JP2005168420A - Method for preparing recombinant animal by using fertilized egg derived from new nucleus-transplanted egg - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for activating a new nucleus-transplanted unfertilized egg for creating recombinant animals. <P>SOLUTION: A method for activating the new nucleus-transplanted unfertilized egg comprising starting maturation division of the nucleus-transplanted unfertilized egg by fertilizing an egg with a sperm in the activation method of nucleus-transplanted unfertilized egg. Also a nucleus-transplanted fertilized egg obtained by the activation method and a method for creating the recombinant animal comprising using the nucleus-transplanted fertilized egg. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an efficient method for obtaining a transgenic animal. More specifically, a method for activating a nuclear-implanted unfertilized egg characterized by initiating mature division of a nuclear-implanted unfertilized egg by fertilization of a nuclear-implanted unfertilized egg, and a nuclear-transplanted fertilization obtained by the activation method The present invention relates to a method for producing a genetically modified animal characterized by using an egg, and further, the nuclear transfer fertilized egg.

  In recent years, transgenic animals have been applied not only to biological research but also to production of useful proteins such as pharmaceuticals and regenerative medicine. Production of transgenic animals was reported in Gordon et al. In 1980 in mice, and subsequently in other animals in succession (see, for example, Non-Patent Document 1). The method of Gordon et al. Is a method of injecting foreign DNA directly into the pronucleus of a fertilized egg, and is most commonly used as a method for producing a transgenic animal. However, with this method, it is difficult to perform advanced gene recombination such as gene homologous recombination. In mice, embryonic stem cells that can be cultured for gene homologous recombination and have the ability to differentiate into individuals have been established (for example, see Non-patent Document 2). Cells have not been established.

Against this background, it has recently been reported that cloned animals can be obtained by nuclear transfer using somatic cells (see, for example, Non-Patent Document 3). This technology is called somatic cell nuclear transfer, and genetically modified operations can be performed on somatic cells that can be cultured in the same way as embryonic stem cells, so it is possible to produce transgenic animals that have undergone advanced operations such as gene homologous recombination. (For example, refer nonpatent literature 4). However, this technology has not been well established, and its techniques and efficiency have not reached the level of technology for producing transgenic animals using ES cells in mice. Improvement is eagerly desired.
Gordon, JW, Scangos, GA, Plotkin, DJ, Barbosa, JA & Ruddle, FH: Genetic transformation of mouse embryos by microinjection of purified DNA.Proceedings of the National Academy of Science of USA, 77: 7380-7384, 1980 Thompson, S., Clarke, AR, Pow, AM, Hooper, ML & Melton, DW: Germ line transmission and expression of a corrected HPRT gene produced by gene targeting in embryonic stem cells.Cell, 56: 313-321, 1989 Wilmut, I., Schnieke, AE, McWhir, J., Kind, AJ & Campbell, KH: Viable offspring derived from fetal and adult mammalian cells.Nature, 385: 810-813, 1997 Polejaeva, IA & Campbell, KH: New advances in somatic cell nuclear transfer: application in transgenesis. Theriogenology, 53: 117-126, 2000

  The embryo-derived cell nuclear transfer and somatic cell nuclear transfer techniques are roughly divided into a process of replacing the nucleus of an unfertilized egg with the nucleus of a target cell (this process is called “initialization”), and a nuclear transfer egg (or nuclear transfer) produced. It consists of a step (embryogenesis) of an embryo) for the formation of an individual (this step is called “activation”). In general, embryo-derived cell nuclear transfer and somatic cell nuclear transfer are strictly distinguished by the type of cell used for transfer, but in the present invention, the cell type is not particularly concerned, and embryonic cells and germline cells are not particularly concerned. In the following, embryo-derived cell nuclear transfer and somatic cell nuclear transfer are collectively referred to as “nuclear transfer”.

  In the first half of the nuclear transplantation technique, it is important to return the transplanted nucleus to an undifferentiated state so that it can be ontogenized. The factor that returns the transplanted nucleus to an undifferentiated state is called “reprogramming factor” and is known to be present in the cytoplasm of the unfertilized egg, but its body has not yet been identified.

  “Activation”, which is the latter half of the nuclear transfer technology, is a process that gives a stimulus to initiate the maturation of an egg (embryo) reconstructed by nuclear transfer, and is generally a technique that artificially induces parthenogenesis It is done in Here too, the mechanism of the initiation of maturation division has many unexplained parts, and artificial parthenogenesis is performed based on known knowledge.

  As described above, the nuclear transfer technology consists of “initialization” of the transplanted nucleus and “activation” of the reconstructed egg. However, as described above, this technology has not been established yet. This is the reason why sufficient production efficiency cannot be obtained.

  Furthermore, one of the problems of this method is that the method, conditions, and production efficiency differ depending on the animal species, strain, and cells used for nuclear transfer.

  The present invention relates to a method for improving nuclear transfer technology, and more particularly to a method for improving the latter step of “activation”.

  We have developed a system for fertilizing sperm in the same way as unfertilized eggs using reconstructed eggs produced by nuclear transfer technology, and creating new genetically modified animals that have undergone advanced operations such as gene homologous recombination. Led to a complete system.

  The present invention is provided as a production method when a high-level genetic recombination such as gene homologous recombination is required when a useful substance such as a pharmaceutical product is produced from a genetically modified animal. Moreover, it is useful not only for substance production but also for producing transgenic animals that cannot be obtained by the conventional pronuclear injection method.

  The present invention comprises the steps of enucleation of a second meiotic metaphase unfertilized egg, transplantation of a nucleus derived from a genetically modified cell, fertilization to a nuclear transplanted egg, and an embryo development operation of a fertilized egg derived from a nuclear transplanted egg.

  The present invention relates to production of a transgenic animal using a system for fertilizing a sperm into a reconstructed egg produced using a nuclear transfer technique. The general nuclear transfer technique aims at obtaining a cloned animal of the transplanted cells, but in the present invention, the purpose is to obtain an animal having genetic characteristics obtained by genetic recombination. There is no particular problem with sex. In other words, the present invention is greatly different from conventional clone production in that contamination and lack of genetic traits due to fertilization are not a problem.

  In a general nuclear transfer technique, the reconstructed egg is activated by an artificial parthenogenetic method. The current fertilization mechanism is understood as follows. The ovulatory unfertilized egg is stopped in the second meiotic metaphase state until fertilization is performed by the action of the metaphase promoting factor (MPF) and the cell division inhibitory factor (CSF) contained in the egg cytoplasm. The same applies to enucleated eggs and nuclear transplanted eggs. Maturation division is initiated by the repeated release of calcium ions from the endoplasmic reticulum in the egg cytoplasm by repeated fertilization, resulting in degeneration of MPF and destruction of CSF. In other words, artificial parthenogenesis methods are carried out by physically and chemically imitating these phenomena, and drug treatment methods (drugs such as alcohol, ionomycin, inositol phosphate, DMAP), Methods using electrical stimulation are widely used.

  The present inventors have developed a method for activating a nuclear transfer egg by fertilizing a sperm, not by a parthenogenetic method commonly practiced as an activation method. If the activation efficiency is further improved, it is possible to combine a parthenogenetic method with sperm fertilization.

  The transplanted cells of the nuclear transfer egg used in this method may be any, but the nuclear phase is limited to 2n or 1n cells. In the present invention, for example, M-phase cells having a nuclear phase of 4n cannot be used as transplanted cells for the production of nuclear transplanted eggs. The method for producing the nuclear transfer egg may be a generally used method.

  The timing of activation of the nuclear transplanted egg by fertilizing sperm may be any time after the nuclear transfer, but it is preferable that at least the transplanted nucleus aggregates as a chromosome, strictly speaking, for 8 hours after the nuclear transfer. Is preferred.

  Fertilization methods include transplanting a nuclear transfer embryo into a recipient animal and fertilizing in the body (including fertilization by mating, artificial insemination by vaginal or oviduct, etc.), in vitro fertilization, microinsemination The method according to any one of the above is carried out. In order to adjust the timing of fertilization, ie, activation, in vitro fertilization and microinsemination are preferred, and in order to prevent multiple fertilization, microinsemination is preferred.

  Sperm used for artificial insemination may be either fresh sperm, frozen sperm or lyophilized sperm. It can also be performed with spermatogonia and spermatocytes. However, in the present invention, the sperm (or comparable cell) can only be a cell having a nuclear phase of 1n, preferably fresh or frozen sperm and mature sperm. The method for collecting sperm is not particularly limited as long as it is carried out by an appropriate method for each animal species.

  The first point of the present invention is to perform maturation division closer to nature by using sperm as described above. The second point of the present invention is to adjust the nuclear phase. In other words, while the conventional nuclear transfer technique generally maintains the number of chromosomes at the time of nuclear transfer, this technique creates a nuclear transfer egg having a nuclear phase of 2n, and fertilizes the nuclear phase of the chromosome derived from the transplanted cell by 1n. It is clarified that this is an important point and can be implemented only in the present invention. That is, a nuclear transplanted egg releases the second polar body by fertilization in the same manner as a normal ovum, so that the nuclear phase of the egg becomes 1n, forming a pronucleus (female pronucleus), and a pronucleus derived from sperm (male male) To form a fusion nucleus of nuclear phase 2n and initiate maturation (see FIG. 1).

  In the present invention, cells used for nuclear transfer may basically be either male or female, but are preferably female. The use of female-derived cells is expected to be able to avoid the influence of “imprinting of genes”, which is considered to be a cause of abnormalities in the oncogenesis of nuclear-transplanted eggs and developmental cessation. “Gene imprinting” is a phenomenon in which the expression of genes derived from males and females is biased, and can be cited as one factor that prevents parthenogenesis of mammals from occurring to individuals. In the present invention, the male and female genes are aligned by using the nuclear transplanted ovum derived from female cells. Therefore, the use of the present invention makes it possible to avoid a failure caused by “gene imprinting”.

  In the present invention, when a cell used for nuclear transfer is a recombinant cell, a transgenic rabbit individual can be obtained by embryo transfer of the obtained nuclear transfer fertilized egg to a surrogate pregnant rabbit.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail according to an Example, these Examples do not limit the scope of the present invention.

Example 1 Preparation of fertilized eggs derived from nuclear transplanted eggs >>
A Japanese white rabbit was used as the animal species for producing the nuclear transfer embryo. Nuclear transfer embryos were prepared according to the method of Matsuda et al. (Cloning Stem Cells, 4: 9-19, 2002).
(1) Egg collection of unfertilized eggs Unfertilized eggs were administered to Japanese white female rabbits at a dose of 0.5 units / unit subcutaneously from 6 days before egg collection 6 times at 12-hour intervals, and hCG was administered at 100 units / day in the evening before egg collection. One animal was superovulated by intravenous administration, and eggs were collected 15-16 hours after hCG administration. After egg collection, cumulus cells were removed from the egg by hyaluronidase treatment and pipetting. The eggs were kept in M2 solution (Sigma) during the nuclear transfer operation.
(2) Enucleation The unfertilized egg collected in (1) above is exposed to M2 solution containing cytochalasin D at a final concentration of 10 μg / ml for 15 minutes, then placed in a micromanipulator, and the cytoplasm immediately below the polar body is removed with a pipette. Removed by suction. After the operation, the aspirated cytoplasm was stained with acridine orange (5 μg / ml) to confirm enucleation.
(3) Injection of genetically modified cells into enucleated unfertilized ovum follicles As the genetically modified cells, rabbit embryo-derived fibroblasts into which a GFP (green fluorescent protein) gene was introduced were used. The recombinant cells were cultured for several generations, and then the cell cycle was synchronized with the G1 / G0 phase by serum starvation (D-MEM containing 0.5% fetal bovine serum) or by long-term culture in a confluent state. Cells were dispersed and collected with 0.1% trypsin solution, and donor cells were injected into the surrounding space of the enucleated unfertilized egg of (2) above under a micromanipulator.
(4) Fusion Fusion of an enucleated unfertilized egg and a recombinant cell is performed at 300 V (3 kV / cm) using a chamber of 1 mm interval in 0.3 M mannitol, 0.05 mM calcium chloride, 0.01 mM magnesium chloride (FM solution). ), By applying a pulse of 100 μs once (LF101, BEX).
(5) Collection of sperm Sperm was collected using an artificial vagina. After removing the glue, dilute 2 to 5 times with physiological saline, centrifuge at 800 rpm for 5 minutes at room temperature, and discard the supernatant twice to remove seminal plasma and resuspend in physiological saline. (Concentration of about 5 × 10 ⁶ cells / ml) and used for in vitro fertilization.
(6) In vitro fertilization In vitro fertilization was performed by the intracytoplasmic sperm injection (ICSI) method using a piezoelectric manipulator (PMAS-CT150, Prime Tech). Sperm suspended in 12% PVP (Polyvinylpyrrolidone, Irvine Scientific) was sucked with a pipette having an inner diameter of about 20 μm and injected into the cytoplasm of the ovum.
In addition, the timing of the spermatozoon was performed between 30 minutes and 3 hours after the fusion.
(7) Culture Oocytes subjected to in vitro fertilization were cultured in CELGROSSER-H (Sumitomo Pharmaceutical Co., Ltd.) supplemented with 10% KNOCKOUT Serum Replacement (Gibco BRL) in a 38.5 degree, 5% carbon dioxide incubator.
As described above, a fertilized egg derived from a nuclear transfer egg was prepared.

<< Test Example 1 Preparation of in vitro fertilized eggs >>
In vitro fertilized eggs were prepared according to the methods described in Example 1 (1) “Collecting unfertilized eggs”, (5) “Collecting sperm”, (6) “In vitro fertilization”, and (7) “Culture”. .

<< Example 2: Early development of fertilized eggs derived from in vitro fertilized eggs and nuclear transplanted eggs >>
About 92% of rabbit in vitro fertilized eggs formed a pronucleus, and a further 92% mature division was observed in the 2-cell stage. In addition, about 83% of fertilized eggs derived from nuclear transplanted eggs prepared by fertilizing a GFP gene-introduced rabbit fetal fibroblast into an enucleated unfertilized ovum, about 83% form a pronucleus and further enter the 2-cell stage. % Maturation was observed (see Table 1).

  Furthermore, in the fertilized egg derived from the nuclear transplanted egg of the present invention, the release of the second polar body and the formation of male and female pronuclei were observed about 4 hours after insemination (see FIG. 2-a). Further, about 1 day after insemination, mature division from the 2 to 4 cell stage was observed (see FIG. 2-b), and GFP fluorescence due to the transplanted cells of the nuclear transplanted egg was observed (FIG. 2-c). reference).

  In this way, as a result of diligent efforts, we have demonstrated for the first time that sperm can be fertilized into a nuclear-transplanted egg, and have completed the invention by establishing that method. This is a method for producing a genetically modified animal derived from a new nuclear transfer cell, which is not found in the conventional method, and is expected to contribute to the advancement of the technology for producing a transgenic animal.

Schematic of a method for producing a transgenic animal using a fertilized egg derived from a nuclear transplanted egg. A drawing-substituting photograph showing the initial development of a fertilized egg derived from a nuclear transfer embryo. a: Egg at 4 hours after insemination (arrow: second polar body, *: male and female pronucleus, **: male and female pronucleus immediately before fusion), b: 2-cell stage (about 1 day after insemination), c: 4 cell stage (about 1 day after insemination, left figure: bright field observation, right figure: GFP fluorescence observation)

Claims (8)

A method for activating a nuclear-implanted non-fertilized egg, comprising activating maturation of a nuclear-implanted non-fertilized egg by fertilization in a method for activating a nuclear-implanted un-fertilized egg. The nuclear transfer unfertilized egg is obtained by transferring a nucleus derived from a cell subjected to a desired genetic recombination operation after enucleation from the unfertilized egg to the enucleated site. The activation method as described. 3. The activation method according to claim 1 or 2, wherein fertilization is performed by using any one of sperm, spermatogonia or spermatocytes. Fertilization is performed by using a sperm, The activation method in any one of Claim 1 to 3 characterized by the above-mentioned. The activation method according to any one of claims 1 to 4, wherein fertilization is performed by combining artificial parthenogenesis with any one of sperm, spermatogonia, or spermatocytes. The activation method according to claim 5, wherein the artificial parthenogenesis is performed by chemical treatment or electrical stimulation. A nuclear transfer fertilized egg which is activated by the activation method according to any one of claims 1 to 6. A method for producing a transgenic animal, comprising using the nuclear transfer fertilized egg according to claim 7.

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