagai, A. Ogura, Nat Biotechnol 22, 25-26 (2004)).

[Example 4] Effect of specific siRNA on Xist RNA level of parthenogenetic activated embryos introduced with siRNA at different times The present inventors have developed parthenogenetic embryos expressing the Xist gene after the morula stage The effectiveness of siRNA constructs was examined. As a result, it was found that siRNA effectively reduces the level of Xist RNA in the resulting blastocyst when introduced into the oocyte 6 hours after activation (FIG. 5). Therefore, in the next series of experiments, a similar dosing regimen was used for somatic cell nuclear transfer embryos reconstructed with nuclei from neonatal Sertoli cells.

[Example 5] Transient suppression of ectopic Xist expression in embryos generated by somatic cell nuclear transfer by introduction of Xist-siRNA One-cell cloned embryos introduced with Xist-specific siRNA (one-cell cloned) Approximately 66% (53/80) of embryos) developed into blastocysts after 96 hours in culture. This trend was not statistically significant compared to those introduced with control siRNA (52%, 44/84; P> 0.05, Fisher's exact test). Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis showed that Xist transcripts were significantly reduced in Xist-siRNA embryos compared to control siRNA embryos in morula stages, but blastocysts It became clear that such a decrease was not recognized in the period (FIG. 6A). The effect of RNAi on ectopic Xist expression was further confirmed by Xist RNA fluorescence in situ hybridization (RNA FISH). In the mulberry stage, a clustered Xist RNA signal called “cloudy” was found in the majority of the nuclei of control siRNA embryos (FIGS. 6B, C). In contrast, in Xist-siRNA embryos, most blastomeres showed a “localized” pinpoint signal. This signal probably represents the early Xist RNA on the X chromosome. From this fact, it was confirmed that large-scale degradation of Xist was induced by the introduced Xist-siRNA (FIGS. 6B and 6C). At the blastocyst stage, in several Xist-siRNA embryos, ectopic Xist expression was clouded, which was indistinguishable from the pattern of control siRNA embryos (FIGS. 6B, C). . This finding suggests that, at least under the conditions of this experiment, the introduced Xist-siRNA is effective up to about 72 hours, but then the effectiveness decreases.

[Example 6] Effect of Xist-siRNA on post-implantation development of cloned embryo Next, the present inventors conducted embryo transfer experiments in order to evaluate the developmental ability of cloned embryos introduced with siRNA after implantation. Went. On day 5.5, siRNA had no effect on implantation rate as assessed by endometrial shedding and embryo recovery. However, the morphology of embryos recovered from the implantation site was significantly improved. That is, 75% (9/12) of Xist-siRNA embryos show normal morphology with distinguishable embryo and extraembryonic compartments, and only 5% (1/20) show normal morphology in the control group (FIG. 7B, P <0.005, Fisher's exact test). These rates correspond to 16% and 1%, respectively, in the transplanted embryos of the Xist-siRNA group and the control group (FIG. 7A). The remaining embryos, as reported for embryos cloned from embryonic stem cells (Jouneau, A. et al., Development 133, 1597-1607 (2006)), are developmentally delayed in the embryo or extraembryonic area And various developmental disorders (FIG. 7B). The distribution of Oct4-positive cells, which are normally localized only in the upper blastoderm, was also abnormally controlled (FIG. 7D). The inventors then born the recipient female at term. As a result, the fertility rate has been greatly improved. That is, 12% of the Xist knockdown embryos developed into pups that looked normal. This reached a 10-fold or higher birth rate compared to the control siRNA group (1%; P <0.005, Fisher's exact test; FIG. 7A). Interestingly, the rate of normal development investigated on days 5.5 and 19.5 (late pregnancy) was not significantly different in the same group (16% and 12% for Xist-siRNA embryos, 1% for controls) And 1%; P> 0.05, Fisher's exact test). This fact clearly shows that the developmental fate of cloned embryos is largely determined at an early stage after implantation at 5.5 days. Importantly, this birth rate could be further improved to about 20% when combined with 50 nM trichostatin A treatment (FIGS. 7A, C). Trichostatin A is a potent inhibitor of histone deacetylation, by mitigating histone-related repression of donor chromatin during genome reprogramming in vitro and in vivo, It is known to promote the development of cloned embryos (Kishigami, S. et al., Biochem. Biophys. Res. Commun. 340, 183-189 (2006)). This finding indicates that trichostatin A treatment and Xist knockdown have a synergistic effect on the development of cloned embryos. This is a mouse somatic cell nuclear transfer cloning since the first success by Wakayama et al. (Wakayama, T., Perry, AC, Zuccotti, M., Johnson, KR & Yanagimachi, R., Nature 394, 369-374 (1998)). Is the highest birth rate ever reported.

The next question of the present inventors was why such a remarkable improvement in cloning efficiency was achieved by simple introduction of Xist RNA at the 1-cell stage. The fact that Xist-siRNA embryos surviving on day 5.5 showed a characteristic normal phenotype suggests the importance of a short pre-implantation period in the survival of cloned mouse embryos To do. Consistent with this, despite the ectopic recurrence of Xist expression, significant upregulation of several developmentally important genes (eg, Fras1, Car2 and Tet3) in Xist-siRNA blastocysts was there. This was revealed by DNA microarray analysis using a single embryo.

In the table, “*” indicates one-way analysis of variance (ANOVA) with Tukey's post-hoc test, IVF (n = 4), Xist-siRNA treated embryo (n = 7) and control siRNA treated embryo ( It is applied to the microarray data from n = 5). “**” indicates an average value of two probes.

These genes may guarantee the correct crosstalk of embryos and extraembryonic parts in the fetus, essential for subsequent normal fetal development (Ang, S.L. & Constam, D.B. , Cell Dev. Biol. 15, 555-561 (2004)). Therefore, the remaining issue to address was the state of X chromosome inactivation in the cloned embryo after implantation. This is because they may have acquired X-chromosome inactivation through the restored Xist expression seen in the blastocyst stage (see FIG. 6B). Both X chromosomes in cells of the inner cell mass are reactivated in female mouse embryos (peripheral of X chromosome inactivation) and then undergo de novo random X chromosome inactivation in epiblast cells However, it is generally known that imprinted X chromosome inactivation is maintained in trophectoderm lines and placenta. Random X-chromosome inactivation occurs normally in cloned fetuses, and imprinted X-chromosome inactivation persists in the placenta, demonstrating the same for female somatic cell nuclear transfer embryos It was done. Thus, we hypothesized that ectopic X-chromosome inactivation specific to cloned embryos is also deleted from their embryonic tissues but maintained in extraembryonic tissues. In order to verify this process, we have identified the state of X-chromosome inactivation in implanted clone embryos as a marker of suppressed chromatin state in the inactivated X-chromosome (Huynh, K D. & Lee, J. T., Nature 426, 857-862 (2003)), and histone H3 (H3K27me3) trimethylated at position 27 of lysine was stained. In both blastocysts of Xist-siRNA and control embryos, the majority of the inner cell mass cells and trophectoderm cells (> 65%) were positive for punctate H3K27me3 staining (FIG. 7D). This is consistent with the findings of RNA FISH analysis (FIG. 6B). In the upper layer of the blastoderm of the cloned embryo on day 5.5, there were only a few cells with punctate H3K27me3 signal. This indicates that the X chromosome is reactivated exactly as expected (FIG. 7D). However, unexpectedly, cells in the extraembryonic compartment were also negative for such H3K27me3 signals, typical for X chromosome inactivation of both the Xist-siRNA and the control group (FIG. 7D). Together, these findings suggest that ectopic Xist expression in cloned embryos is corrected immediately after implantation in both embryos and extraembryonic tissues by some unknown mechanism. Therefore, the abnormal regulation of somatic cell nuclear transfer-specific X-chromosome inactivation caused by ectopic Xist expression is limited to a limited time before implantation, and in the rescue of cloned embryos, Xist knockout is It is thought that a large effect is shown in a very short time (Fig. 8). In fact, the disappearance of many embryos during this period strongly suggests how important this period is for the survival of embryos generated by somatic cell nuclear transfer (ouneau, A. et al. ., Development 133, 1597-1607 (2006), Wakisaka-Saito, N. et al., Biochem. Biophys. Res. Commun. 349, 106-114 (2006), Rielland, M., Brochard, V., Lacroix , M. C., Renard, J. P. & Jouneau, A., Dev. Biol. 334, 325-334 (2009)).

As described above, according to the present invention, it has become possible to dramatically increase the success rate in a method for producing a cloned animal by somatic cell cloning technology. According to the present invention, it is possible to efficiently produce an individual having the same gene as the parent individual, and the present invention has many applications in a wide range of fields such as biological pharmaceutical production, regenerative medicine, and agriculture. Be expected.

SEQ ID NOs: 1-4, 9-12 <223> Sequences of artificially synthesized primers SEQ ID NOs: 5-8
<223> Artificially synthesized siRNA sequences

Claims

A method for producing a cloned animal by somatic cell nuclear transfer, characterized by suppressing abnormal gene expression of an active X chromosome in a reconstructed embryo formed by transplanting a somatic cell nucleus into an enucleated oocyte how to.
The method according to claim 1, wherein the abnormal gene expression of the active X chromosome is increased expression of the Xist gene.
The method according to claim 2, wherein somatic cells in which the expression of the Xist gene on the active X chromosome is artificially suppressed are used.
The method according to claim 2, wherein the suppression of the increase in expression of the Xist gene is performed using siRNA against the Xist gene.
Furthermore, the method according to any one of claims 1 to 4, wherein trichostatin A treatment is performed on somatic cells or reconstructed embryos.
A kit for carrying out the method according to claims 1 to 5, which suppresses abnormal gene expression of active X chromosome in a reconstructed embryo formed by transplanting a somatic cell nucleus into an enucleated oocyte. A kit comprising a molecule having the activity of
A cloned animal produced by the method according to any one of claims 1 to 5.