CN1424870A - Nuclear transfer with selected donor cells - Google Patents

Abstract

The present invention provides a method of nuclear transfer by selecting and segregating G1 cells from a donor cell population. This method is advantageous over the prior art as it provides certainty as to the stage of the cell cycle which the donor nuclei are in and allows for the production of cloned transgenic or non-transgenic embryonic cells, reconstituted embryos and whole animals for agricultural, pharmaceutical, nutraceutical and biomedical applications.

Description

Nuclear transfer with selected donor cells

The present invention relates to a novel method of nuclear transfer, specifically, but in no way exclusive, for use in cloning techniques for the production of mammalian embryos, fetuses and offspring, including genetically engineered or transgenic mammalian embryos, fetuses and offspring.

Background of the invention

The timing of the cell cycle phase of the donor nucleus and recipient cytoplasm at the time of embryo reconstitution is a key determinant of successful development after nuclear transfer. A specific combination of the two "cells" is necessary to ensure a diploid set of chromatin after the first embryogenic cell cycle and to maximize the chances of nuclear reprogramming and subsequent development. When the donor nucleus during cell division fuses with the enucleated metaphase-arrested oocyte (called the cytoplasm or recipient cell), nuclear envelope disintegration (NEBD) occurs immediately and the donor chromatin undergoes advanced chromosome condensation (PCC) (Barnes et al., 1993). These effects are induced by a cytoplasmic activity called maturation-promoting factor (MPF, also called meiotic or mitogenic factor, reference, review, Campell et al., 1996a). In oocyte maturation, the activity of MPF was highest at metaphase and decreased rapidly after fertilization or artificial activation. Therefore, two types of cytoplasm can be used for reconstitution; use of non-activated or activated metaphase II (M II) cytoplasm corresponds to a cytoplasm with sequentially higher or lower MPF content.

Early work that contributed to the understanding of the importance of cell cycle coordination for maintenance of chromosomal integrity and consequent developmental potential in mammalian nuclear transfer used preimplantation embryos from species such as rabbit, sheep, and cattle. Undifferentiated blastomeres (ie, unspecialized embryonic cells). These studies reveal that the cell cycle phase of the donor nuclei and the length of exposure to cytoplasmic MPF have a significant effect on the degree of PCC observed. S-phase nuclei exposed to MPF had a typical comminuted appearance of chromatin, and chromosome studies indicated a high incidence of malformations. (Collas et al., 1992b). In contrast, the chromatin of nuclei in Gl or G2 phase condenses to form elongated chromosomes with sequentially single- or double-stranded chromatids (Collas et al., 1992b). Following appropriate stimuli that liberate the reconstructed embryo from metaphase arrest and activate development, the nuclear envelope reorganizes around the donor chromatin, which subsequently undergoes DNA synthesis regardless of its previous cell cycle stage. Thus, donor nuclei in G1 initiate DNA synthesis compatible with normal development, while nuclei in G2 or S phase fully or partially re-replicate the DNA that has already been replicated, so that, to the first embryogenic At the end of the cell cycle, the DNA content of the two daughter cells is incorrect, leading to abnormal early embryonic development.

Instead, these earlier studies showed that after a sufficiently long interval after cytoplasmic activation, the blastomere nuclei transplanted into the cytoplasm after MPF disappearance, NEBD (and thus PCC) did not occur and the donor nuclei, based on their presence in The phase of the cell cycle at the time of transplantation controls DNA replication. Therefore, the nucleus in G1 phase or S phase initiates or continues to replicate, respectively, while the nucleus in G2 phase cannot induce or enter another DNA synthesis. This pre-activated cytoplasm has been named the "universal receptor" (Compbell et al., 1994), which has the ability to coordinate the development of the donor cell at any stage of the cell cycle. This is particularly important for cloning preimplantation embryos, where the majority of undifferentiated blastomere nuclei are in S phase at any one time (80-90%, Barnes et al., 1993; Campbell et al., 1994), so it is most Suitable for transplantation into cytoplasm with low MPF content.

Following nuclear transfer, normal development depends on factors in the oocyte cytoplasm (or exogenously induced cofactors) that remodel chromatin structure and appropriately reprogram the expression patterns of genes in the donor nucleus. The mature cytoplasm contains RNA transcripts and proteins that guide the development of a normally fertilized cleavage-stage embryo to the time of normal genome activation, when the embryonic nucleus begins to synthesize its own RNA and direct embryonic development. Therefore, donor nuclei from embryos or cell types that have passed the normal activation time point of the genome must cease their RNA synthesis after reconstitution and remain inactive until the transition to the newly reprogrammed maternal embryo genome occurs. After transplantation, the donor nuclei are forced to reprogram the zygotic state and subsequently activate the appropriate genes at the correct level, in the appropriate spatio-temporal manner, required for normal embryogenesis to occur. The mechanism by which this nuclear reprogramming is achieved is not well understood.

Given the recent interest in nuclear transfer using culture-preserved cells, reprogramming and cell cycle coordination have become important topics. The cultured cells were more differentiated (ie, had more specialized cellular functions) than cells cloned with embryonic blastomeres for earlier studies. These cells can be isolated from embryos, fetuses or adult animals. Since larger numbers of cells can be obtained, high-efficiency nuclear transfer techniques using differentiated cell cultures may achieve large-scale proliferation of desired genotypes in livestock and will accelerate the production of transgenic animals following genetic manipulation of cultured cells.

Early studies using actively growing, unsynchronized sheep embryonic cell (cell cycle unclear) cultures of early (Campbell et al., 1995) but not later passage cells (Campbell et al., 1996b) fusion with preactivating cytoplasm actually produced The farrowing lamb. 5 days after deprivation of serum, cells in a quiescent state (i.e., cells so treated are considered to have exited the normal cell division cycle and entered the so-called G0 state) fused with cytoplasmic cells activated before, after, or at the same time with a similar overall efficiency Subsequent studies produced live lambs (Campbell et al., 1996b). These authors (Campbell et al., 1996b; Schnieke et al., 1997; Wilmut et al., 1997; patent WO 97/07669) proposed the use of cells exiting the normal cell division cycle and synchronized to a quiescent or G0 state to facilitate nuclear reprogramming and differentiation from The importance of cells to generate cloned animals. The preferred method for synchronization of cell quiescence proposed by the above authors (based on the absence of proliferating cell nuclear antigen (PCNA), a condition that indicates no cells in S phase) is serum starvation at the appropriate time. Recently, however, questions have been raised about the proportion of cells actually in the G0 state among serum-starved cells. Using a dual-parameter flow cytometer to simultaneously measure cellular DNA and protein content (in order to differentiate between G0 and G1 phase cells in a diploid population, quiescent cells contain less RNA and protein), Boquest et al. (1999) studied cultured Cell cycle characteristics of porcine fetal fibroblasts. They demonstrated that despite 5 days of serum starvation, less than 50% of the cells were actually in the G0 state by their definition. The share of GO cells in serum starved cultures was increased to 72% by selection of "small" cells in the cell population (Boquest et al., 1999).

As an alternative to the use of quiescent cells, Cibelli and co-workers (1998; and patent specification WO 99/01163) reported the use of non-serum-starved free-growing bovine cell cultures with subsequent use of ionomycin and 6-dimethylamino Purine (6-DMAP)-activated metaphase II (M II) cytoplasmic fusion. However, these announcements do not address the cell cycle of the donor cells that ultimately yield cloned calf embryos using these methods at the time of nuclear transfer.

Likewise, other reports that cloned calves (Vignon et al., 1998, 1999; Zakhartchenko et al., 1999) and mice (Wakayama and Yangimachi, 1999) have been generated from non-serum-starved free-growing cell cultures. However, as before, the cell cycle stage of the donor cells that give rise to the cloned progeny at the time of nuclear transfer cannot be determined.

Therefore, none of the above-mentioned studies established exactly which phase or phases of the cell cycle resulted in such a low proportion of reconstructed embryos ultimately developing viable offspring.

The above discussion emphasizes that, to date, the cell cycle in cultured donor cells at the time of nuclear transfer has generally been described poorly, making the state of the art unstable and not easily reproducible.

Therefore, it is desirable to have a nuclear transfer method that accurately knows the cell cycle phase of the donor cell nucleus.

It is an object of the present invention to move forward in order to fulfill this desire or at least to provide the public with a useful choice.

Summary of the invention

According to a first aspect, the present invention provides a method of nuclear transfer, the method comprising selecting and isolating cells in G1 phase from a population of proliferated or non-proliferating donor cells and transplanting a cell nucleus from the cells in G1 phase thus separated into an enucleated recipient cell. The donor cell population can be in one or more known or unknown phases of the cell cycle.

This method provides certainty about the stage of the cell cycle in which the donor nuclei are at the time of embryo reconstruction and is therefore superior to existing techniques.

The present invention contemplates the use of cell cycle inhibitors to arrest freely growing cells at specific phases of the cell cycle in order to generate non-proliferating synchronized cell populations. Preferably, cell growth is arrested in mitosis, and once the inhibitor is removed, cells entering G1 phase are used for nuclear transfer.

Therefore, in a second aspect, the present invention provides a method of nuclear transfer comprising transferring a nucleus of a cell isolated from a population of non-proliferating cells synchronized to the G1 phase of the cell cycle to an enucleated recipient in cells.

Preferably, said cells in G1 phase are isolated singly from a population of freely proliferating cells or from a population of non-proliferating synchronized cells in early G1.

Alternatively, the non-proliferating cell population can include senescent cells.

Isolated G1 donor cells are isolated from an animal, or, more preferably, from in vitro cell culture. Suitable cells can be derived from embryos, fetuses, juvenile animals, up to fully grown adult animals. In fact, any karyotypically normal diploid cell or senescent cell capable of cell proliferation can be used in the present invention. Cells can be in an undifferentiated state or in varying degrees of cell differentiation, so long as they can be stimulated to enter the cell cycle and proliferate. Cells in quiescent phase can be stimulated to enter the cell cycle with appropriate culture conditions (eg addition of serum or specific growth factors) and used for nuclear transfer in early post-mitotic G1. Some cell types may prove more effective than others, but adult and fetal fibroblasts and adult follicle cells have been found to be satisfactory. To illustrate the invention, the results of experiments using two follicular cell lines, four skin fibroblast cell lines and two genetically modified fetal fibroblast cell lines in cattle (Examples 1-7) are presented below.

Preferably, the recipient cell comprises an enucleated oocyte from the same species as the nucleus of the donor cell. Enucleated oocytes can have higher or lower MPF activity upon embryo reconstitution because both states are compatible with G1-phase donor nuclei with 2C-content DNA.

Alternatively, the recipient cells may comprise an enucleated stem cell or a fused mass of enucleated stem cells. Preferably, these stem cells are embryonic stem cells. Embryonic stem cells used as recipient cells in nuclear transfer can themselves be isolated from growing embryos or from established cultured stem cell lines. In this case, nuclear transfer of donor nuclei from cells in G1 phase selected by the method of the present invention can be used for the purpose of "therapeutic cloning".

According to a third aspect, the present invention provides a method for producing cloned animal embryos by transplanting isolated G1 donor cell nuclei, preferably early G1 , into enucleated recipient cells.

The methods of the present invention can be used to produce embryos of any animal, relevant species including birds, amphibians, fish and mammals. Preferably, the relevant animal embryo is a mammalian embryo including, but not limited to, primates including humans, rodents, rabbits, cats, dogs, horses, pigs and most preferably ungulates such as cattle, sheep, deer and goats.

Cloned animal embryos obtained preferably using genetically modified nuclei by methods known in the art have desired genetic traits.

According to a fourth aspect, the present invention provides a recombinant animal embryo prepared by the method of the present invention, including a recombinant transgenic animal embryo. Embryos so formed can then be cloned again to further increase the number of embryos or undergo serial nuclear transfer to further aid nuclear reprogramming and/or developmental potential.

According to a fifth aspect, the present invention provides a method for cloning a non-human animal, the method comprising (1) producing a cloned animal embryo according to the method provided by the above-mentioned invention; (2) using a known method to make the animal from Embryo development to parturition; and (3) random breeding of animals thus formed by conventional methods or cloned according to the method of the present invention.

The methods of the invention can be used to produce non-human animals, and relevant species include birds, amphibians, fish and mammals. Preferably, said non-human animal is selected from the group consisting of non-human primates, rodents, rabbits, cats, dogs, horses, pigs and most preferably ungulates such as cattle, sheep, deer and dogs.

According to a sixth aspect, the present invention provides cloned non-human animals produced by the method of the invention described above, and the progeny and descendants of such cloned non-human animals.

The methods of the invention can be used to produce non-human animals, preferably mammals, having a desired genetic trait obtained by genetically modifying the donor nucleus using methods well known in the art. Transgenic animals produced in this way also form part of the invention.

The invention can also be used to produce embryonic cell lines, embryonic stem cell lines, fetuses or offspring suitable for, eg, cell, tissue and organ transplantation.

Accordingly, in another embodiment, the present invention provides a method of producing an embryonic cell line comprising the steps of: a) proliferating from a cell cycle-agnostic donor cell population or from a synchronized G1 phase cell population selecting and isolating cells in the G1 phase and transplanting the nuclei of the isolated cells into enucleated recipient cells; b) culturing to the blastocyst stage; c) recovering embryonic cells; and d) establishing immortalized cells in vitro using methods known in the art Tie.

In another embodiment, there is provided a method of producing animal embryonic stem cells comprising the steps of: a) selecting from a growing population of animal donor cells at different stages of the cell cycle or from synchronized G1 phase cultures Isolating G1 phase cells and transplanting the nuclei of the isolated cells into enucleated animal recipient cells; b) culturing to blastocyst stage; c) recovering embryonic stem cells.

Preferably, the animal donor cells used in the above methods are of human origin. Most preferably, both the animal donor and recipient cells used in the above methods are of human origin.

Most preferably, the donor cell is an adult or fetal cell selected from any karyotypically normal cell type, and the recipient cell is selected from any cell type capable of reprogramming gene expression, including enucleated oocytes or embryonic stem cells.

The embryonic stem cells produced by the method of the present invention are pluripotent cells, which can be induced to differentiate in culture by methods known in the art to form purified populations of specialized types of human cells, including nerve cells such as neurons, astrocytes and Oligodendrocytes; Hepatocytes; Muscle cells such as myogenic cells; Cardiac cells such as cardiomyocytes; Hematopoietic cells; Pancreatic cells and other cells of interest.

These specialized human cells and tissues can then be used for transplantation therapy in specific diseases or injuries where damaged cells cannot self-renew or renew effectively. Where the human donor cells come from the patient in need of such a transplant, since the transplanted tissue is genetically identical to the patient, the transplanted tissue will not be rejected by the patient.

Alternatively, these differentiated cells and tissues can be used to treat diseases or injuries, for example, various neurological disorders (such as Parkinson's disease), diabetes, heart disease, muscle wasting, various genetic diseases, certain cancers ( such as leukemia), spinal cord injuries, burns and other afflictions.

These methods are known in the art as "therapeutic cloning".

In vitro differentiation of human embryonic stem cells into specific cell types is also useful for drug screening and human drug toxicology studies.

Therefore, in another aspect, the present invention provides a method for using the method of the present invention to produce in vitro differentiated human embryonic stem cells for drug screening and drug toxicology experiments.

According to a further aspect, the present invention provides methods of xenotransplantation wherein cells, tissues and organs can be isolated from non-human cloned animals produced according to the methods of the present invention and their progeny for use in human diseased patients in need of such treatment being transplanted. If such cells, tissues or organs include transgenes, such cells, tissues or organs can be used for gene therapy, or to modulate a patient's immune response to tissues containing foreign genes.

Description of drawings

The invention will now be described with reference to the accompanying drawings:

Figure 1 shows the survival rate of cloned bovine embryos reconstituted with follicular cells in G0 or G1 phase of the donor cell cycle during whole pregnancy;

Figure 2 shows the survival rate during whole gestation of cloned bovine embryos reconstituted with female adult skin fibroblasts in G0 or G1 phase of the donor cell cycle;

Figure 3 shows the survival rate during whole gestation of cloned bovine embryos reconstituted with female adult skin fibroblasts (3XTC cells) in G0 or G1 phase of the donor cell cycle;

Figure 4 shows the embryo survival rate during the whole gestation of cloned bovine embryos reconstituted with male adult skin fibroblasts (LJ801 cells) in the G0 or G1 phase of the donor cell cycle;

Figure 5 shows embryo survival throughout gestation of cloned transgenic bovine embryos reconstituted with genetically modified female fetal lung fibroblasts (casein+5110 cells) in the G0 or G1 phase of the donor cell cycle; and

Figure 6 shows the embryo survival rate during whole gestation of cloned transgenic bovine embryos reconstituted with non-proliferating senescent female fetal fibroblasts (561 cell line);

Detailed description of the invention

The present invention relates to the improvement of the prior art of cloning animal embryos by nuclear transfer. Although it is contemplated that the embryo cloning method of the present invention may be used in a variety of mammals and other animal species, the method will be described with reference to bovines. An essential feature of the invention is that the donor cell nucleus is in G1 phase, preferably, in early G1 phase.

One method of identifying cells in G1 phase from a freely growing population of cultured cells is to individually pick mitotic cells from the culture surface and place them in a medium containing 10% fetal calf serum (FCS) to complete mitosis. This donor cell then fuses with the recipient cell (ie cytoplasm) within a short time after mitosis and before entering S phase, typically three hours, detectable with 5-bromodeoxyuridine labeling. In this way, cells are ensured to be in early G1 and possess a 2C content of DNA. Therefore, circulating cells are available for nuclear transfer before they enter the G1/S junction. Selected cells are maintained in high serum content medium throughout the manipulation, at least until fusion with the cytoplasm is complete. Therefore, cells cannot be induced to exit the cell division cycle and cannot be quiescent for any desired moment.

Although the present invention contemplates the production of synchronized G1 phase cell populations for nuclear transfer, an advantage in the methods of the present invention is that it is not necessary to use agents that are potentially cytotoxic or interfere with cell synchronization, such as thicarbazol or colchicine, For example, presynchronizing a higher proportion of cells in M phase and later releasing from this arrest allows selection of early G1 cells after cell division. However, in order to select a large number of cells in G1 phase or to reversibly suppress G1 phase cells for nuclear transfer at a specific time point, according to the present invention, a suitable reagent, namely a cell cycle inhibitor, is used at a suitable drug concentration and incubation time. is beneficial. Such reagents and methods are known to those skilled in the art. These methods can include presynchronization to reduce drug exposure time, such as using serum starvation to temporarily induce cells into G0 phase before the second addition of serum, allowing cells to re-enter the G0/G1 junction or checkpoint and carry out the cell division cycle . Reagents suitable for reversibly inhibiting cells at different time points in G1 phase (reference, Gadbois et al., 1992) include: (1) staurosporine (a non-specific kinase inhibitor, very low concentrations); (2) more specific kinase inhibitors that act on cell cycle progression, such as cAMP-dependent protein kinase and cGMP-dependent protein kinase inhibitors; (3) lovastatin; (4) isobright (5) Aphidicolin or hydroxyurea, used to prevent cells from entering the S phase and arresting at the G1/S junction.

Since the G1 donor nucleus contains a 2C content of DNA (ie it is diploid), they can be reconstituted with cytoplasm with either high or low MPF content. That is, G1 nuclei can be brought into the cytoplasm before, after, or simultaneously with activation. However, for better development of cloned embryos, preferably, the donor nuclei (whether introduced after electrical induction of cell fusion or direct nuclei injection) are exposed to factors present in the cytoplasm of the enucleated oocyte for an appropriate period of time so that Promotes nuclear reprogramming. This is called "fusion before activation" or FBA. Previous research work has shown the benefits of this approach compared to essentially "simultaneous fusion and activation" or AFS (Stice et al., 1996; Wells et al., 1998; 1999). Furthermore, it is recommended to expose the cytoplasm for at least more than one hour, preferably between 3-6 hours, in order to increase the proportion of development to the blastocyst stage. However, in order to maintain the correct ploidy chromosomes in the final embryo, using this method, it is important to prevent the formation of micronuclei with appropriate methods, which occur when fusion precedes activation (Czolowska et al., 1984).

The following is an overview of methods for reconstituting and generating cloned embryos in bovine from cultured G0 or G1 donor cells. In the detailed examples below, the results of the use of bovine follicle cells collected from follicles are shown in Examples 1-3 (the use of fibroblast cell lines is illustrated in Examples 4-7). In practice, the technique can be used to demonstrate that essentially any cell type with a normal diploid karyotype is totipotent, including embryonic, fetal, juvenile and adult cells, whether they are proliferating or capable of being induced into the cell cycle or the senescence process . Also, other methods known in the art as recognized by those skilled in the art can be used to recombine and produce cloned embryos.

in vitro maturation of oocytes

The ovaries of the female cattle were collected in the slaughterhouse, placed in normal saline (30°C), and transported to the laboratory within 2 hours. Cumulus-oocyte complexes (COCs) were recovered by aspirating 3-10 mm of follicles with an 18-gauge needle and negative pressure. (Alternatively, immature oocytes can be collected from donor cows by egg extraction followed by in vitro maturation). COCs were placed in N-2 supplemented with 50 μg/ml heparin (Sigma, St. Louis, MO) and 0.4% w/v bovine serum albumin (BSA) (Immuno-Chemical Products (ICP), Auckland, New Zealand). -Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffered tissue culture medium 199 (H199; Life Technologies, Auckland, New Zealand). Before proceeding to in vitro maturation, only COCs with compact non-atresisting cumulus coronas and homogeneous oocysts were selected. These COCs were first washed twice with H199 medium + 10% fetal calf serum (FCS) (Life Technologies, New Zealand), and then washed once with bicarbonate-buffered tissue culture medium 199 + 10% FCS. Ten COCs were transferred to 10 microliters of this medium, added to 40 microliters of maturation medium placed in a 5 cm Petri dish (Falcon, Becton Dickinson Labware, Lincoln Park, NJ), and washed with paraffin oil (Squibb , Princeton, NJ) coverage. Maturation medium was supplemented with 10% FCS, 10 μg/ml ovine follicle stimulating hormone (FSH) (Ovagen; ICP), 1 μg/ml ovine luteinizing hormone (LH) (ICP), 1 μg/ml ovine estradiol (Sigma ), and tissue culture medium 199 at 0.1 mmol/L cystamine (Sigma). Microtiter dishes were incubated at 39°C for 18-20 hours in a humidified atmosphere containing 5% _CO2 . After maturation, COCs were placed in HEPES-buffered synthetic oviduct fluid (HSOF; Thompson et al., 1990) containing 0.1% hyaluronidase (from bovine testes; Sigma) and vortexed for 3 minutes, then washed three times with HSOF+10% FCS, Completely remove the corona cumulus.

nuclear transfer of cultured cells

a) Media Matured oocytes, cytoplasm and reconstructed embryos were maintained or manipulated in H199-based media for the period of time after maturation until the end of fusion assessment 15-30 minutes after pulse shock. Embryos reconstituted by fusing donor cells and M II cytoplasm (FBA treatment) 3–6 hours prior to activation were grown in AgResearch Synthetic Oviduct Liquid Medium (AgR SOF; this medium was developed by Gardner et al. , 1994, a modified formulation of the formulation described by AgResearch, Hamilton, New Zealand, which has been commercialized) until the eve of activation, ie after 3-6 hours of incubation. Calcium was present in all media formulations used thereafter.

b) Enucleation The nuclei of oogonia matured for approximately 18-20 hours were removed using a 15-20 micron (outer diameter) glass pipette by aspirating the first polar bodies and metaphase plates in the surrounding cytoplasm. Oogonia were first stained with 10% FCS, 5 μg/ml Hoechst 33342 and 7.5 μg/ml cytochalasin B (Sigma) for 5-10 minutes and then manipulated in this medium without Hoechst 33342. Enucleation was confirmed by visualization of the nuclei under UV light. After enucleation, the resulting cytoplasm was washed roughly in H199 + 10% FCS and then kept in this medium until injected into donor cells.

c) Preparation of quiescent donor cells (ie cells in G0 phase) Cultured follicle cells were induced into quiescence by serum deprivation (Campell et al., 1996b). On one day of routine passaging, the medium was aspirated, the cells were washed three times with fresh phosphate-buffered saline (PBS), and fresh medium containing only 0.5% FCS was added. The follicle cells are further cultured in low serum medium for 9-23 days (usually 10 days), and then used for nuclear transfer. Just prior to injection, single-cell suspensions of donor cells were prepared by standard trypsinization. Cells were pelleted, resuspended in H199+0.5% FCS, and kept in this medium until injection.

d) Preparation of Donor Cells in G1 Phase Proliferating follicle cells were cultured on glass coverslips in a petri dish with a suitable medium (eg DMEM/F12 supplemented with 10% FCS). Coverslips with cells grown on their surface are aseptically picked from a Petri dish and placed in a suitably configured micromanipulator chamber that allows collection and injection of cells or nuclei. A small drop of HEPES buffered medium containing 10% FCS was dropped on the cells and then covered with mineral oil. As long as the cell concentration on the coverslip is not too high, mitotic cells can be distinguished and carefully physically picked from the coverslip with a handling pipette, because in mitosis, cells become round and only loosely attached to the culture surface. If this is not easy, cells can be washed very quickly with PBS before using a diluted insulin solution containing 1.5 µg/mL cytochalasin B (e.g., one-tenth of the strength commonly used for transplanting cultured cell lines), mainly Reduces mechanical damage from physically picking cells from coverslips. Using an appropriate microscope (e.g., phase-contrast microscopy or DIC light microscopy), identify mitotic cells on coverslips primarily by seeing condensed chromosomes in the spindle or identifying condensed chromosomes in doublets of cells in telophase that are still connected by cytoplasmic bridges. Therefore, it is not necessary to use DNA-specific fluorescent dyes such as Hoechst and to expose cells to UV light. Mitotic cells are individually picked from the coverslip by means of an injection dropper fixed to the manipulator and placed into an adjacent droplet of HEPES buffered medium containing 10% FCS to complete mitosis and eventually cell division to form two cells. cells. The diameter of the pipette should be appropriate, depending on the cell line, so that the manipulation does not physically damage the cells or the spindle. Therefore, preferably, mitotic cells in anaphase or telophase are single-picked, removed and allowed to complete mitosis and divide in two. The paired cells resulting from these divisions can then be gently dissociated into single cells by a facile method of short-term exposure to a suitable enzyme solution. Each intact cell is then injected and fused into the cytoplasm. Alternatively, nuclei can be isolated and injected directly into the cytoplasm of the nucleated oocyte. Preferably, the transfer of donor cell nuclei to the cytoplasm is accomplished within three hours of first picking the mitotic cells from the culture surface. This ensures that cultured cells are confluent early in G1 of the cell cycle and before S phase occurs. For each cell type or cell line used here it should be confirmed using, for example, negative 5-bromodeoxyuridine labeling of selected cell samples. Also, it is advisable to confirm that cells picked using this method are actually in the cell cycle and enter S phase at some later point in time.

e) Microinjection of recipient cytoplasm dehydrated in H199 medium containing 10% FCS and 5% sucrose. This medium is also used as a micromanipulation medium. Piercing the zona pellucida with an appropriately sized pipette (eg, 30-35 micron outer diameter) to accommodate the donor cells, where the cells are wedged between the zona pellucida and the cytoplasmic membrane, will promote tight cell membrane contact that facilitates subsequent fusion. After injection, the reconstructed embryos were dehydrated again in two steps: first in H199 medium with 10% FCS and 2.5% sucrose for 5 min, then in H199+10% FCS until confluent.

f) Cell fusion Embryos were reconstituted using the FBA strategy (fusion before activation) for G0 and G1 cell treatments. Approximately 24 hours (hpm) after the start of the maturation process, the reconstituted embryos were treated with 0.3 mol/L mannitol, 0.5 mmol/L HEPES, and 0.05 fatty acid-free (FAF) containing 0.05 mmol/L calcium and 0.1 mmol/L magnesium. ) for electrofusion in a buffer composed of BSA. Fusion was performed at room temperature in a chamber with two stainless steel electrodes covered with fusion buffer 500 microns apart. Align the reconstructed embryo by hand with a slender mouth-controlled Pasteur pipette so that the contact surface of the cytoplasm and donor cells is parallel to the electrode. For follicle cells, cell fusion is performed with two shots by a BTX electrocytomanipulator 200 (BTX, San Diego, CA) (appropriate electrical parameters are required to differentiate each cell line), each 15 microsecond intensity 2.25-2.50 kV/ centimeter induced by pulsed direct current. After electrical stimulation, the reconstructed embryos were washed with H199+10% FCS. Fusion was then checked microscopically within 15-30 minutes.

Do not expect the electrofusion parameters described above to produce significant activation ratios for young cytoplasm used at 24hpm, because less than 1% of control oocytes (n=112) of the same age formed pronuclei after the same electrical stimulation (Wells et al., 1999). This is important for FBA processing in order for NEBD and PCC to occur, allowing donor chromosomes in G0 and G1 nuclei to be exposed to factors in the oocyte cytoplasm for chromatin remodeling and nuclear reprogramming.

Alternatively, nuclei from cells in G1 phase can be isolated and injected directly into the oocyte cytoplasm, as known to those skilled in the art.

g) Activation There are many ways to achieve manual activation. A unique approach involves ionomycin (Sigma) and 6-dimethylaminopurine (6-DMAP; Sigma) (Susko-Parrish et al., 1994). Following fusion, the embryo is preferably activated 3-6 hours after exposure of the donor nucleus to the oocyte cytoplasm. This preferred method has been termed "fusion before activation" (FBA; Wells et al., 1998). Thirty minutes before activation, fused embryos obtained by the FBA treatment method were washed with HSOF (with calcium) + 1 mg/ml FAF BSA and stored. Activation was induced by incubating 30 μl 5 μmol/L ionomycin (Sigma) dropwise in HSOF + 1 mg/ml FAF BSA at 37°C for 4 minutes. Activation usually occurs between cytoplasmic age 27-30hpm. Embryos were washed roughly for 5 min with HSOF + 30 mg/ml FAF BSA and then washed in AgR SOF (plus calcium) + 10% Incubate in FCS for 4 hours.

As described in the FBA method (Stice et al., 1996; Wells et al., 1998; 1999), the increased rate of embryonic development resulting from prolonged exposure of nuclei to oocyte cytoplasm must be related to appropriate prevention of micronucleus formation after delayed activation (Czolowska et al., 1984) a combination of processing methods occurred. Serine-threonine kinase inhibitors, such as 6-DMAP, appear to be suitable agents. Thus, after the initial activating stimulus, 6-DMAP allowed the formation of a single intact nucleus, thus maintaining the correct ploidy in the reconstructed embryo.

In vitro culture of nuclear transfer embryos

Embryo culture was performed in 20 microliters of AgR SOF (available from AgResearch, Hamilton, New Zealand) overlaid with mineral oil. AgR SOF is a modified formulation of SOFaaBSA (containing 8 mg/ml of FAF BSA; as described by Gardner et al., 1994). Whenever possible, up to 10 embryos were cultured together in droplets of medium. Embryos were incubated at 39°C in a humidified modular incubation chamber (ICN Biomedicals, Aurora, Ohio) in a gas mixture consisting of 5% CO2, 7% O2 and 88% N2. On days 4-5 of development (day 0 = day of embryo reconstitution), embryos were transferred to 20 μl of fresh AgR SOF supplemented with 10 μmol/l 2,4-dinitrophenol as described in published patent specification WO 00/38538, wherein 2,4-dinitrophenol acts as an uncoupler of oxidative phosphorylation to improve bovine embryo development in vitro, the specification of which is incorporated herein by reference. On day 7 after fusion, the developmental characteristics of transplantable blastocysts were evaluated.

Embryo transfer, pregnancy diagnosis and calving

Embryo transfer, pregnancy diagnosis and calving management are performed using techniques well known in the art.

Sequential nuclear transfer and recloning

In additional embodiments of the invention, it may be desirable to re-clon the first generation of cloned embryos derived from the reconstitution of G1 donor cells with suitable recipient cells. Recloning can be achieved by isolating preimplantation embryos into individual cells and fusing each of them with the appropriate recipient cytoplasm. As will be appreciated by those skilled in the art, this form of embryonic cell (or blastomere) cloning requires coordination of the cell cycles of the donor and recipient cells in order to avoid chromosomal abnormalities and to optimize development. Preferably, the donor cells are taken at the morula stage (approximately 32 cells in bovines) of the first generation cloned embryos, but the embryos can also be early or late in development. Alternatively, G1 donor cells from culture can be used to generate a cloned fetus first, the fetal cell line can then be derived again, and the new cell line used to re-clon the embryo. This recloning approach may facilitate nuclear reprogramming by prolonging the exposure of the original donor nuclei to the oocyte cytoplasm during the preimplantation period. The present invention also has advantages in multiplying the number of cloned embryos from original embryos available at the first generation to produce second, third, etc. cloned embryos.

Serial embryo transfer involves the serial transfer of nuclei to a suitable recipient cytoplasmic environment. As described in embodiments of the present invention, it is desirable to first reconstitute a one-cell embryo with donor cells in G1 phase and non-activated recipient cytoplasm with higher MPF. This will allow the NEBD and PCC phenomena to occur and when followed by activating stimuli, fully diploid nuclei will form. This nucleus (which has undergone nuclear remodeling and to some extent nuclear reprogramming) can then be aspirated from a single-cell cloned embryo as a nucleus and subsequently transplanted into an enucleated zygote at the appropriate time post-fertilization. Embryo development is then allowed to proceed. This sequence of sequential nuclear transfer processes improves nuclear reprogramming. This may also result in developmental improvements, since the second nuclear transfer step introduces the nucleus into a cytoplasmic environment where fertilization with sperm makes it more suitable for activation into embryonic development.

Production of transgenic animals

Transgenic livestock produced by nuclear transfer using donor nuclei genetically modified in the G1 phase of the cell cycle may be a more efficient method than injection of DNA into the pronucleus of fertilized eggs (Wall et al., 1997) or DNA sperm involving intracytoplasmic injection of spermatozoa and tethering of foreign genes. Mediated gene transfer (Perry et al., 1999) is a more efficient and versatile approach. Advantages of nuclear transfer with cultured cells include: (1) the scope of genetic manipulation may be broader; (2) the use of cell lines allows genetic manipulation to be more easily performed on a high genetic background relative to the collection of oogonia or fertilized eggs ; (3) all cloned offspring produced are transgenic and of the desired sex; and (4) the present method has the opportunity to produce a useful product in the short term compared to pronuclear injection methods or sperm-mediated gene transfer to produce a single original animal direct herds and herds.

Cultured cells can be genetically altered by inserting, removing or altering appropriate DNA. Induced mutations include random insertion of new DNA sequences (which may be heterologous), site-specific insertions of DNA, and homologous recombination that allow insertion, deletion, or alteration of DNA sequences at specific sites in the genome. From karyotypically normal transgenic cells, cells in the G1 phase of the cell cycle can then be selected for nuclear transfer to generate clones/transgenes after cell selection and DNA analysis confirm the appropriate genetic alterations using methods known in the art animal.

Depending on the particular gene being manipulated, there are a variety of factors applicable to genetic modification to alter livestock for biomedical and agricultural use. The range of factors includes; (1) production of medicinal proteins in milk, blood or urine; (2) production of nutritious products and medical foods such as milk; (4) production of proteins for industrial use, such as in milk; (5) xenotransplantation; (6) generation of livestock as models of human diseases, such as cystic fibrosis and Huntington's disease.

therapeutic cloning

A significant impact of nuclear transfer and embryonic stem cell technologies may be in the field of human cell-based therapeutics (Pederson, 1999). Patients with certain diseases or tissue damage that cannot be repaired or effectively renewed on its own (e.g., diabetes, muscle wasting, spinal cord injury, certain cancers, various neurological disorders, including Parkinson's disease) have the potential to generate their own therapeutic tissue for transplantation potential to provide long-acting or life-long therapy. First the method will use human cell nuclear transfer. This involves collecting a sample of healthy tissue from a diseased or injured patient and growing it to stimulate cell proliferation. Nuclei reprogramming is possible by selecting donor cells in the G1 phase of the cell cycle and fusing them with suitable recipient cells. If the recipient cells are enucleated human oocytes, then, following appropriate activation stimuli, the reconstructed embryos can grow to the blastocyst stage in suitable embryo culture media. Under appropriate culture conditions (Thomson et al., 1998), human embryonic stem cells can be derived from the inner cell population of such cloned embryos, which will be genetically identical to the patient who donated the cultured cells.

"Embryonic stem cells" literally means any pluripotent cell type isolated from an embryo, preferably from the inner cell population of the blastocyst stage by methods well known in the art.

Embryonic stem cells produced by the methods of the present invention will be undifferentiated, pluripotent (potential to differentiate into any cell type within the cell) and essentially capable of immortality in vitro. Cell lines isolated from internal cell populations may also be somewhat differentiated and have a limited ability to differentiate into multiple cell types, but may still be of therapeutic value. Based on experience with mouse embryonic stem cells, suitable conditions can be developed that can produce purified populations of specific differentiated cell types, such as nerve cells, blood cells, cardiomyocytes, etc., for the treatment of specific diseases (such as insulin-producing cells for diabetes or Dopamine-producing cells to treat Parkinson's disease). These genetically compatible cells can then be administered back to the patient to regenerate normal tissue in situ following transplantation. Because the cells are genetically identical to the patient, they will not be rejected and there is little or no need for immunosuppressive drugs. By systematically modifying loci, such as major histocompatibility complex genes that play an important role in foreign cell recognition by the immune system, it may be beneficial to generate "universal" embryonic stem cell lines for allogeneic transplantation.

Some applications may involve differentiation and genetic modification of embryonic stem cells prior to implantation. This may be because the goal of gene therapy is to deliver therapeutic drugs or to correct genetic defects in somatic cells such as the dystrophin gene in the skeletal muscle of patients with Duchenne muscular dystrophy.

In addition to transplantation therapy with cells, tissues or organs, differentiation of hESCs into specific cell types may also be beneficial for drug discovery and toxicology studies of human drugs.

Also, cells, tissues and organs can be isolated from the progeny of cloned non-human animals for use in transplantation in human patients in need of such treatment (exogenous transplantation). When such cells, tissues and organs contain a transgene, such cells, tissues and organs can be used for gene therapy or to modulate a patient's immune response to foreign tissue.

Recipient cells in human applications

In the method of nuclear transfer according to the present invention, the preferred recipient is an enucleated oocyte prepared by the disclosed method. However, for human applications, this is difficult.

An alternative source of recipient cells capable of reprogramming differentiated somatic nuclei are embryonic stem cells. Thus, somatic cells from patients in need of some form of cell therapy can be dedifferentiated in culture, rather than human oocytes. This can be achieved by fusing a healthy somatic cell in the G1 phase of the cell cycle with an enucleated ES cell or a pool of enucleated ES cells (in order to provide a large amount of cytoplasm for reprogramming). It is necessary to control the cell cycle state of recipient stem cells, preferably in M phase or G1 phase at the time of fusion. In this approach, differentiated nuclei of somatic cells can be dedifferentiated following exposure to stem cytoplasm. The reconstituted cells can have developmental potential for pluripotency, potentially inducing a panel of additional specialized cell types for therapeutic use. This notion was previously demonstrated in hybrid cells resulting from the fusion of thymic lymphocytes and embryonic germ cells (Tada et al., 1997).

The present invention is now illustrated in detail by the following examples, which are not intended to limit the scope of the present invention as known to those skilled in the art.

Example 1 Effect of Follicle Donor Cell Synchronization in G0 or G1 Phase on In Vitro Development after Nuclear Transfer

In this particular experiment, a primary cell line of follicular granulosa cells was used for nuclear transfer. The cell line, denoted J1, was derived from a New Zealand Jersey heifer. The J1 cells used in this experiment are the 7-8 generation cells in culture.

For comparison, donor cells are synchronized in two phases of the cell cycle:

(1) G0 phase cells were cultured in a medium containing 0.5% FCS and obtained after 10-11 days of serum deficiency.

(2) Cells in G1 phase fuse with cytoplasm within 1-3 hours after completing mitosis.

Negative 5-bromodeoxyuridine labeling assay confirmed that confluent J1 cells did not enter the S phase of the cell cycle, but the G1 phase of the cell cycle within 3 hours of mitosis.

Embryos reconstructed in this experiment with donor cells from both cell cycle treatment groups were cultured in vitro in AgR SOF medium supplemented with bovine serum albumin (BSA, Life Technologies Prod. No. 30036-578).

The results of in vitro development are shown in Table 1. When electrofusion was used, donor cells in the G0 (control) and G1 phases of the cell cycle fused with cytoplasm (recipient cells) with equal efficiency. As shown in Table 1 below, there was no difference in the proportion of fusion-reconstructed embryos that developed in vitro to the blastocyst stage between the G0 and G1 donor cell treatment groups.

Table 1: In vitro development of cloned embryos reconstituted with J1 follicular cells in G0 or G1 phase of the cell cycle and cultured in AgR SOF medium supplemented with Life Technologies bovine serum albumin (BSA). The stage fusion rate of the cell cycle The total development rate of blastocyst stage 1-2 stage G0 (n=152) 86±5.2% 24±1.1% 77±6.4% G1 (n=186) 78±5.0% 25±6.2% 77± Effect of 6.9% Example 2 Follicle Donor Cells (J1 and EFC Cell Lines) Synchronized in G0 Phase or G1 Phase on In Vitro Development after Nuclear Transplantation Cultured in Medium Supplemented with Sigma Bovine Serum Albumin

Further evidence is provided in Table 2 to support that follicle donor cells in the G0 and G1 phases of the cell cycle have no effect on the development of cloned embryos cultured in vitro to the blastocyst stage after 7 days of culture.

Experiments were performed with two independent follicular cell lines:

(1) "EFC"; a follicular cell line from a New Zealand Frisian cow and

(2) "J1" is a young cow from New Zealand's Jersey breed.

In this experiment, EFC cells only used the G0 phase, while J1 cells only used the G1 phase of the cell cycle. However, the data in this example were combined because both cell lines were derived from follicular cells and the reconstructed embryos were cultured on the same media formulation. In these experiments, the medium was standard AgR SOF medium, but supplemented with Sigma's bovine serum albumin (Sigma Chemical Company; Product No. A-7073) instead of Life Technologies' bovine serum albumin as in Example 1 above.

J1 cells are used for nuclear transfer at passage 3-6, and donor cells are fused within 1-3 hours after the completion of mitosis. EFC cells were used between passages 3-8 in culture and synchronized in G0 phase with serum containing 0.5% between days 9-18 in culture.

The in vitro development results are presented in Table 2, showing that for follicular donor cells, the proportion of fused embryos that developed to the blastocyst stage did not differ between the G0 and G1 phases of the cell cycle.

Table 2 The in vitro developmental cell line cell cycle stage fusion rate of cloned embryos reconstituted with follicular cells in G0 or G1 phase of the cell cycle and cultured in AgR SOF medium supplemented with Sigma bovine serum albumin The total development rate of blastocyst J1 G1 (n=576) 82±3.8% 38±3.6% 67±1.8% EFC G0 (n=1108) 78±2.3% 37±2.3% 59±3.3%

Example 3 After nuclear transfer, the effect of synchronization of follicle donor cells in G0 or G1 phase on development in vivo after nuclear transfer.

The data in Figure 1 represent the survival of cloned bovine embryos reconstituted with follicular cells in G0 or G1 phase of the cell cycle throughout gestation after transplantation into recipient cows. The data in Figure 1 were compiled from data on embryos generated from the experiments set forth in Examples 1 and 2 above. This includes cloned embryos generated from two follicular cell lines, namely J1 and EFC. Also, cloned embryos were generated in the same AgR SOF medium formulation supplemented with either Sigma's bovine serum albumin or Life Technologies' bovine serum albumin. These data were pooled because the two follicular cell lines or the two sources of bovine serum albumin had no effect on post-transplant viability (although there was a significant effect on development to the blastocyst stage).

The data in Figure 1 represent 85 embryos reconstituted with G0 donor cells and 95 embryos reconstituted with G1 early donor cells transplanted into the reproductive tract of synchronized recipient cows.

Figure 1 illustrates the percentage of embryos/fetus present throughout pregnancy from day 7 of embryo transfer to delivery as determined by conventional sonography, rectal palpation, and calving. Most notably, the use of G1 follicular cells resulted in the birth of live calves at full farrow. Compared with G0 donor cells, there was a trend toward decreased embryo survival in cloned embryos reconstituted with G1 cells starting from day 30 of gestation until completion of full parturition, however, the number of transfers reported here did not reach statistical significance .

For G1 follicular cells, 9% of transferred embryos (9/95) resulted in the birth of fully delivered calves. However, 4 of these calves died at or after birth, resulting in an overall efficiency of 5% for live cloned calves generated from G1 cells. In contrast, GO donor follicular cells resulted in 20% development to full farrowing (17/85), resulting in an overall efficiency of 16% (14/85) of live calves born as 3 calves died at birth.

Example 4 Synchronization of Female Adult Skin Fibroblasts (Age± and Age− Cell Lines) in G0 or G1 Pairs After Nuclear Transfer in Cultured Media Supplemented with Life Technologies Bovine Serum Albumin for In Vitro and In Vivo Development Influence.

Data are from two independent primary cell lines of adult skin fibroblasts. These two cell lines are defined as "Age+" and "Age-". They are all derived from female lines of Angus beef cattle that have been selected to enter puberty later or earlier. Since these two similar cell lines did not differ significantly in in vivo and in vitro development, the data are compiled in Table 3 and Figure 2 below.

Two phases of the cell cycle are compared in these experiments:

(1) G1, within 1-3 hours after the completion of mitosis, the donor cell and the cytoplasm fuse.

(2) G0, the donor cells were cultured in a medium supplemented with 0.5% FCS for 3-12 days. For reconstructed embryos transplanted into recipient cows, cells were subjected to serum starvation for 4-5 days.

Cells from two cell lines and two cell cycle treatments were used for nuclear transfer at passage 7 in culture. Reconstructed embryos were cultured in standard AgR SOF medium supplemented with Life Technologies bovine serum albumin (Life Technologies product number 30036-578).

The data presented in Table 3 demonstrate the effect of the length of time G0 cells were in low serum on fusion efficiency, however, once fused to the cytoplasm there was no effect on subsequent in vitro development, therefore, the G0 cytoplasm data were pooled. G0 and G1 cell cycle phases had no effect on development to the blastocyst stage.

Table 3 In vitro development of cloned bovine embryos reconstituted with female adult skin fibroblasts (Age+ and Age- cell lines) at G0 or G1, cultured on AgR SOF medium supplemented with Life Technologies bovine serum albumin. Stage of cell cycle Fusion rate 1-2 blastocyst stage Total development rate G0 3-5 days (n=411) 66±1.9% ^a

19±3.0% 58±4.5% 12 days (n=107) 41±6.9% ^b G1 (n=401) 59±2.5%a ^b 19±5.0% 67±6.6%

ab P＜0.05

The data in Figure 2 demonstrate that selected G1 adult skin fibroblasts are capable of producing live calves at full farrow. Similar to the data in Figure 1, there was a trend towards greater survival in complete delivery than in G0, however, this was not statistically significant with respect to the number of transplants performed here. Using G1 donor cells, 4% of the embryos transferred eventually produced live calves (1/25). In contrast, GO donor cells resulted in an eventual 14% of viable embryos developing to farrowing (3/22). In this experiment, all 4 calves produced underwent postnatal development.

Example 5 Effect of Synchronization of Adult Skin Fibroblasts (LJ801 and 3XTC Cell Lines) in G0 or G1 Phase on In Vivo and In Vitro Development After Nuclear Transfer, Cultured in Medium Supplemented with ICP Bovine Serum Albumin

Additional experiments comparing G0 and G1 cell cycle phases were performed using two independent adult skin fibroblast cell lines. These two cell lines are denoted "LJ801" and "3XTC". LJ801 was derived from a male cell line of a Limousine X Jersey bull, while 3XTC was derived from a crossbreed cow that had produced trisomic calves three times.

Two cell cycle phases are compared:

(1) G0 cells, the donor cells of the two cell lines were cultured for 4-5 days in a medium containing 0.5% FCS; and

(2) For G1 cells, within 1-3 hours after the completion of mitosis, the donor cells fuse with the cytoplasm.

Negative 5-bromodeoxyuridine labeling confirmed that confluent adult skin fibroblasts did not enter S phase within 3 hours of mitosis.

Both LJ801 and 3XTC cell lines were used for nuclear transfer experiments at passage 3-4.

Since there was no difference in the efficiency of development to the blastocyst stage between LJ801 and 3XTC cells, and the reconstructed embryos from both cell lines were grown in the same standard AgR SOF medium supplemented with bovine serum albumin, but this time with bovine serum Protein was from ICP (Immuno-Chemical Products, Auckland, New Zealand; product number ABFF-002). After embryo transfer, embryo survival throughout gestation is shown in Figures 3 and 4 for the corresponding cell lines 3XTC and LJ801.

The data in Table 4 demonstrate that cell cycle synchronization of adult skin fibroblast donor cells at G0 or G1 has no effect on development to the blastocyst stage.

Table 4 In vitro development of cloned embryos reconstituted with adult skin fibroblasts (LJ801 and 3XTC) in cell cycle G0 or G1 and cultured in AgR SOF medium supplemented with ICP bovine serum albumin. The stage fusion rate of the cell cycle The total development rate of blastocyst stage 1-2 stage G0(n=145) 72±7.8% 36±6.1% 61±5.0%G1(n=200) 60±3.1% 37±6.0% 57± 5.0%

The data in Figure 3 indicate that embryos reconstituted with 3XTC did not differ in survival to at least day 150. Embryo survival at day 150 was 25% (3/12) for donor cells in cell cycle Gl and 27% (3/11) for G0 donor cells.

The data in Figure 4 indicate that embryos reconstituted with LJ801 fibroblasts had a greater trend towards gestational day 210 survival of embryos using G0 donor cells, but this was not statistically significant. Embryo survival at day 210 was 23% (3/13) for donor cells in cell cycle G1 and 39% (7/18) for G0 donor cells.

Example 6 Effect of synchronization of genetically modified bovine fetal fibroblasts in G0 or G1 on development in vitro and in vivo after nuclear transfer.

The effect of cell cycle phase on development after nuclear transfer was also studied using genetically modified female bovine fetal lung fibroblasts. Genetic alterations include the random insertion of additional copies of the bovine beta and κ casein genes. Transgenic cell lines are indicated as "Casein+5110".

Two cell cycle phases are compared:

(3) G0 cells, the donor cells of the two cell lines were cultured in a medium containing 0.5% FCS for 3-6 days; and

(4) For G1 cells, within 1-3 hours after the completion of mitosis, the donor cells fuse with the cytoplasm.

Reconstructed embryos were cultured in standard AgR SOF medium supplemented with ICP bovine serum albumin (Immuno-Chemical Products, Auckland, New Zealand; product number ABFF-002).

The data of embryonic development in vitro are shown in Table 5. When the casein+5110 donor cells used for nuclear transfer were in the G1 phase, development to the blastocyst stage was significantly reduced compared to the G0 phase. This result is in contrast to other cell lines described above in previous examples.

Table 5 In vitro development of cloned bovine embryos reconstituted with transgenic female fetal lung fibroblasts (Casein+5110 cell line) in the G0 or G1 phase of the cell cycle and cultured in AgRSOF medium supplemented with ICP bovine serum albumin . Stages of cell cycle Fusion rate Grade 1-2 blastocyst stage Total development rate G0(n=150) 90±8.1% 52±1.8% ^a 72±2.9%°G1(n=73) 77±6.5% 26±5.1% ^b 43±2.2%°

ab P<0.0b; cd P<0.01

Embryo survival data presented in Figure 5 indicate that for the casein+5110 cell line, the phase of the cell cycle has no effect on development at least up to day 90. Embryo survival at day 90 was 38% (9/24) for donor cells in cell cycle G1 and 27% (6/22) for G0 donor cells.

Example 7 Effect of non-proliferating, senescent donor cells (at late G1 stage) on in vitro and in vivo development after nuclear transfer

Nuclear transfer experiments were conducted to study the effects of non-proliferating, senescent bovine donor cells on development. The cells used in this experiment were female fetal lung fibroblasts and were genetically modified (labeled 561 cells). Initially, the cells are actively growing, but during the culture, the growth becomes increasingly slow, and at a later stage the cells enter a non-proliferative phase, known to those skilled in the art as senescence, when cell division ceases. Senescent cells are arrested at G1 of the cell cycle, specifically at the G1/S phase boundary (Sherwood et al., 1988). When cells enter senescence, they are arrested in late G1 and unable to enter S phase in response to physiological mitogens. Thus, the senescent phase is distinct from the quiescent phase, in which cells can be induced to re-enter the cell cycle and proliferate once they return to suitable conditions (for example, by adding serum to serum-deficient cell cultures).

Embryos reconstituted with transgenic senescent cells were cultured in AgRSOF medium supplemented with ICP bovine serum albumin.

The data of in vitro embryo development are shown in Table 6. The rate of development to the blastocyst stage using senescent donor cells was lower than expected for early G0 or G1 as illustrated in the previous examples.

Table 5 In vitro development of cloned bovine embryos reconstituted with non-proliferating, senescent transgenic fibroblasts (561 cells) cultured in AgR SOF medium supplemented with ICP bovine serum albumin. Cell Line Phase of Cell Cycle Fusion Rate Grade 1-2 Blastocyst Stage Total Developmental Rate 561 Senescent (n=158) 88% 12% 35%

The embryo survival data shown in Figure 6 suggest that the cloning efficiency of suppressing non-proliferating senescent cells at late G1 is low, with only 4% of embryos developing to day 90 (1/26).

in conclusion

1. It is possible to select single cells in a certain phase of the cell cycle, preferably early G1. This has an advantage over previous techniques using so-called proliferating cells, where the actual cell cycle phase of the individual cells used for nuclear transfer was not precisely known.

2. Cells in the early G1 phase of the cell cycle after mitosis are totipotent due to the generation of viable calves after nuclear transfer.

3. Therefore, G0 is not the only phase of the cell cycle that is compatible with development after nuclear transfer, with differentiated cultured cells and G1 cells, preferably early G1 cells, nuclei can also be functionally reprogrammed.

4. Early post-mitotic G1 cells promote blastocyst development to a similar level as G0 cells after nuclear transfer.

5. There was no significant difference in the survival rate of cloned embryos reconstituted with G0 or G1 early donor nuclei after transplantation.

industrial application

The present invention may be useful in establishing cloned herds/herds of animals, including transgenic animals that produce pharmaceutically useful proteins, agriculturally useful products such as meat, milk and fiber, and human applications, particularly in the field of clonality therapy .

It should be understood that the present description is not intended to limit the scope of the invention to the above-described examples, and that many variations, obvious to those skilled in the art, are possible without departing from the scope of the appended claims.

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Claims (55)

1. the method for a nuclear transplantation, this method comprise to be selected from donorcells group propagation or that do not breed and separates the G1 cell, and a nucleus transplantation that will obtain from the G1 cell of separation like this is in the stoning recipient cell.

2. the method for claim 1, wherein described donorcells group is the one or more known or unknown stage that is in the cell cycle.

3. method as claimed in claim 1 or 2, wherein, described donorcells group be do not breed and synchronization in any one time point in cell cycle G1 stage.

4. as each described method of claim 1-3, wherein, described G1 cell is in the early stage separation of G1.

5. as each described method of claim 1-3, wherein, described donorcells group be do not breed and comprise senile cell.

6. as each described method of claim 1-5, wherein, described donorcells group is from animal body or the embryo who separates the vitro cell culture, fetus, childhood or become somatic cell.

7. method as claimed in claim 6, wherein, described donorcells group comprises anyly can enter the normal cell of dliploid caryogram of cell cycle and propagation by irriate.

8. method as claimed in claim 7, wherein, described donorcells group is in the neoblast state or is in differentiation or arbitrary degree of resting stage or declining period.

9. each described method in the claim as described above, wherein, described donorcells is fibroblast or the follicule cell of adult or fetus.

10. each described method in the claim as described above, wherein, described donorcells comprises the modification cell.

11. method as claimed in claim 10, wherein, described donorcells comprises transgenic cell.

12. each described method in the claim as described above, wherein, described recipient cell comprises an enucleation oocyte.

13. method as claimed in claim 12, wherein, described enucleation oocyte is to obtain from the species consistent with donorcells nuclear source.

14. as each described method of claim 1-11, wherein, described recipient cell comprises a stoning stem cell or a stoning stem cell that merges.

15. method as claimed in claim 14, wherein, described stem cell is from the embryo who is growing or the embryonic stem cell that separates from a kind of cultured cell system that has set up.

16. a method of producing the cloned animal embryo, this method comprise the donor nuclei in the G1 stage that is in the cell cycle of a separation is transplanted in the recipient cell of stoning.

17. method as claimed in claim 16, wherein, described donorcells nuclear carries out genetic modification with the known method in present technique field so that produce the cloning embryo with desired genetic character.

18., when it is used to produce the cloning embryo of a kind of animal, be selected from: birds, amphibian animal, fish and mammal as claim 16 or 17 described methods.

19. method as claimed in claim 18, wherein, described cloning animal embryo is a kind of mammal, is selected from: comprise human primate, rodent, rabbit, cat, dog, horse, ox, sheep, deer, goat and pig.

20. reconstruction animal embryo with method preparation as claimed in claim 16.

21. the described reconstruction animal embryo of method as claimed in claim 17 comprises transgenic embryo.

22. one kind as claim 20 or 21 described reconstruction animal embryos, its again time cloning further increase embryo's quantity or it is transplanted continuously through cell nucleus, helper cell nuclear is rearranged and/or is grown.

23., comprise being selected from the mammal that comprises human primate, rodent, rabbit, cat, dog, horse, ox, sheep, deer, goat and pig as each described reconstruction animal embryo of claim 20-22.

24. a method of cloning the non-human animal, this method comprise the steps: that (1) allows the non-human animal embryo grow term from embryonic period, embryonic phase according to claim 16 or 17 each described method production cloning non-human animal embryos (2) use known methods; (3) arbitrarily breed the non-human animal who forms thus with conventional method or further PCR cloning PCR.

25. method as claimed in claim 24, wherein, described cloning non-human animal is selected from: the non-human mammal that comprises human primate, rodent, rabbit, cat, dog, horse, ox, sheep and deer.

26. as claim 24 or 25 described methods, wherein, it is a kind of transgenic nonhuman animal with expectation genetic character that described clone talks about the non-human animal.

27. method as claimed in claim 26, wherein, described transgenic nonhuman animal is a kind of transgenic cow or sheep.

28. cloning non-human animal with the preparation of method as described in claim 24.

29. cloning non-human animal as claimed in claim 28 comprises being selected from: the mammal of non-human primates, rodent, rabbit, cat, dog, horse, ox, sheep and deer.

30. as claim 28 or 29 described cloning non-human animals, comprising transgenic nonhuman animal with desired genetic character.

31. cloning non-human animal as claimed in claim 30 comprises transgenic cow or sheep.

32. as claim 30 or 31 described cloning transgenic animal, wherein, described desired genetic character is selected from: insert, lack or change one or more genes, it can produce pharmaceutical protein in milk, blood or urine; Produce nourishing milk meat product; Produce useful agricultural production proterties so that improve the quality that milk, meat and fiber are produced; Raising is to the resistance of insect and disease; Manufacture albumen in milk; Heterograft; And be used to produce transgenic animal as the human diseases model.

33. as claim 28-32 each described cloning non-human animal offspring and descendant thereof.

34. method that produces embryo cell line, this method comprises the steps: a) to select from donorcells propagation group or G1 cell synchronization cell mass or senile cell group separates the G1 cell, then from isolated cells like this in a nucleus transplantation to the stoning recipient cell; B) cultivate blastocyst stage; C) reclaim embryonic cell; And d) use method well-known in the art to set up external unlimited propagated cell system.

35. method as claimed in claim 34, wherein, described embryonic cell is an embryonic stem cell.

36. as claim 34 or 35 described methods, wherein, described donorcells is people's cell.

37. as each described method of claim 34-36, wherein, described donorcells and recipient cell all are people's cells.

38. as each described method of claim 34-37, wherein, described donorcells is adult or the fetal cell that is selected from the normal cell type of any caryogram, recipient cell is to be selected from any cell type of gene expression of can rearranging.

39. one kind with the embryo cell line of producing as each described method of claim 34-36.

40. a human embryonic stem cell of producing with method as claimed in claim 36 when according to claim 35, has using value on treatment is used.

41. method of producing embryonic stem cell, this method comprises the steps: a) to select from donorcells propagation group or G1 cell synchronization cell mass or senile cell group separates the G1 cell, then from isolated cells like this in a nucleus transplantation to the stoning recipient cell; B) cultivate blastocyst stage; And c) reclaims embryonic stem cell.

42. method as claimed in claim 41, wherein, described donorcells is people's cell.

43. as claim 41 or 42 described methods, wherein, described donorcells and recipient cell all are people's cells.

44. as each described method of claim 41-43, wherein, described donorcells is adult or the fetal cell of selecting from the normal cell type of any caryogram, recipient cell is to select from any cell type of gene expression of can rearranging.

45. use the embryonic stem cell that method is produced as described in each as claim 41-43.

46. embryonic stem cell as claimed in claim 45 is comprising human embryo stem cell.

47. purposes as claim 39,40 and 45 embryonic cell as described in each, wherein, specialized cell or the types of organization that is selected from nerve cell, muscle cell, heart cell, liver cell, pneumonocyte, kidney cell or other a kind of relevant cell types cultivates with method well-known in the art.

48. a purposes as claimed in claim 47, wherein, described embryonic cell is as claim 40 or 46 described human embryo stem cells.

49. therapeutic cloning method, wherein, embryonic stem cell is that each is described according to claim 35 and 41-43, produce from the recipient cell of curee's gained, and cultivate and produce the described curee that is used for this treatment of needs or other curees transplanting with specialized cell or tissue.

50. method as claimed in claim 49, wherein, described embryonic stem cell comprises one or more transgenosiss, gives the genetic character of the resulting noble cells expectation that is used to transplant.

51. a methods of treatment that is used for passing through disease, obstacle or the damage of transplanting specialized cell or tissue treatment, this method comprise specialized cell or tissue to the claim 49 of patient's administering therapeutic effective dose of this kind of needs treatment or 50 described methods productions.

52. as claim 49 or 50 described methods, wherein, described disease, obstacle or damage are selected from: multiple nervous system disorders (for example Parkinson's), diabetes, cardiopathy, amyotrophy, multiple hereditary disease, particular cancers (for example leukemia), spinal cord injury, burn and other miseries.

53. a vitro differentiation human embryo stem cell that uses the described method of claim 47 to produce carries out the method for drug screening or drug toxicology test.

54. a heteroplastic transplantation method, wherein, cell, tissue and organ are from as separating each described inhuman cloning animal of claim 28-32, and are used for the human diseases patient's of this treatment of needs transplanting.

55. a gene therapy method, wherein, cell, tissue and organ comprise transgenosis, and are separated and are used for as claim 30 or 31 described inhuman cloning animal embryos.

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