WO2021200768A1 - Procédé de dispersion et procédé d'isolation de chromosome humain, et procédé de transplantation de chromosome humain dans un embryon animal - Google Patents

Procédé de dispersion et procédé d'isolation de chromosome humain, et procédé de transplantation de chromosome humain dans un embryon animal Download PDF

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WO2021200768A1
WO2021200768A1 PCT/JP2021/013163 JP2021013163W WO2021200768A1 WO 2021200768 A1 WO2021200768 A1 WO 2021200768A1 JP 2021013163 W JP2021013163 W JP 2021013163W WO 2021200768 A1 WO2021200768 A1 WO 2021200768A1
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human
cells
chromosome
cell
chromosomes
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Japanese (ja)
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啓光 中内
山口　智之
英二 水谷
直彬 水野
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国立大学法人 東京大学
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells

Definitions

  • 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.
  • micronucleus cell fusion method (microcell mediated chromosome transfer: MMCT) has been developed.
  • MMCT microcell mediated chromosome transfer
  • human cells are treated with corsemido to form intracellular micronuclei containing a single chromosome, and the micronuclei are purified and transplanted into non-human mammalian cells.
  • micronuclei can be formed only in a very limited number of established cells such as skin fibroblast A9 cell line and Chinese hamster ovary cell (CHO cell) line.
  • the chromosomes are usually unstable and it is difficult to maintain full length.
  • the present inventors have found that a human cell nucleus containing a human chromosome can be introduced into a rodent cell having an M-phase cell cycle and dispersed in each chromosome in the cell.
  • the present inventors have also found that intracellularly dispersed chromosomes can be isolated one by one using a micromanipulator. Furthermore, the present inventors can stably maintain the isolated chromosome in the rodent cell even if the isolated chromosome is introduced into the rodent cell having the cell cycle of the M phase to culture or generate the cell. , And found that it could include the entire length from one end to the other of the chromosome. In this way, we have completed a method for maintaining one or more human chromosomes in non-human mammalian cells.
  • a method for dispersing human chromosomes A step of introducing human nuclei obtained from human cells into non-human mammalian M-phase cells; and a step of culturing human nuclei-introduced M-phase cells in the presence of a microtubule formation inhibitor.
  • a method comprising the step of dispersing the chromosomes contained in the human nucleus in the cell thereby.
  • the method according to the above [1], wherein the step of culturing M-phase cells into which a human nucleus has been introduced is a step of culturing in the presence of okadaic acid in addition to a microtubule formation inhibitor.
  • a method for producing a non-human mammalian cell having a human chromosome comprising the step of introducing an isolated human chromosome obtained by the method according to the above [5] into non-human mammalian cells in the M phase.
  • a method for producing non-human mammalian embryonic stem cells containing a human chromosome A method for producing non-human mammalian embryonic stem cells containing a human chromosome.
  • a method comprising culturing an embryo obtained by developing a fertilized egg obtained by the method according to the above [9], or an inner cell mass derived from the embryo.
  • a method for producing a non-human mammal containing a human chromosome A method comprising introducing and developing embryonic stem cells obtained by the method according to the above [10] into the embryo of the non-human mammal.
  • a method for producing a non-human mammalian cell containing a human chromosome By culturing the embryonic stem cells obtained by the method according to the above [10] in vitro, A method comprising differentiating embryonic stem cells into cells of interest.
  • FIG. 1 shows a state in which chromosomes are dispersed in unfertilized mouse eggs.
  • FIG. 2 shows that one of the chromosomes dispersed in an unfertilized mouse egg is isolated by a micromanipulator.
  • FIG. 3 shows a series of steps in which a single human chromosome is isolated in an unfertilized mouse egg, and the isolated single human chromosome is introduced into an unfertilized mouse egg and fertilized.
  • FIG. 4 shows an example of a scheme for establishing embryonic stem cells from mouse embryos having isolated human chromosomes. In FIG. 4, markers and / or drug resistance genes are selectively introduced into human chromosomes during the process of establishing ES cells.
  • FIG. 4 shows an example of a scheme for establishing embryonic stem cells from mouse embryos having isolated human chromosomes. In FIG. 4, markers and / or drug resistance genes are selectively introduced into human chromosomes during the process of establishing ES cells.
  • FIG. 5 shows the results of a chromosomal test on mouse cells having an isolated human chromosome 4.
  • FIG. 5 is also the result of a PCR experiment showing that various parts of the human chromosome 4 are maintained.
  • FIG. 6 shows the appearance of a chimeric mouse prepared by using an ES cell having an isolated human chromosome and an ICR mouse embryo. Black hair is derived from the ES cell, and white hair is derived from the embryo.
  • FIG. 7 shows the ratio (chimerism) of embryo-derived cells and cells having a human chromosome in various organs of the obtained chimeric mouse. The left panel of FIG.
  • FIG. 7 shows the chimerism values
  • the dark gray bar shows the chimerism of the donor cell
  • the light gray shows the chimerism of the donor cell having human chromosome 4.
  • the right panel of FIG. 7 is a stained image showing that cells having human chromosomes are evenly present in the tissue.
  • FIG. 8 shows the results of a whole genome sequence of two mouse cell clones transplanted with human chromosome 4. The number of human-derived reads detected from Clone # 16 and Clone # 26 is shown on the human genome reference sequence.
  • FIG. 9 shows RNA of mRNA in the tissues of mouse ES cells having human chromosome 4 and chimeric mouse organs (ES cells, brain, lung, heart, stomach, liver, pancreas, and kidney) prepared from these cells.
  • FIG. 10 shows the expression level of human GRIA2 mRNA in the brain tissue of a chimeric mouse prepared from ES cells having a human chromosome. In FIG. 10, expression of the human GRIA2 gene was observed in a tissue-specific manner only in the brain having a human chromosome.
  • FIG. 11 shows the results of a PCR experiment showing that various parts of human chromosome 21 are maintained in cells into which human chromosome 21 has been introduced.
  • FIG. 12 shows the results of flow cytometry fractionation of mouse ES cells having human chromosome 15 using an antibody against CD156c expressed from human chromosome 15.
  • the "human chromosome” means a human chromosome. Chromosomes are rod-like structures that are formed before division in the M phase and are observable under a microscope.
  • the human cell nucleus also referred to as the "human nucleus" contains 22 pairs of autosomal chromosomes and a pair of sex chromosomes. That is, the human nucleus contains 46 chromosomes.
  • single chromosome means one of 46 chromosomes.
  • having a human chromosome does not mean having all 46 human chromosomes, but means having a part of 46, unless otherwise specified.
  • having a human chromosome includes a human chromosome, wherein the human chromosome is a human chromosome (preferably one to several, one to three, one to two, or more preferably one. It can mean that it consists of a book).
  • isolation means separation from at least one other component.
  • isolated a human chromosome means separating a particular chromosome (s) from another.
  • isolatedation of a single chromosome or similar expression means separating one particular chromosome from another.
  • non-human mammal means a mammal other than human.
  • mammals include rodents such as rats and mice, non-rodent mammals such as horses, goats, cows, pigs, sheep, dogs and cats, and non-human primates.
  • rodents which are often used as experimental animals, are preferred as non-human mammals.
  • the "M phase” is one phase of the cell cycle.
  • the M phase is the phase in which cells undergo mitosis and cell division. In mitosis, sister chromosomes are divided into both ends of the cell, and subsequent cell division divides the cytoplasm to produce two cells. In the pre-cell division phase of the M phase, the environment in which chromosomes are formed is in place.
  • unfertilized egg means an egg before fertilization. Unfertilized eggs contain half the number of chromosomes as a result of meiosis. Unfertilized eggs are considered to be M-phase cells.
  • a human cell nucleus when introduced into an unfertilized egg (M stage cell) of a non-human mammal and cultured in the presence of a microtubule formation inhibitor, 46 chromosomes contained in the human cell nucleus are contained. Was found to be widely dispersed in unfertilized eggs.
  • Dispersion method of human chromosomes According to the present invention A method of dispersing human chromosomes The step of introducing human nuclei obtained from human cells into non-human mammalian M-phase cells (eg, unfertilized eggs); and the M-phase cells into which human nuclei have been introduced (eg, unfertilized eggs) are microscopic. A method of culturing in the presence of a tube formation inhibitor, which comprises a step of dispersing chromosomes contained in human nuclei in cells (hereinafter, may be referred to as "dispersion method of the present invention"). Is provided.
  • the human cell nucleus (or human nucleus) can be obtained from a human cell.
  • Human cells include human pluripotent cells (eg, pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells)), human tissue stem cells, human progenitor cells, and human somatic cells. And human germ cells. Human cells may be immortalized cells or primary cells. Human cells have hereditary disorders (eg, chromosomal abnormalities, monogenic disorders, autosomal recessive inheritance disorders, autosomal dominant inheritance disorders, X-linked recessive disorders, X-linked recessive inherited disorders, X-linked dominant inheritance). It may be a cell of a patient having a disease (drinking genetic disease).
  • the human nucleus can be obtained from, for example, a human cell that has formed a nucleus by a method of a technique well known to those skilled in the art used in, for example, somatic cell nuclear transfer.
  • human cell nuclei can be obtained by separating from the nucleated human cells using a micromanipulator.
  • the significance of introducing human nuclei into M-phase cells is to promote chromosome formation from the genome contained in human nuclei.
  • DNA promotes chromosome formation. Therefore, human nuclei are introduced into non-human mammalian M-phase cells (eg, pre-cell division M-phase cells).
  • non-human mammalian M-phase cells eg, pre-cell division M-phase cells.
  • unfertilized eggs can be preferably used from the viewpoint of size. Since the cytoplasm of an unfertilized egg is huge, it is advantageous to disperse the chromosomes internally because the degree of chromosome dispersion is good.
  • Unfertilized eggs can be obtained from female non-human mammals by a method well known to those skilled in the art.
  • unfertilized eggs are obtained from female non-human mammals that have induced hyperovulation.
  • Hyperovulation can be induced, for example, by administering equine chorionic gland stimulating hormone (PMSG) and human chorionic gonadotropin (hCG) to non-human mammals.
  • Unfertilized eggs can be collected from the oviduct.
  • the unfertilized egg to be collected may have cumulus cells attached to it. Cumulus cells can be removed from unfertilized eggs by hyaluronidase treatment.
  • the cells After introducing human nuclei into M-phase cells of non-human mammals, the cells can then be cultured in the presence of microtubule formation inhibitors. In the presence of microtubule formation inhibitors, chromosomes are unable to undergo mitosis. As a result, the chromosomes are out of control of intracellular orientation and are dispersed in the cytoplasm. Chromosomes can be dispersed in different places for each chromosome, and each chromosome can be isolated by a micromanipulator.
  • microtubule formation inhibitor examples include a microtubule polymerization inhibitor and a microtubule depolymerization accelerator.
  • microtubule formation inhibitors include vinca alkaloid microtubule formation inhibitors such as nocodazole, colchicine, colsemido; vincristine, vinorelbine, vinblastine, and vindesine.
  • chromosomes Whether or not human chromosomes are dispersed can be confirmed by, for example, fluorescent staining of chromosomes. Chromosome staining can be performed by a variety of well-known techniques by those skilled in the art. In the following examples, a mechanism can be used in which a fusion protein of human histone H2B and a fluorescent protein is introduced into a cell, and when the fusion protein is incorporated into a chromosome, the chromosome emits fluorescence.
  • the fluorescent protein is not particularly limited, but GFP, RFP, YFP, BFP, and modified fluorescent proteins thereof can be used.
  • fluorescent proteins superfolder GFP (sfGFP), EGFP, Citrine, Venus, mVenus, YFP, mApple, mOrange, mCherry, BFP, TagBFP, mTurqueoise, and Cerulean GFP-like fluorescent proteins such as mTangerine, mStrawbury, mPlum, mScarlet, mNeonGreen, mNeptune and NirFP, and circularly ordered variants of these modified fluorescent proteins (eg, circularly ordered variants having a helix structure at the C-terminal).
  • sfGFP superfolder GFP
  • EGFP Citrine
  • Venus Citrine
  • mVenus e.g., mVenus
  • YFP mVenus
  • mApple mOrange
  • mCherry mCherry
  • BFP TagBFP
  • mTurqueoise mTurqueoise
  • 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 the step of staining human chromosomes (particularly, fluorescent staining).
  • the fusion protein may be expressed intracellularly by injecting a nucleic acid encoding the fusion protein (eg, mRNA) into the cytoplasm of the egg.
  • the step of culturing M-phase cells into which a human nucleus has been introduced may be a step of culturing in the presence of a chromosome formation promoter in addition to a microtubule formation inhibitor.
  • Chromosome formation promoters include, for example, okadaic acid and caliculin A.
  • a method for isolating a human chromosome (hereinafter, may be referred to as “method for isolating the present invention”) is provided.
  • Human chromosomes dispersed in cells in the M phase can be isolated on a chromosome-by-chromosome basis by a micromanipulator. Then, by isolating in this way, the isolated chromosomes can be isolated one by one in chromosome units.
  • the obtained isolated chromosome is the total length of the chromosome (or most of the chromosome, for example, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, It may contain 80% or more, 85% or more, 90% or more, or 95% or more base pairs).
  • a single chromosome can also be isolated using a micromanipulator.
  • the isolation method of the present invention may include a step of sucking at least one of the human chromosomes dispersed in the cells of the M phase by the above-mentioned dispersion method of the present invention using a micromanipulator.
  • the isolation method of the present invention may include a step of sucking one of the human chromosomes dispersed in the cells of the M phase by the above-mentioned dispersion method of the present invention using a micromanipulator.
  • a composition (or human) containing an isolated human chromosome (particularly a single chromosome) and the human chromosome and the cytoplasm of a non-human mammal obtained by the isolation method of the present invention.
  • a cytoplasmic solution containing chromosomes is provided.
  • This composition comprises a human chromosome, which is a composition consisting of human chromosomes (preferably one to several, one to three, one to two, or more preferably one). could be.
  • an isolated human chromosome can have at least 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or full length of the chromosome.
  • Non-human mammalian cells having human chromosomes and methods for producing them According to the present invention, human chromosomes or cytoplasmic solutions or compositions containing human chromosomes are further introduced into M-phase non-human mammalian cells. Non-human mammalian cells having the human chromosome can be obtained. In this way, non-human mammalian cells with a single or multiple isolated human chromosomes are obtained.
  • a method for producing a non-human mammalian cell having a human chromosome wherein the cytoplasmic solution or composition containing the human chromosome (for example, containing the human chromosome, and the human chromosome is a human chromosome (preferably).
  • a method comprising the step of introducing a composition) consisting of one to several, one to three, one to two, or more preferably one) into cells of a non-human mammalian in stage M. Is provided.
  • a human chromosome or a cytoplasmic solution or composition containing a human chromosome is further introduced into an unfertilized egg of a non-human mammal to produce an unfertilized egg of a non-human mammal having the human chromosome.
  • the cytoplasmic solution or composition containing the human chromosome for example, containing the human chromosome, and the human chromosome is a human chromosome (preferably).
  • the human chromosome comprises a human chromosome, the human chromosome being a human chromosome (preferably one to several, one to three, one to two, or more preferably one).
  • An unfertilized egg consisting of is provided.
  • Fertilized eggs can be obtained by fertilizing unfertilized eggs with sperms. Therefore, the present invention may further include a step of fertilizing an unfertilized egg and a sperm and a step of obtaining a fertilized egg thereby.
  • a fertilized egg having a human chromosome By developing a fertilized egg having a human chromosome, a non-human mammal having a human chromosome can be obtained.
  • the non-human mammal has cells with human chromosomes.
  • the non-human mammal having a human chromosome includes a human chromosome
  • the human chromosome is a human chromosome (preferably one to several, one to three, one to two, or more preferably one.
  • non-human mammal consisting of a book
  • a rodent for example, a rodent.
  • the non-human mammalian cell having a human chromosome includes a human chromosome
  • the human chromosome is a human chromosome (preferably one to several, one to three, one to two, or more preferably one. It can be a non-human mammalian cell consisting of the book), for example, a rodent cell.
  • a non-human mammal having only one human chromosome (for example, a model animal, for example, a rodent such as a mouse or a rat) can be a trisomy model animal.
  • telome-specific polymerase chain reaction PCR
  • Northern blot Southern blot
  • genome sequencing RNA sequencing
  • FISH fluorescent in situ hybridization
  • a cell into which a human chromosome having a chromosome number of interest has been introduced can be selected from cells of a non-human mammal into which a human chromosome has been introduced.
  • the method of the present invention is from a non-human mammalian cell into which a human chromosome has been introduced to a human chromosome having a chromosome number of interest (eg, chromosome 1, chromosome 2, chromosome 3, chromosome 4, 4).
  • a human chromosome having a chromosome number of interest eg, chromosome 1, chromosome 2, chromosome 3, chromosome 4, 4
  • Non-human mammalian embryonic stem cells containing human chromosomes and methods for producing them are generated by generating fertilized eggs having human chromosomes. ) Can be produced.
  • 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 blastocysts. Therefore, ES cells having a human chromosome can be obtained by culturing a blastocyst or an inner cell mass thereof. Therefore, according to the present invention A method for producing non-human mammalian embryonic stem cells containing human chromosomes.
  • a method comprises culturing an embryo obtained by developing a fertilized egg having a human chromosome, or an inner cell mass derived from the embryo. Culturing can be performed under conditions suitable for culturing ES cells.
  • ES cells having a human chromosome include a human chromosome, and the human chromosome is an ES composed of human chromosomes (preferably one to several, one to three, one to two, or more preferably one). Can be a cell.
  • ES cells having human chromosomes are similar to normal ES cells in non-human mammalian embryos (eg, for example. , Morula, blastocyst) and then embryos can be obtained to obtain non-human mammals.
  • non-human mammalian embryos eg, for example. , Morula, blastocyst
  • embryos can be obtained to obtain non-human mammals.
  • Those skilled in the art can introduce ES cells into the fertilized blastocoel to generate animals. Therefore, according to the present invention
  • a method for producing non-human mammals containing human chromosomes comprises introducing and developing embryonic stem cells having a human chromosome into the embryo of the non-human mammal. Embryos can engraft in the uterus of pseudopregnant foster females and develop in individuals.
  • 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.
  • an ES cell having a human chromosome can also be obtained by culturing an ES cell having a human chromosome and differentiating it into a target cell.
  • Target cells can be, for example, tissue stem cells, progenitor cells, somatic cells, and germ cells.
  • the produced cells can be grown under conditions suitable for the growth of the cells (original cells).
  • the cells produced contain isolated human chromosomes.
  • the cells produced can express surface antigens derived from isolated human chromosomes. Therefore, cells into which a human chromosome has been introduced can be isolated, concentrated, or purified using a molecule specific to the surface antigen (for example, an antibody). Whether or not a cell expresses a surface antigen can be tested in advance using an antibody against the antigen. By determining the expressed surface antigen, it is possible to infer which human chromosome was introduced into the cell and to concentrate the cell into which the target chromosome was introduced.
  • a method of examining a cell which comprises contacting the cell with one or more antibodies against a human surface antigen to form a complex of the cell and the antibody.
  • the cell can be a non-human cell into which an isolated human chromosome has been introduced by the method of the invention.
  • Detection of cell-antibody complexes can be performed by flow cytometry. In flow cytometry, for example, a fluorescently labeled antibody can be used to optically detect or fractionate a cell-antibody complex.
  • the non-human mammal can be a rodent, preferably a mouse.
  • Example 1 Dispersion of chromosomes and recovery of individual chromosomes
  • Equine chorionic gland stimulating hormone (PMSG) and human chorionic gonadotropin (hCG) were administered to 8-week-old BDF1 (B6 ⁇ DBA2) female mice to induce hyperovulation.
  • the cumulus cell-egg complex was recovered from the oviduct 15 to 17 hours after the administration of hCG in KSOM medium.
  • Eggs were stripped by 0.1% hyaluronidase treatment, washed several times with KSOM medium, and then mature eggs were transferred to fresh KSOM medium and cultured at 37 ° C. under 5% CO 2 conditions.
  • Preparation of human cells Medium (StemFlex: Gibco) was aspirated from a 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 DMEM medium containing 10% fetal bovine serum (FCS) was added, and then the cells were isolated by pipetting. After transferring to a centrifuge tube, the supernatant was removed after centrifugation at 1200 rpm for 3 minutes.
  • FCS fetal bovine serum
  • FCS-free DMEM was added, centrifugation was performed at 1200 rpm for 3 minutes, and supernatant removal was repeated twice, and then resuspended in a medium containing a small amount of FCS-free DMEM supplemented with a ROCK inhibitor (Y27632). The cell suspension was placed on ice until injection.
  • H2B and mRFP Chromosome Label Fluorescent Probe and Injection of Human Cell Nuclei
  • human cell nuclei were introduced into the cytoplasm of mature mouse eggs.
  • a fluorescent fusion protein of H2B and mRFP was expressed in the egg to dye the nucleus. Specifically, it was as follows. Hepes-CZB medium (PVP drop) containing 10-12% polyvinylpyrrolidone (PVP), a drop of fluorescent probe for chromosome labeling (mRNA of H2B-mRFP1 fusion protein having the amino acid sequence of SEQ ID NO: 1) and Hepes-CZB medium.
  • Each drop was prepared on the culture dish lid and placed on an inverted microscope stage equipped with an epi-fluorescent observation unit equipped with a micromanipulator equipped with a piezodrive.
  • a human cell suspension was added to the PVP drop and stirred to obtain a uniform cell suspension.
  • the collected unfertilized eggs were transferred to Hepes-CZB medium.
  • a glass capillary with a diameter of 2 to 3 ⁇ m is set in the microinjector, and after sucking the mRNA solution, the zona pellucida of the egg fixed with the holding glass capillary is pierced by applying several piezopulses, and the glass capillary is attached to the egg cell membrane.
  • the egg cell membrane was punctured by contacting it and pushing it all the way in, and applying a weak piezopulse once.
  • a small amount of mRNA solution was injected into the egg cytoplasm, and the glass capillary was slowly withdrawn from the egg.
  • the injected eggs were allowed to stand at room temperature for about 15 minutes and then transferred to 37 ° C. and 5% CO 2 conditions for culturing.
  • the cell membrane was destroyed by sucking and exhaling human cells with a microinjector set with a glass capillary having a diameter of 7 to 8 ⁇ m.
  • the egg cell membrane was similarly pierced after penetrating the zona pellucida with piezopulse.
  • the glass capillary was slowly withdrawn from the egg.
  • the prepared human nucleus-injected ovum was left at room temperature for about 15 minutes to promote the repair of the cell membrane.
  • KSOM medium As a medium for chromosomal dispersion chromosome dispersion, to produce a drop in KSOM medium supplemented with okadaic acid (50 [mu] M) causing nocodazole (50 [mu] g / ml) and premature chromosome aggregation microtubule formation inhibitor, 37 °C, 5% CO 2 It was placed under the conditions. It was confirmed that the human nucleus-injected ovum prepared in the above process was transferred to a medium for chromosomal dispersion and the chromosomes were dispersed by culturing for 4 hours or more. Human nucleus-injected eggs were cultured at 37 ° C. under 5% CO 2 conditions until the next day. As shown in FIG. 1, by observing RFP fluorescence with a fluorescence microscope, human chromosomes dispersed in the egg cytoplasm could be confirmed.
  • Chromosome transplantation In order to isolate human chromosomes from human nucleus-injected ova with confirmed chromosomal dispersion, they were transferred to KSOM medium supplemented with cytochalasin B (5 ⁇ g / ml), which is an actin polymerization inhibitor, and 37 for 30 minutes to 1 hour. The cells were cultured under 5 ° C. and 5% CO 2 conditions. Drops of Hepes-CZB medium containing 10-12% PVP, Hepes-CZB medium supplemented with cytochalasin B, and Hepes-CZB medium were prepared on the culture dish lid and set in an inverted microscope.
  • cytochalasin B 5 ⁇ g / ml
  • the human nucleus-injected ovum was transferred to Hepes-CZB medium supplemented with cytochalasin B, a glass capillary having a diameter of about 7 to 8 ⁇ m was set in a microinjector, and the zona pellucida was penetrated in the same manner as in the case of cell nucleus injection.
  • a single chromosome was selected from the dispersed chromosomes using RFP fluorescence as an index, and the selected chromosome with a small amount of egg cytoplasm was sucked into a glass capillary (see FIG. 2). At this time, the egg cell membrane was not broken, and the egg cytoplasm containing a single chromosome was torn off like a rice cake and collected.
  • Microinsemination Next, unfertilized mouse eggs having a human chromosome were subjected to microinsemination (see FIG. 3).
  • Sperm were collected from the epididymal tail of a 10-week-old albino 129sv strain male mouse and suspended in TYH medium. A small amount of sperm suspension was added to the PVP drop set in an inverted microscope and stirred well. A glass capillary with a diameter of 7 to 8 ⁇ m was newly set in a microinjector, and the tail of sperm having a normal morphology was cut by several piezopulses, and only the sperm head was collected.
  • Chromosome-transplanted eggs were transferred to Hepes-CZB medium drops and one sperm head was injected into the egg cytoplasm.
  • the injection procedure was the same as when injecting human cell nuclei.
  • the eggs were allowed to rest at room temperature, transferred to KSOM medium, and continuously cultured at 37 ° C. under 5% CO 2 conditions. This gave a mouse embryo with an additional human chromosome.
  • ES cell line A mouse embryo having an additional human chromosome was cultured in KSOM medium to develop a blastocyst, and an ES cell line was established (see FIG. 4). Specifically, the zona pellucida of the embryo that had developed up to the blastocyst was removed by an acidic tie load treatment. An ES cell culture medium containing one of MEK and Src inhibitors and a GSK3 inhibitor was placed in a 96-well plate previously sprinkled with feeder cells (MEF treated with mitomycin C). Embryos from which the zona pellucida had been removed were transferred one by one to a 96-well plate, and culture was continued under 37 ° C. and 5% CO 2 conditions.
  • Chromosome transfer confirmation and introduction of marker gene and comfort drug resistance gene Design specific primers for each human autosomal chromosome and sex chromosome, perform PCR for each established ES cell line, and determine the number of chromosome in each ES cell line. I confirmed whether it was introduced. As a result, the number of established cell lines was 134, and the number of cell lines including human chromosomes was 33. In addition, human chromosome numbers: 1, 2, 3, 4, 5, 6, 7, 9, 12, 13, 14, 15, 17, 18, 20, 21, and 22 are intracellular as a single human chromosome. It was found that it was introduced in. From these results, it was clarified that any human chromosome can be evenly isolated and introduced into other cells.
  • a plasmid vector in which the expression cassettes of the drug resistance gene and the fluorescent reporter gene were sandwiched between genomic DNA sequences near the target site was prepared, and double-stranded DNA cleavage by CRISPR / Cas9 was used in combination and inserted into the locus by homologous recombination. ..
  • Human chromosome-bearing ES cells cultured in a drug-selective medium are peeled off by trypsin treatment and introduced into the fertilized egg surroundings cavity of ICR mice from the 8-cell stage to the morula stage to obtain chimeric embryos. Made.
  • the chimeric embryo was transplanted into the uterus of a female mouse on the 3rd day of pseudopregnancy the day after the cell injection, and a chimeric individual was obtained by spontaneous delivery or cesarean section on the 20th day of pregnancy (see FIG. 6).
  • mice tyrosinase genome copies were absolutely quantified for each of the wild-type and albino mutant types.
  • Human chromosome-bearing ES cells which are donor cells at the time of chimera production, have a wild-type mouse tyrosinase genome, whereas recipient ICR mouse embryos have only an albino mutant mouse tyrosinase genome.
  • the contribution rate of origin can be estimated. From these genome copy absolute quantification values, the contribution rate of human chromosome-carrying ES cells in each organ of the chimeric individual and the human chromosome retention rate in the cells after terminal differentiation were calculated. The results were as shown in FIG. The left panel of FIG.
  • RNA-seq library An Illumina high-throughput sequencer-compliant RNA-seq library was prepared from the obtained total RNA, and the sequence was performed with Novaseq or Hiseq X10. The sequence 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 by the kmer method or the deconvolution method. Furthermore, the expression level of the human gene whose tissue-specific expression was estimated from the RNA-seq results was confirmed by the quantitative RT-PCR method. The results were as shown in FIG. As shown in FIG. 9, it was found that mouse cells having the full length of human chromosome 4 transcribed mRNA from the entire human chromosome 4.
  • a cell into which a chromosome has been introduced can present a translation product from the chromosome on the cell surface. Therefore, cells into which chromosomes have been introduced can be selected based on their binding affinity for products expressed on the cell surface.
  • mouse ES cells into which human chromosome 15 was introduced were obtained according to the above example, and an attempt was made to select the introduced cells with an antibody against CD156c encoded on the same chromosome.
  • Mouse ES cells into which human chromosome 15 was introduced were confirmed by amplification of PCR products of hOCA2, hB2M, hUACA, and hSYNM.
  • the chromosome-introduced ES cells could be maintained under the same culture conditions as the original cells (ie, mouse ES cells). It has been found that in such human chromosome-introduced cells, the human gene on the introduced human chromosome is transcribed and translated. Human CD156c-expressing cells present on chromosome 15 were fractionated using a cell sorter.
  • the proportion of CD156c-expressing cells was measured by flow cytometry. The results were as shown in FIG. As shown in FIG. 12, almost no CD156c-expressing cells were observed in wild-type mouse ES cells, whereas a large amount of CD156c-expressing cells were observed in mouse ES cells into which human chromosome 15 was introduced.

Abstract

La présente invention concerne un procédé de dispersion et un procédé d'isolation de chromosome humain, et un procédé de transplantation d'un chromosome humain dans un embryon animal. La présente invention propose un procédé d'isolation d'un chromosome humain unique à partir de chromosomes restants, et une cellule de mammifère non humaine ayant le chromosome humain isolé.
PCT/JP2021/013163 2020-03-30 2021-03-29 Procédé de dispersion et procédé d'isolation de chromosome humain, et procédé de transplantation de chromosome humain dans un embryon animal WO2021200768A1 (fr)

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Citations (3)

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JPH0394626A (ja) * 1988-12-21 1991-04-19 Univ Pennsylvania トランスジェニックな生物体および細胞、ならびにトランスジェニックな生物体および細胞の製造法
JP2001521617A (ja) * 1997-03-27 2001-11-06 シメグ リミテッド 臨床学的診断方法
WO2018062392A1 (fr) * 2016-09-28 2018-04-05 国立大学法人鳥取大学 Rat de modèle du syndrome de down et son procédé de production

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JPH0394626A (ja) * 1988-12-21 1991-04-19 Univ Pennsylvania トランスジェニックな生物体および細胞、ならびにトランスジェニックな生物体および細胞の製造法
JP2001521617A (ja) * 1997-03-27 2001-11-06 シメグ リミテッド 臨床学的診断方法
WO2018062392A1 (fr) * 2016-09-28 2018-04-05 国立大学法人鳥取大学 Rat de modèle du syndrome de down et son procédé de production

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