Methods for dispersing, isolating, and transferring human chromosomes into animal embryos

Abstract

According to the present invention, there are provided a human chromosome dispersion method and isolation method, and a method for transplanting a human chromosome into an animal embryo. Through the present invention, there are provided a method for isolating a single human chromosome from remaining chromosomes, and a nonhuman mammalian cell having the isolated human chromosome.

Description

Technical Field

[0001] The present invention relates to a method for dispersing human chromosomes, a method for isolating them, and a method for transplanting them into animal embryos.

Background Art

[0002] As a method for introducing isolated human chromosomes into non-human mammalian cells, a microcell mediated chromosome transfer (MMCT) method has been developed. In the MMCT method, human cells are treated with colcemid to form micronuclei containing a single chromosome inside the cells, the micronuclei are purified, and then transplanted into non-human mammalian cells. However, micronuclei can only be formed in a very limited number of established cell lines, such as the skin fibroblast A9 cell line and the Chinese hamster ovary (CHO) cell line. However, in established cells, chromosomes are usually unstable and it is difficult to maintain their full length. In the MMCT method, human chromosomes are introduced into the A9 cell line or the CHO cell line, and only the chromosomes that can be stably maintained in these cells are transplanted into non-human mammalian cells. Using this technique, rats having chromosomes derived from human chromosome 21 have been produced (Patent Document 1).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

[0004] The inventors have discovered that a human cell nucleus containing human chromosomes can be introduced into a rodent cell having an M-phase cell cycle and dispersed chromosome by chromosome within the cell. The inventors have also discovered that the dispersed chromosomes can be isolated one by one using a micromanipulator. Furthermore, the inventors have found that even when the isolated chromosomes are introduced into a rodent cell having an M-phase cell cycle and the cells are cultured or developed, they can be stably maintained within the rodent cell, and that the entire length of the chromosome, from one end to the other, can be included. In this way, the inventors have perfected a method for maintaining one or more human chromosomes within a non-human mammalian cell.

[0005] The following inventions may be provided according to the present invention. [1] A method for dispersing human chromosomes, The process of introducing human nuclei obtained from human cells into M-phase cells of non-human mammals; and A step of culturing M-phase cells into which a human nucleus has been introduced in the presence of a microtubule formation inhibitor, thereby dispersing the chromosomes contained in the human nucleus within the cell. Methods that include... [2] The method according to [1] above, wherein the step of culturing M-phase cells into which human nuclei have been introduced is a step of culturing in the presence of okadaic acid in addition to a microtubule formation inhibitor. [3] The method according to [1] or [2] above, wherein the cells are unfertilized eggs. [4] The method according to any of the above [1] to [3], wherein the non-human mammal is a rodent. [5] A method for isolating a human chromosome, A method comprising the step of aspirating at least one human chromosome dispersed within a cell in the M phase by any of the methods described in [1] to [4] above using a micromanipulator. [6] Cytoplasm of a non-human mammal containing isolated human chromosomes, obtained by the method described in [5] above. [7] A method for producing non-human mammalian cells having human chromosomes, A method comprising the step of introducing isolated human chromosomes obtained by the method described in [5] above into M-phase non-human mammalian cells. [8] The method according to [7] above, wherein the cells of a non-human animal are unfertilized eggs of a rodent. [9] The method according to [8] above, further comprising the step of fertilizing an unfertilized egg containing human chromosomes with sperm to obtain a fertilized egg.

[10] A method for producing embryonic stem cells of a non-human mammal containing human chromosomes, A method comprising culturing an embryo obtained by developing a fertilized egg obtained by the method described in [9] above, or an inner cell mass derived from such embryo.

[11] A method for producing a non-human mammal containing human chromosomes, A method comprising introducing embryonic stem cells obtained by the method described in

[10] above into an embryo of a non-human mammal and causing it to develop.

[12] A method for producing non-human mammalian cells containing human chromosomes, Culture embryonic stem cells obtained by the method described in

[10] above in vitro, A method comprising differentiating embryonic stem cells into target cells. [Brief explanation of the drawing]

[0006] [Figure 1] Figure 1 shows the state in which chromosomes are dispersed in an unfertilized mouse egg. [Figure 2] Figure 2 shows the isolation of one chromosome dispersed in an unfertilized mouse egg using a micromanipulator. [Figure 3] Figure 3 shows the process of isolating a single human chromosome in an unfertilized mouse egg, introducing the isolated single human chromosome into the unfertilized mouse egg, and then fertilizing it. [Figure 4] Figure 4 shows an example of a scheme for establishing embryonic stem cells from mouse embryos containing isolated human chromosomes. In Figure 4, during the process of establishing ES cells, markers and / or drug resistance genes are selectively introduced into the human chromosomes. [Figure 5]Figure 5 shows the results of chromosome analysis of mouse cells containing isolated human chromosome 4. Figure 5 also shows the results of PCR experiments demonstrating the preservation of various regions of the human chromosome 4. [Figure 6] Figure 6 shows the appearance of a chimeric mouse created using isolated human chromosome-containing ES cells and ICR mouse embryos. The black fur originates from the ES cells, and the white fur originates from the embryo. [Figure 7] Figure 7 shows the proportion of embryo-derived cells and cells with human chromosomes (chimerism) in various organs of the obtained chimeric mice. The left panel of Figure 7 shows the chimerism values, with dark gray bars indicating chimerism in donor cells and light gray bars indicating chimerism in donor cells with human chromosome 4. The right panel of Figure 7 shows staining images indicating that cells with human chromosomes are evenly distributed throughout the tissue. [Figure 8] Figure 8 shows the results of whole-genome sequencing of two mouse cell clones transplanted with human chromosome 4. The number of human-derived reads detected from Clone #16 and Clone #26 is plotted on the human genome reference sequence. [Figure 9] Figure 9 shows the results of RNA sequencing of mRNA in mouse ES cells containing human chromosome 4 and in various organs of chimeric mice (ES cells, brain, lungs, heart, stomach, liver, pancreas, and kidney) derived from these cells. It was confirmed that transcription from human chromosome 4 occurs in both undifferentiated ES cells and the differentiated chimeric mouse organs derived from these cells. [Figure 10] Figure 10 shows the expression level of human GRIA2 mRNA in the brain tissue of chimeric mice generated from ES cells containing human chromosomes. In Figure 10, tissue-specific expression of the human GRIA2 gene was observed only in the brain tissue containing human chromosomes. [Figure 11] Figure 11 shows the results of a PCR experiment demonstrating that various regions of human chromosome 21 are maintained in cells into which human chromosome 21 has been introduced. [Figure 12]Figure 12 shows the results of fractionation of mouse ES cells containing human chromosome 15 using flow cytometry with an antibody against CD156c expressed from human chromosome 15. Specific description of the invention

[0007] In this specification, “human chromosome” means a human chromosome. A chromosome is a rod-shaped structure formed before cell division during the M phase, and is a structure that can be observed under a microscope. The human cell nucleus (also called the “human nucleus”) contains 22 pairs of autosomes and 1 pair of sex chromosomes. That is, the human nucleus contains 46 chromosomes. In this specification, “single chromosome” means one of the 46 chromosomes. In this specification, “having human chromosomes” does not mean having all 46 human chromosomes, unless otherwise specified, but rather having some of the 46. In this specification, “having human chromosomes” may mean that human chromosomes include human chromosomes, and that human chromosomes consist of human chromosomes (preferably one to several, one to three, one to two, or more preferably one).

[0008] In this specification, “isolation” means separating from at least one other component. In this specification, “isolation of human chromosomes” means separating a specific chromosome(s) from other chromosomes. In this specification, “isolation of a single chromosome” or similar expression means separating a specific single chromosome from other chromosomes.

[0009] In this specification, "non-human mammal" means mammals other than humans. Examples of mammals include rodents such as rats and mice, non-rodent mammals such as horses, goats, cattle, pigs, sheep, dogs, and cats, and primates other than humans. Throughout this specification, rodents, which are commonly used as laboratory animals, are preferred as non-human mammals.

[0010] In this specification, the "M phase" is one of the phases in the cell cycle. The M phase is the phase in which the cell undergoes mitosis and cell division. In mitosis, sister chromosomes are separated at both ends of the cell, and subsequent cell division divides the cytoplasm to produce two cells. In the phase before cell division in the M phase, the environment for chromosome formation is ready.

[0011] In this specification, the "unfertilized egg" refers to an egg before fertilization. As a result of meiosis, the unfertilized egg contains a haploid number of chromosomes. The unfertilized egg is considered to be a cell in the M phase.

[0012] According to the present invention, it has been clarified that when a human cell nucleus is introduced into an unfertilized egg of a non-human mammal (which is a cell in the M phase) and cultured in the presence of a microtubule formation inhibitor, 46 chromosomes contained in the human cell nucleus are widely dispersed in the unfertilized egg.

[0013] Methods of dispersal of human chromosomes According to the present invention, A method for dispersing human chromosomes, comprising: introducing a human nucleus obtained from a human cell into a cell in the M phase of a non-human mammal (e.g., an unfertilized egg); and culturing the cell in the M phase (e.g., an unfertilized egg) into which the human nucleus has been introduced in the presence of a microtubule formation inhibitor, thereby dispersing the chromosomes contained in the human nucleus within the cell (hereinafter sometimes referred to as "the dispersion method of the present invention") is provided.

[0014] In the present invention, a human cell nucleus (or a human nucleus) can be obtained from a human cell.

[0015] Examples of human cells include human pluripotent cells (e.g., pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells)), human tissue stem cells, human progenitor cells, human somatic cells, and human germ cells. Human cells may be immortalized cells or primary cells. Human cells may also be cells from patients with hereditary diseases (e.g., chromosomal abnormalities, single-gene disorders, autosomal recessive disorders, autosomal dominant disorders, X-linked disorders, X-linked recessive disorders, X-linked dominant disorders, polycystic disorders).

[0016] Human nuclei can be obtained, for example, from nucleated human cells by methods well known to those skilled in the art, such as those used in somatic cell nuclear transplantation. For example, human cell nuclei can be obtained by separating them from nucleated human cells using a micromanipulator.

[0017] In this invention, the significance of introducing a human nucleus into M-phase cells lies in promoting chromosome formation from the genome contained in the human nucleus. In M-phase cells, DNA promotes chromosome formation. Therefore, the human nucleus is introduced into M-phase cells of non-human mammals (for example, M-phase cells before cell division). Among M-phase cells of non-human mammals, unfertilized eggs are preferably used from the viewpoint of size. Unfertilized eggs are advantageous because their large cytoplasm allows for good chromosome dispersion when chromosomes are dispersed within them.

[0018] Unfertilized eggs can be obtained from female non-human mammals by methods well known to those skilled in the art. For example, unfertilized eggs can be obtained from female non-human mammals in which superovulation has been induced. Superovulation can be induced, for example, by administering pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) to a non-human mammal. Unfertilized eggs can be collected from the fallopian tubes. The collected unfertilized eggs may have cumulus cells attached. Cumulus cells can be removed from the unfertilized eggs by hyaluronidase treatment.

[0019] After introducing human nuclei into M-phase cells of non-human mammals, these cells can then be cultured in the presence of a microtubule formation inhibitor. In the presence of the microtubule formation inhibitor, chromosomes cannot undergo mitosis. As a result, the chromosomes are freed from the control of their orientation within the cell and disperse in the cytoplasm. The chromosomes can be dispersed to separate locations, and each chromosome can be isolated using a micromanipulator.

[0020] Examples of microtubule formation inhibitors include microtubule polymerization inhibitors and microtubule depolymerization promoters. Examples of microtubule formation inhibitors include vinca alkaloid microtubule formation inhibitors such as nocodazole, colchicine, colsemid; vincristine, vinorelbine, vinblastine, and vindesine.

[0021] Whether human chromosomes are dispersed can be confirmed, for example, by fluorescent staining of the chromosomes. Chromosome staining is possible by various well-known techniques for those skilled in the art. In the following examples, a mechanism is used in which a fusion protein of human histone H2B and a fluorescent protein is introduced into cells, and when the fusion protein is incorporated into a chromosome, the chromosome begins to fluoresce. The fluorescent protein is not particularly limited, but GFP, RFP, YFP, BFP, and modified fluorescent proteins thereof can be used. More specifically, for example, as the fluorescent protein, superfolder GFP can be used. GFP-like fluorescent proteins such as (sfGFP), EGFP, Citrine, Venus, mVenus, YFP, mApple, mOrange, mCherry, BFP, TagBFP, mTurquoise, and Cerulean, mHoneydew, mBanana, tdTomato, mTangerine, mStrawberry, mPlum, mScarlet, mNeonGreen, mNeptune, and NirFP, as well as circular permutation variants of these modified fluorescent proteins (e.g., with a helix at the C-terminus). Fluorescent proteins that are circular permutation mutants with a specific structure; the prefix "cp-" is added to the protein name to indicate that the protein is a circular permutation mutant; mTurquiose, for example, mTurquiose-DR, mTurquiose-GL, mTurquiose-GV, mTurquiose-RA, mTurquiose2, mTurquiose2-G, mTurquiose-146G, and mTurquiose-146S, and their modified fluorescent proteins can be used. The method of the present invention may further include a step of staining (in particular by fluorescent staining) human chromosomes. The fusion protein may be expressed in a cell by injecting the nucleic acid (e.g., mRNA) encoding the fusion protein into the oocyte cytoplasm.

[0022] According to the method of the present invention, the step of culturing M-phase cells into which human nuclei have been introduced may be a step of culturing in the presence of a chromosome formation promoter in addition to a microtubule formation inhibitor. Examples of chromosome formation promoters include okadaic acid and calcium A. By culturing in the presence of a chromosome formation promoter, chromosome aggregation within the cell can be further promoted.

[0023] Methods for isolating human chromosomes The present invention provides a method for isolating human chromosomes (hereinafter sometimes referred to as "the isolation method of the present invention"). Human chromosomes dispersed within M-phase cells can be isolated chromosome by chromosome unit using a micromanipulator. This isolation allows for the isolation of individual chromosomes. The resulting isolated chromosome may contain the entire length of the chromosome (or a large portion of the chromosome, e.g., 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more base pairs). Using a micromanipulator, it is also possible to isolate a single chromosome, or several single chromosomes. Therefore, the isolation method of the present invention may include the step of aspirating at least one of the human chromosomes dispersed in M-phase cells by the above-described dispersion method of the present invention using a micromanipulator.

[0024] Cytoplasmic solution containing human chromosomes In the aspiration step, the human chromosome is aspirated together with the surrounding cytoplasm. Therefore, the present invention provides a composition (or a cytoplasmic solution containing a human chromosome) comprising an isolated human chromosome (particularly a single chromosome) obtained by the isolation method of the present invention, and the human chromosome and the cytoplasm of a non-human mammal. The composition may be composed of a human chromosome, wherein the human chromosome consists of a human chromosome (preferably one to several, one to three, one to two, or more preferably one).

[0025] According to the present invention, an isolated human chromosome may have at least 50%, 60%, 70%, 80%, 90%, or the entire length of the chromosome.

[0026] Non-human mammalian cells containing human chromosomes and methods for producing them. According to the present invention, human chromosomes or a cytoplasmic solution or composition containing human chromosomes can be further introduced into M-phase non-human mammalian cells to obtain non-human mammalian cells having said human chromosomes. In this way, one or more isolated non-human mammalian cells having human chromosomes can be obtained. Therefore, the present invention provides a method for producing non-human mammalian cells having human chromosomes, comprising the step of introducing a cytoplasmic solution or composition containing human chromosomes (for example, a composition containing human chromosomes, wherein the human chromosomes consist of human chromosomes (preferably one to several, one to three, one to two, or more preferably one)) into M-phase non-human mammalian cells.

[0027] According to the present invention, human chromosomes or a cytoplasmic solution or composition containing human chromosomes can be further introduced into an unfertilized egg of a non-human mammal to obtain an unfertilized egg of a non-human mammal having said human chromosomes. Therefore, the present invention provides a method for producing non-human mammalian cells having human chromosomes, the method comprising the step of introducing a cytoplasmic solution or composition containing human chromosomes (for example, a composition containing human chromosomes, wherein the human chromosomes consist of human chromosomes (preferably one to several, one to three, one to two, or more preferably one)) into an unfertilized egg of a non-human mammal. The introduction of said solution or composition into the unfertilized egg can be performed using a micromanipulator. In this way, the present invention provides an unfertilized egg containing human chromosomes, wherein the human chromosomes consist of human chromosomes (preferably one to several, one to three, one to two, or more preferably one).

[0028] An unfertilized egg can be fertilized with sperm to obtain a fertilized egg. Therefore, the present invention may further include the steps of fertilizing an unfertilized egg with sperm and obtaining a fertilized egg therefrom. A non-human mammal having human chromosomes can be obtained by generating a fertilized egg having human chromosomes. The non-human mammal has cells having human chromosomes. Here, a non-human mammal having human chromosomes includes human chromosomes, and the human chromosomes may be non-human mammals consisting of human chromosomes (preferably one to several, one to three, one to two, or more preferably one), for example, rodents. Also, a non-human mammalian cell having human chromosomes includes human chromosomes, and the human chromosomes may be non-human mammalian cells consisting of human chromosomes (preferably one to several, one to three, one to two, or more preferably one), for example, rodent cells.

[0029] Non-human mammals that possess only one human chromosome (e.g., model animals, such as rodents like mice and rats) can be used as trisomy model animals.

[0030] The type of human chromosome introduced into a cell can be determined by methods well known to those skilled in the art. For example, the type of chromosome introduced into a cell can be determined by chromosome-specific polymerase chain reaction (PCR), Northern blotting, Southern blotting, genome sequencing, RNA sequencing, and fluorescence in situ hybridization (FISH). According to the present invention, it is possible to select cells from non-human mammalian cells into which human chromosomes of a desired chromosome number have been introduced. Accordingly, in some embodiments, the method of the present invention may further include selecting cells from non-human mammalian cells into which human chromosomes have been introduced, into which a human chromosome of a desired chromosome number (for example, one or more selected from chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X chromosome, and Y chromosome) has been introduced.

[0031] Embryonic stem cells of non-human mammals containing human chromosomes and methods for producing them According to the present invention, embryonic stem cells (ES cells) of non-human mammals containing human chromosomes can be produced by generating fertilized eggs containing human chromosomes. ES cells can be obtained from embryos by methods well known to those skilled in the art. ES cells are contained, for example, in the inner cell mass of a blastocyst. Therefore, ES cells containing human chromosomes can be obtained by culturing a blastocyst or its inner cell mass. Therefore, according to the present invention, A method for producing embryonic stem cells of non-human mammals containing human chromosomes, A method comprising culturing an embryo obtained by developing a fertilized egg containing human chromosomes, or an inner cell mass derived from such embryo. This is provided. Culturing can be carried out under conditions suitable for culturing ES cells. ES cells having human chromosomes contain human chromosomes, and the human chromosomes may be ES cells consisting of human chromosomes (preferably one to several, one to three, one to two, or more preferably one).

[0032] Method for producing non-human mammals from ES cells containing human chromosomes, and the produced non-human mammals According to the present invention, ES cells possessing human chromosomes can be transplanted into non-human mammalian embryos (e.g., morula, blastocyst) in the same way as ordinary ES cells, and non-human mammals can be obtained by subsequently developing the embryos. Those skilled in the art can also introduce ES cells into the periuterocoel to develop animals. Therefore, according to the present invention, A method for producing a non-human mammal containing human chromosomes, A method comprising introducing embryonic stem cells having human chromosomes into the embryo of a non-human mammal and causing development. It will be provided. The embryo can be implanted in the uterus of a false pregnant surrogate mother and developed into an individual.

[0033] Method for producing non-human mammalian cells containing human chromosomes and the produced cells According to the present invention, isolated human chromosomes can be introduced into M-phase non-human mammalian cells. This makes it possible to obtain non-human mammalian cells containing isolated human chromosomes. According to the present invention, target cells possessing human chromosomes can also be obtained by culturing ES cells possessing human chromosomes and differentiating them into target cells. The target cells may be, for example, tissue stem cells, progenitor cells, somatic cells, and germ cells. Various in vitro methods have been established for differentiating each type of cell, and those skilled in the art can use them as appropriate. The produced cells can be grown under conditions suitable for the proliferation of those cells (the original cells). The cells produced contain isolated human chromosomes. Furthermore, the produced cells may express surface antigens derived from isolated human chromosomes. Therefore, cells into which human chromosomes have been introduced can be isolated, concentrated, or purified using molecules specific to the surface antigen (e.g., antibodies). Whether cells express a surface antigen can be tested beforehand using antibodies against that antigen. By determining the expressed surface antigen, it is possible to infer which human chromosome has been introduced into the cell, or to concentrate cells into which the target chromosome has been introduced. For example, the present invention provides a method for examining cells, comprising contacting a cell with one or more antibodies against human surface antigens, wherein the formation of a complex between the cell and the antibody indicates that the cell expresses a human surface antigen or has a human chromosome carrying the surface antigen. Here, the cell may be a non-human cell into which isolated human chromosomes have been introduced using the method of the present invention. Detection of the cell-antibody complex can be performed by flow cytometry. In flow cytometry, for example, a fluorescently labeled antibody can be used to optically detect or separate the cell-antibody complex.

[0034] Mouse as a non-human mammal In all embodiments of the present invention, the non-human mammal may be a rodent, preferably a mouse. [Examples]

[0035] Example 1: Chromosome dispersion and individual chromosome recovery

[0036] Collection of unfertilized mouse eggs Eight-week-old BDF1 (B6×DBA2) female mice were administered pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) to induce superovulation. 15-17 hours after hCG administration, cumulus cell-oocyte complexes were collected from the fallopian tubes in KSOM medium. Oocytes were denatured by 0.1% hyaluronidase treatment, washed several times in KSOM medium, and then mature oocytes were transferred to fresh KSOM medium and cultured at 37°C under 5% CO2 conditions.

[0037] Preparation of human cells The culture medium (StemFlex: Gibco) was aspirated from the human iPS cell culture dish and washed with PBS. Cells were detached from the culture dish by adding 0.5% or 0.25% trypsin-EDTA solution, and cells were isolated by pipetting after adding 10% fetal bovine serum (FCS)-containing DMEM medium. After transferring to a centrifuge tube, the cells were centrifuged at 1200 rpm for 3 minutes, and the supernatant was removed. FCS-free DMEM was added, and the centrifugation at 1200 rpm for 3 minutes and supernatant removal was repeated twice. The cells were then resuspended in medium containing a small amount of FCS-free DMEM with the ROCK inhibitor (Y27632). The cell suspension was kept on ice until injection.

[0038] Fluorescent probes for chromosome labeling and injection of human cell nuclei In the following, human cell nuclei were introduced into the cytoplasm of mature mouse oocytes. To stain the nuclei, a fluorescent fusion protein of H2B and mRFP was expressed in the oocytes. Specifically, the procedure was as follows: Drops of 10-12% polyvinylpyrrolidone (PVP)-containing Hepes-CZB medium (PVP drops), drops of a fluorescent probe solution for chromosome labeling (mRNA of the H2B-mRFP1 fusion protein having the amino acid sequence of SEQ ID NO: 1), and drops of Hepes-CZB medium were prepared on the lids of culture dishes and placed on an inverted microscope stage equipped with an epifluorescence observation unit and a piezo-drive-equipped micromanipulator. Human cell suspension was added to the PVP drops and mixed to obtain a homogeneous cell suspension. The collected unfertilized oocytes were transferred to the Hepes-CZB medium. A glass capillary with a diameter of 2-3 μm was set in a microinjector. After aspirating the mRNA solution, the zona pellucida of the oocyte, fixed with a holding glass capillary, was penetrated by several piezoelectric pulses. The glass capillary was then brought into contact with the oocyte membrane and pushed in, and a single weak piezoelectric pulse was applied to create a hole in the oocyte membrane. A small amount of mRNA solution was injected into the oocyte cytoplasm, and the glass capillary was slowly withdrawn from the oocyte. After injection, the oocyte was left at room temperature for about 15 minutes, then transferred to 37°C and cultured under 5% CO2 conditions. Next, human cells were aspirated and expelled using a microinjector equipped with a glass capillary with a diameter of 7-8 μm to disrupt the cell membrane. After collecting several human cell nuclei in the capillary, the zona pellucida was penetrated by piezoelectric pulses and a hole was created in the oocyte membrane in the same manner. After releasing a single human cell nucleus into the egg cytoplasm, the glass capillary was slowly withdrawn from the egg. The prepared human nucleus-injected egg was left at room temperature for about 15 minutes to promote cell membrane repair.

[0039] Chromosome dispersion As a medium for chromosome dispersion, drops were prepared in KSOM medium supplemented with nocodazole (50 μg / ml), a microtubule formation inhibitor, and okadaic acid (50 μM), which induces early chromosome condensation, and placed under conditions of 37°C and 5% CO2. Human nuclear-injected oocytes prepared using the above process were transferred to the chromosome dispersion medium, and chromosome dispersion was confirmed after more than 4 hours of culture. Human nuclear-injected oocytes were cultured until the following day under conditions of 37°C and 5% CO2. As shown in Figure 1, human chromosomes dispersed in the egg cytoplasm were confirmed by observing RFP fluorescence with a fluorescence microscope.

[0040] Example 2: Transfer of recovered individual chromosomes

[0041] chromosome transplantation To isolate human chromosomes from human nuclear-injected oocytes in which chromosome dispersion was confirmed, the oocytes were transferred to KSOM medium supplemented with cytochalasin B (5 μg / ml), an actin polymerization inhibitor, and cultured for 30 minutes to 1 hour at 37°C under 5% CO2 conditions. Drops of 10-12% PVP-containing Hepes-CZB medium, cytochalasin B-supplemented Hepes-CZB medium, and Hepes-CZB medium were prepared on the lids of culture dishes and placed in an inverted microscope. Human nuclear-injected oocytes were transferred to cytochalasin B-supplemented Hepes-CZB medium, and a glass capillary with a diameter of approximately 7-8 μm was placed in a microinjector and penetrated through the zona pellucida, similar to the time of cell nuclear injection. Using RFP fluorescence as an indicator, a single chromosome was selected from the dispersed chromosomes, and the selected chromosome, along with a small amount of oocyte cytoplasm, was aspirated into the glass capillary (see Figure 2). At this time, the oocyte membrane was not ruptured, and the oocyte cytoplasm containing the single chromosome was torn off like mochi (rice cake) to collect it. Next, newly collected unfertilized mouse eggs were transferred to Hepes-CZB medium drops, and the oocyte cytoplasm containing the single chromosome obtained above was injected using the same procedure as for human cell nucleus injection (see Figure 3). This produced mouse oocytes with additional human chromosomes. The oocytes injected with human chromosomes were left to stand at room temperature for about 15 minutes, and then transferred to an incubator at 37°C and 5% CO2.

[0042] Intracytoplasmic sperm injection (ICSI) Next, unfertilized mouse eggs containing human chromosomes were subjected to intracytoplasmic sperm injection (ICSI) (see Figure 3). Sperm were collected from the epididymal tails of 10-week-old albino 129sv strain male mice and suspended in TYH medium. A small amount of the sperm suspension was added to a PVP drop set up in an inverted microscope and thoroughly mixed. A new glass capillary with a diameter of 7-8 μm was set in a microinjector, and the tails of morphologically normal sperm were cut with several piezo pulses, collecting only the sperm heads. Oocytes with chromosome transplantation were transferred to a Hepes-CZB medium drop, and one sperm head was injected into the oocyte cytoplasm. The injection procedure was the same as when human cell nuclei were injected. After sperm injection, the oocytes were allowed to rest at room temperature, then transferred to KSOM medium and continued cultured at 37°C under 5% CO2 conditions. This resulted in obtaining mouse embryos with additional human chromosomes.

[0043] ES cell line establishment Mouse embryos with additional human chromosomes were cultured in KSOM medium and developed to the blastocyst stage to establish ES cell lines (see Figure 4). Specifically, the zona pellucida of embryos that had developed to the blastocyst stage was removed by acidic Tyrode's treatment. ES cell culture medium containing either MEK or Src inhibitor, selected from the latter, and GSK3 inhibitor was added to 96-well plates pre-soaked with feeder cells (MEF treated with mitomycin C). Embryos from which the zona pellucida had been removed were transferred one by one to the 96-well plates and cultured at 37°C under 5% CO2 conditions. After 1 to 10 days, the growing cell clumps were dispersed by trypsin treatment and then seeded again into 96-well plates. Subculturing was repeated every few days until the cells reached approximately 6 wells. This yielded multiple ES cell lines.

[0044] Confirmation of chromosome translocation and introduction of marker genes and drug resistance genes. Specific primers were designed for each human autosome and sex chromosome, and PCR was performed on each established ES cell line to confirm which chromosomes were introduced into each ES cell line. As a result, 134 cell lines were established, and 33 of them contained human chromosomes. Furthermore, it was found that human chromosome numbers 1, 2, 3, 4, 5, 6, 7, 9, 12, 13, 14, 15, 17, 18, 20, 21, and 22 were introduced into the cells as single human chromosomes. These results demonstrate that any human chromosome can be isolated and introduced into other cells in a uniform manner.

[0045] As shown in Table 1, the ratio of cells retaining human chromosomes to the total number of cells established was high, and human chromosome-retaining mouse cells were obtained in 9 out of 34 cell lines. Furthermore, as shown in Table 1, it became clear that various human single chromosomes could be transplanted into mouse cells.

[0046] Furthermore, karyotype analysis was performed on cell lines in which human chromosome retention was confirmed, and it was confirmed that they were maintained as independent chromosomes (see the left photograph in Figure 5). In addition, primers were designed for several locations on both the short and long arms of the introduced chromosome, and PCR was performed to confirm that the entire chromosome had been introduced (see an example on the right in Figure 5). Among the gene loci on the introduced human chromosome, a locus expected to be a safe harbor was selected. A plasmid vector was created by flanking the expression cassettes of the drug resistance gene and the fluorescent reporter gene with genomic DNA sequences near the target site, and this was inserted into the gene locus by homologous recombination using CRISPR / Cas9 double-strand DNA cleavage.

[0047] Confirmation of the chimeric formation ability of cells containing human chromosomes Human chromosome-containing ES cells cultured in drug-selective medium were detached by trypsin treatment and introduced into the periuterocoel of ICR mouse embryos at the 8-cell to morula stage to create chimeric embryos. The chimeric embryos were transplanted into the uterus of female mice on day 3 of pseudopregnancy the day after cell injection, and chimeric individuals were obtained by natural delivery or cesarean section on day 20 of gestation (see Figure 6).

[0048] Chimerism analysis and human chromosome retention analysis in chimeric tissues Adult chimeric individuals of human chromosome-carrying embryonic stem cells and ICR mice were euthanized, and organs from the entire body, including the brain, lungs, heart, liver, pancreas, spleen, kidneys, stomach, intestines, skin, and bone marrow, were collected. The collected samples were lysated, and genomic DNA was purified using either the phenol-chloroform-isoamyl alcohol method or the silica membrane method. Using the purified genomic DNA, the absolute number of human chromosomes was determined by Bio-Rad QX200 droplet digital PCR. In addition, the absolute number of mouse tyrosinase genomes was determined for both wild-type and albino mutant types. Human chromosome-carrying ES cells, which are the donor cells used in chimera creation, possess the wild-type mouse tyrosinase genome, while recipient ICR mouse embryos possess only the albino mutant mouse tyrosinase genome. Therefore, the contribution rate from donor cells can be estimated from the tyrosinase genome mutant ratio. From these absolute genomic copy values, the contribution rate of human chromosome-carrying ES cells in each organ of the chimeric individual and the human chromosome retention rate in cells after terminal differentiation were calculated. The results are shown in Figure 7. The left panel of Figure 7 shows data for the heart and pancreas. As shown in the left panel of Figure 7, the chimera rate of donor cells and the chimera rate of donor cells containing human chromosome 4 were almost the same in the heart and pancreas, indicating that almost all terminally differentiated cells derived from donor cells possessed human chromosomes. Similar results were observed in other organs. Furthermore, as shown in the right panel of Figure 7, human chromosomes were evenly distributed throughout the tissues of the heart and pancreas. Similar results were observed in other organs. Furthermore, whole-genome sequencing was performed on ES cell lines containing human chromosome 4. The results are shown in Figure 8. As shown in Figure 8, mouse ES cells containing human chromosome 4 retained the full length of human chromosome 4. Thus, it became clear that human chromosomes can be stably maintained within mouse cells.

[0049] Confirmation of transcription from transchromosome Adult chimeric individuals of human chromosome-carrying embryonic stem cells and ICR mice were euthanized, and organs from the entire body, including the brain, lungs, heart, liver, pancreas, spleen, kidneys, stomach, intestines, skin, and bone marrow, were collected. The collected samples were immersed in RNAlater at 4°C for 16-24 hours, then disrupted, and total RNA was obtained by acid guanidine phenol chloroform extraction silica membrane purification. Similarly, total RNA was obtained from human chromosome-carrying ES cells by acid guanidine phenol chloroform extraction silica membrane purification. From the obtained total RNA, an RNA-seq library compliant with Illumina high-throughput sequencer standards was created, and sequencing was performed using Novaseq or Hiseq X10. The sequencing results were mapped to the human reference genome sequence GRCh38 and the mouse reference genome sequence GRCm38, and human chromosome-derived gene expression was identified using the k-mer method or deconvolution method. Furthermore, the expression levels of human genes whose tissue-specific expression was predicted by RNA-seq results were confirmed by quantitative RT-PCR. The results are shown in Figure 9. As shown in Figure 9, it was found that mouse cells possessing the full length of human chromosome 4 transcribed mRNA from the entirety of human chromosome 4. In addition, quantitative PCR was performed on total RNA obtained from brain tissue to confirm the expression of human GRIA2, which is specific to the human brain. The results are shown in Figure 10. As shown in Figure 10, human GRIA2 expression was not observed in wild-type mice, but was observed in the brains of mice possessing human chromosomes.

[0050] For cell lines into which chromosome 21 was introduced, genes located in different regions of the chromosome were amplified by PCR, and it was confirmed that each portion of the chromosome was present in the introduced chromosome. Specifically, LIP1, JAM2, SOD1, and CSTB were amplified by PCR. The results are shown in Figure 11. As shown in Figure 11, amplification of all genes was confirmed in the genomic DNA extracted from cells into which chromosome 21 was introduced, whereas no amplification was observed in cells without chromosome 21. From this, it was considered that the introduced chromosome 21 retained at least almost the entire chromosome. Similarly, for cell lines into which chromosome 18 or other chromosomes were introduced, genes that can be located in different regions of the chromosome were amplified by PCR, and it was confirmed that each portion of the chromosome was present in the introduced chromosome. In the same way, the total byproducts of each amplified gene were confirmed only in cells into which the chromosome was introduced. Cells possessing human chromosome 21 can be used as a Down syndrome model cell.

[0051] Selection of chromosome-transformed cells Cells into which a chromosome has been introduced can display the translation products from the chromosome on their cell surface. Therefore, cells into which a chromosome has been introduced can be selected based on their binding affinity to the products expressed on the cell surface. In this example, as an example, mouse ES cells into which human chromosome 15 has been introduced were obtained according to the above example, and the introduced cells were selected using an antibody against CD156c encoded on the same chromosome. Mouse ES cells into which human chromosome 15 has been introduced were confirmed by amplification of the PCR products of hOCA2, hB2M, hUACA, and hSYNM. This revealed that the entire human chromosome 15 had been introduced into the obtained ES cells. The ES cells into which the chromosome has been introduced could be maintained under the same culture conditions as the original cells (i.e., mouse ES cells). In such human chromosome-introduced cells, it was found that the human genes on the introduced human chromosome were being transcribed and translated. Human CD156c-expressing cells located on chromosome 15 were fractionated using a cell sorter. Specifically, the fractionation was as follows: (1) After washing the transchromosome cells cultured on a 6-well plate with D-PBS, a cell detachment enzyme (Accutase, # AT104, Innovative Cell Technologies, etc.) was added and the cells were allowed to stand at 37°C for about 5 minutes. (2) Cells were harvested in D-PBS supplemented with 3% inactivated FBS. (3) The collected cells were transferred to a 15 ml tube and centrifuged at 300 g for 3 minutes. (4) Remove the supernatant with an aspirator, loosen the cell pellet by tapping, and then resuspend in 50-100 μl of D-PBS with 3% inactivated FBS. (5) APC-labeled anti-human CD156c antibody (200 μg / ml, clone SHM14, # 352706, BioLegend) was added at a rate of 1 μl / 1 × 10^7 cells. (6) The samples were kept in a dark place and left to stand at 4°C for 30 minutes. (7) Add 10 ml of D-PBS and centrifuge at 300 g for 3 minutes. (8) The supernatant was removed with an aspirator, the cell pellet was loosened by tapping, and then suspended in 200-500 μl of culture medium containing transchromosome cells (mouse ES cell medium supplemented with a2i). (9) The cell suspension was dropped onto a 48 μm nylon filter (N-No.305T) and aggregates were removed. (10) Propidium iodide was added at a final concentration of 1 μg / ml. This stained the dead cells. (11) The PI-negative CD156c-APC-positive fraction was separated using BD FACS AriaIII. An appropriate amount (approximately 5 ml / 15 ml tube) of culture medium for transchromosome-transfected cells (mouse ES cell medium supplemented with a2i) was placed in the tubes to be separated.

[0052] The percentage of CD156c-expressing cells was measured by flow cytometry. The results are shown in Figure 12. As shown in Figure 12, very few CD156c-expressing cells were observed in wild-type mouse ES cells, while a large number of CD156c-expressing cells were observed in mouse ES cells into which human chromosome 15 had been introduced.

Claims

1. A method for producing non-human mammalian cells having human chromosomes, A method comprising the step of introducing the entire length of isolated human chromosomes, obtained by a method for isolating human chromosomes, into M-phase non-human mammalian cells, wherein the isolation method includes the step of aspirating at least one of the human chromosomes dispersed within the M-phase non-human mammalian cells into which one to several human chromosomes have been introduced, using a micromanipulator.

2. The method according to claim 1, wherein the M-phase non-human mammalian cell into which human chromosomes have been introduced is an unfertilized rodent egg.

3. The method according to claim 2, further comprising the step of fertilizing the unfertilized egg containing human chromosomes with sperm to obtain a fertilized egg.

4. A method for producing embryonic stem cells of non-human mammals containing human chromosomes, The method involves culturing an embryo obtained by developing a fertilized egg obtained by the method described in claim 3, or an inner cell mass derived from said embryo, A method comprising obtaining embryonic stem cells from an embryo or inner cell mass, wherein the embryo and embryonic stem cells include the full length of human chromosomes introduced into the cells.

5. The method according to claim 4, wherein the embryo and embryonic stem cells contain human chromosomes, and the human chromosomes consist of one to three human chromosomes.

6. A method for producing a non-human mammal containing human chromosomes, A method comprising introducing embryonic stem cells obtained by the method of claim 4 into an embryo of a non-human mammal and developing it to obtain a non-human mammal containing human chromosomes.

7. The method according to claim 6, wherein the non-human mammal contains human chromosomes, and the human chromosomes consist of one to three human chromosomes.

8. A method for producing non-human mammalian cells containing human chromosomes, Culture embryonic stem cells obtained by the method of claim 4 in vitro, Differentiating embryonic stem cells into target cells to obtain non-human mammalian cells containing human chromosomes. Methods that include...

9. A method for producing non-human mammalian cells having human chromosomes, A method comprising the step of introducing isolated human chromosomes, obtained by a method for isolating human chromosomes, into M-phase non-human mammalian cells, wherein the isolation method includes the step of introducing one to several human chromosomes and aspirating at least one of the human chromosomes dispersed within the M-phase non-human mammalian cells using a micromanipulator, wherein the M-phase non-human mammalian cells into which the human chromosomes have been introduced are unfertilized eggs.

10. The process further includes the step of fertilizing the unfertilized egg containing human chromosomes with sperm to obtain a fertilized egg. The method according to claim 9.

11. A method for producing embryonic stem cells of non-human mammals containing human chromosomes, To obtain a fertilized egg by the method of claim 10, A method comprising culturing an embryo obtained by developing a fertilized egg, or an inner cell mass derived from said embryo.

12. A method for producing a non-human mammal containing human chromosomes, To obtain embryonic stem cells by the method of claim 11, A method comprising introducing the obtained embryonic stem cells into the embryo of a non-human mammal and causing it to develop.

13. A method for producing non-human mammalian cells containing human chromosomes, To obtain embryonic stem cells in vitro by the method of claim 11, The obtained embryonic stem cells are cultured, A method comprising differentiating embryonic stem cells into target cells.

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