HK1035991A - Full term development of animals from enucleated oocytes reconstituted with adult somatic cell nuclei - Google Patents

Description

Full term development of enucleated oocytes reconstituted with adult somatic cell nuclei into animals

This application is a partial renewal application of U.S. patent application No.09/132,104 filed on 10.8.1998, which claims priority to U.S. provisional patent application No.60/072,002 filed on 21.1.1998 and No.60/072,002 filed on 19.6.1998.

The U.S. government has a paid-up license in this invention and, in certain instances, has the right to license others under reasonable conditions under the terms of the national institutes of health treaty No. r01-HD-03402, the ministry of public health services.

Background

The present invention relates to a method for cloning an animal by inserting the nucleus of an adult somatic cell into an enucleated oocyte, thereby allowing the host oocyte to form an embryo and to develop into a living animal. In one embodiment of the invention, the insertion of the nucleus is achieved by piezo-electrically driven microinjection.

It is highly desirable to produce large numbers of nearly identical animals quickly. For example, a wide range of medical benefits are expected to be obtained when the nearly identical animal is a genetically engineered (e.g., transgenic) animal. Genetically altered large animals can be used as "factories" of living drugs to produce valuable pharmaceutical preparations in their milk or other body fluids or tissues (this production process is sometimes referred to as "drumming"), or as "factories" of living organs or cells to produce human organs or cells that are not rejected by the human immune system. It is also desirable to produce large numbers of nearly identical research animals such as mice, guinea pigs, rats and hamsters. For example, mice are the primary research model for studying mammalian biology, and thus the availability of nearly identical transgenic or non-transgenic mice is very beneficial for, for example, analyzing embryonic development, analyzing human disease, and testing for new drugs. Thus, for various reasons (e.g., in breeding farm animals, or in interpreting data generated in mice), it is desirable to reliably produce offspring of a particular animal that is genetically nearly identical to its parent.

In addition, in transgenic technology, the methods currently used to produce transgenic animals are not advanced enough to ensure that gene expression is under program control throughout the animal. Although deleterious "location" effects caused by the quasi-random manner in which a transgene is integrated into the host genome can be minimized, differences in transgene expression levels still exist between individuals carrying the same transgene construct of the same copy number inserted at the same locus. Thus, even the production of a small number of transgenic animals capable of producing the desired levels of a given recombinant protein is very time consuming and expensive. These problems may be further exacerbated because the number of transgenic offspring is usually low (usually only one because of inefficiency) and many transgenic animals are non-fertile.

One approach to solving these problems is to "clone" genetically nearly identical animals from transgenic or non-transgenic adult animal cells that have the desired trait or are capable of producing the desired level of the desired product. Therefore, it is possible to generate genetically almost identical animal colonies (clones) from the cells of an adult animal rather rapidly. In addition, selective, reliable cloning of adult animals that produce high yields of emulsion and meat can rapidly produce large numbers of high producers. Cloning animals from adult somatic cells is also beneficial in replicating pets (e.g., dogs, cats, horses, birds, etc.) as well as rare or about-to-be-extinct animals. As used herein, "cloning" refers to complete development to the adult stage of an animal whose non-mitochondrial DNA can be derived from a donor somatic cell by transferring the nuclear chromosomes of the donor somatic cell to a recipient cell (e.g., an oocyte) from which the original chromosomes have been removed.

In normal mammalian development, the oocyte is arrested in the Germinal Vesicle (GV) stage during the first meiotic prophase. Under appropriate stimulation (e.g., a sudden increase in plasma luteinizing hormone), meiosis continues to begin, the blastocyst breaks, the first meiosis is completed, and the oocyte then stagnates in the second metaphase of meiosis ("MetII"). MetII oocytes can then be ovulated and fertilized. Once fertilized, the oocyte completes meiosis, expelling the second polar body, forming the pronuclei of the male and female. The embryo begins to develop, undergoes a series of meioses and then differentiates into specific cells, resulting in constituent tissues and organs. This developmental program ensured successful transition from oocyte to offspring.

Although cells of early embryos are thought to be totipotent in classification (i.e. they can develop themselves into new individuals), this totipotency is lost after several divisions, while the number of divisions differs between animal species (e.g. murine and bovine embryos). The mechanism of this apparent loss of totipotency is rarely known, but it is presumed to reflect subtle changes in the DNA environment that affect gene expression, and is collectively referred to as "reprogramming". Without wishing to be bound by theory, it is believed that cloning techniques may disturb or mimic "reprogramming".

In view of the numerous advantages of cloning in practice, there is great interest in overcoming technical obstacles and the development of new techniques for fusing embryonic or fetal cells (total cells) with enucleated oocytes. However, a method has not been reported so far which reproducibly produces a clone (individual) of full term development (full term development) from adult somatic cells. For example, it has been reported that bovine cumulus cell nuclei are injected into enucleated oocytes and then these oocytes are electrically activated, and 9% of 351 of the injected oocytes develop into blastocysts, but none of them have developed to the end. Similarly, fusion of adult mouse thymocytes with enucleated MetII oocytes mediated by Sendai virus followed by activation with 7% ethanol for 30 to 60 minutes resulted in 70% of 20 oocytes reaching the 2-cell stage, but none developing beyond the 4-cell stage.

A recent report describes the electrofusion of cultured "mammary cells" with enucleated oocytes to produce a live offspring sheep, called "Duoli" (Wilmut, I. et al (1997), Nature 385, 810-813). Dolichen was reported to develop from one of 434 breast-derived cells electrofused by enucleated oocytes cultured for 5 days under serum starvation conditions. According to the reported method for cloning Dolisu, a "mammary gland cell" is inserted into the perivitelline space of an enucleated oocyte by micropipetting. Wilmut reports that the cells are immediately given an electrical pulse to induce membrane fusion and activate the oocyte to trigger the re-start of the cell cycle. The resulting embryos (the other 28 in the experiment) were transferred to suitable recipients and in this unique example, conception resulted in dori sheep. However, since the "mammary gland cell" was taken from a cell line established in a 6 year old sheep three months after third conception, the public suspects to obtain the cell identity of the donor cell nucleus, and even whether the cell originated from an adult sheep.

In our co-owned U.S. patent application No.09/132,104, which is a partial continuation thereof, we disclose and claim a controllably effective method for cloning animals from adult somatic cells, a typical example of which is the successful generation of cloned fertile mice from adult cumulus cell nuclei. We also published that this method can be successfully used to generate clones of this cloned mouse. Since the source of donor cumulus cells is female, all cloned mice produced are female.

Summary of The Invention

The present invention is an extension of the inventive method for successfully producing cloned, live offspring from adult fibroblasts. In particular, the method of the invention provides for obtaining cloned, live offspring from fibroblasts from adult male animals, and shows that the method is not limited to the production of female cloned animals. In one embodiment of the invention, the fibroblasts are incubated for a period of time prior to their use as nuclear donors to produce cloned animals.

The method of cloning animals from adult somatic cells of the present invention is to insert the nucleus (or a portion of nuclear material, including at least minimal chromosomal material capable of supporting development) of a somatic cell directly into the cytoplasm of an enucleated oocyte, to facilitate development of the reconstructed oocyte embryo, and to produce a live offspring. The term "adult somatic cell" as used herein refers to a cell from a post-natal animal, which is therefore neither a fetal cell nor an embryonic cell, and is not of the gametic lineage. The resulting viable offspring are clones of animals that initially provide somatic cell nuclei for injection into oocytes. The invention is applicable to cloning all animals, including amphibians, fish, birds (e.g., chickens, turkeys, geese, etc.), and mammals, such as primates, sheep, cattle, pigs, bears, cats, dogs, horses, rodents, etc.

In one embodiment of the invention, the donor adult somatic cell is "doubled (2 n)"; i.e., two sets of chromosomes as seen at G0 or G1 of the cell cycle. The donor cells may be obtained from an in vivo source, or may be derived from a cultured cell line. An example of an in vivo source of two-fold donor nuclei (i.e., at G0 or G1) is cumulus cells. Cumulus (latin meaning "cumulus") cells are called because they form a tight mass (heap) of follicular cells around the developing ovum before ovulation. Many of these cells remain associated with oocytes (forming cumulus) after ovulation in some animals, such as mice, in which more than 90% of them are in stage G0/G1 and are therefore diploid. Donor nuclei contemplated for use in the present invention are obtained from other in vivo or in vitro (i.e., cultured) sources of diploid adult somatic cells, including, but not limited to, epithelial cells, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes, macrophages, monocytes, nucleated red blood cells, fibroblasts, podocytes, cardiomyocytes, skeletal muscle cells, smooth muscle cells, and other cells from organs including, but not limited to, skin, lung, pancreas, liver, kidney, bladder, stomach, intestine, bone, and the like, as well as suitable progenitors thereof.

In another embodiment of the invention, the donor adult somatic cell is "2C to 4C"; that is, it contains 1 to 2 times as much diploid genomic material (produced by cell cycle S phase replication). The donor cells may be obtained from in vivo or in vitro sources of actively dividing cells, including, but not limited to, epithelial cells, hematopoietic cells, epidermal cells, keratinocytes, fibroblasts, and the like, and suitable progenitor cells thereof.

One embodiment of the method of the present invention comprises the steps of: contacting the nucleus (or part thereof comprising a chromosome) with the cytoplasm of the enucleated oocyte for a period of time (e.g. up to about 6 hours) after insertion into the oocyte, before activation of the oocyte, and (ii) activating the oocyte. In this embodiment, the nucleus is inserted into the cytoplasm of the enucleated oocyte by a method which does not involve activation of the oocyte.

When a donor nucleus having a diploid chromosome is employed, the method further comprises the step of disrupting microtubules and/or microwire assembly (assembly) for a period of time following insertion of the nucleus into the enucleated oocyte, so as to inhibit polar body formation and maintain diploid chromosome number. For example, when a quadruplicate donor nucleus is used, the chromosome magnification of the nucleus-implanted oocyte can be reduced to two-fold by omitting this step in the method, thereby forming a polar body.

In a preferred embodiment of the invention, the nucleus is inserted by microinjection, more preferably by piezo-electrically driven microinjection. The use of a piezoelectric micromanipulator allows for harvesting and injection of donor nuclei with one needle. Moreover, enucleation of oocytes and injection of donor cell nuclei can be performed rapidly and efficiently, and thus, less damage to oocytes than previously reported methods (e.g., fusion of donor cells and oocytes by chemicals that promote fusion, by electricity, or by mediation of fusion viruses).

The introduction of nuclear material by microinjection is temporally and locally anatomically different from cell fusion. Microinjection is used to penetrate the plasma membrane of a donor cell (so that the nucleus can be extracted) in one or more steps separate in time from the step of delivering the nucleus (or a portion thereof, including at least the chromosome) into the enucleated oocyte after penetrating the plasma membrane. The act of separate penetration is not characteristic of cell fusion.

In addition, the spatiotemporal separation of the removal of the nucleus from the introduction makes it possible to control the introduction of substances other than the nucleus. Methods for removing the foreign cytoplasm and introducing additional substances or agents are highly desirable. For example, the supplement may advantageously modulate the embryological development of a nuclear transfer oocyte. Such agents may include antibodies, pharmacological signal transduction inhibitors, or combinations thereof, wherein the antibodies and/or inhibitors target and/or inhibit the action of proteins or other molecules that play a negative regulatory role in cell division or embryonic development. Such agents may include nucleic acid sequences, such as recombinant plasmids or transformation vector constructs capable of expression during embryonic development to encode proteins with potentially positive effects on development, and/or nucleic acid sequences that integrate into the genome of the cell to form transformed cells and genetically altered animals. Introduction of the agent into the cell may occur before, simultaneously with, or after the nucleus has been associated with the enucleated oocyte.

Brief Description of Drawings

The patent application contains at least one drawing executed in color. Copies of this patent application with color drawing(s) will be provided by the U.S. patent and trademark office upon request and payment of the necessary fee.

FIG. 1A is a photomicrograph of ovulated live oocytes surrounded by cumulus cells. The egg membrane (eg coat) (zona pellucida) appears as a relatively transparent region surrounding the oocyte.

FIG. 1B is a photomicrograph taken within 10 minutes of injecting a cumulus cell nucleus into the cytoplasm of an enucleated oocyte, showing an intact cumulus cell nucleus within the cytoplasm of the oocyte. Oocytes injected with cumulus cell nuclei were fixed, stained, and photographed with a phase contrast microscope.

FIG. 1C is a micrograph showing nuclear transformation into chromosomes that appear confused 3 hours after nuclear injection. The confusion reflects an unusual state in which each single-colored agglomerated chromatid is associated with a pole of the spindle and is therefore not aligned on the metaphase plate.

FIG. 1D shows the reaction with Sr^2-Micrographs taken 1 hour after oocyte activation show the chromosomes separated into two groups (mb = intermediate).

FIGS. 1E and 1E' are made of Sr^2-Micrographs taken 5 hours after oocyte activation are shown for two pseudopronuclei, each egg having a different number of identifiable nucleolar-like structures. The size and number of pseudopronuclei varied, suggesting that the chromosomes were "randomly" separated upon oocyte activation.

FIG. 1F is a photomicrograph of a viable blastocyst produced following injection of a cumulus cell nucleus into an enucleated oocyte.

FIG. 2A is a photograph of four week old (cloned mouse) Cumulina (predecessor) and its surrogate mother mouse.

FIG. 2B is a photograph of young mice born after Cumulina mated with CD-1 (albino) male mice at 2.5 months.

Fig. 2C shows two B6C3F 1-derived cloned, grayish-wild (agouti) pups (middle), as well as their surrogate mother mice (CD-1) (B6D2F1 oocyte donor, black, right) and B6C3F1 cumulus cell donor (grayish, left). The central two wild-grey offspring were clones of wild-grey cumulus cell donors (the same "twin" sister) and were two of the series C (see below) and the offspring described in table 2.

FIG. 3 depicts the development of an embryo derived after injection of a podocyte nucleus into an enucleated oocyte, after transfer into uterus. FIG. 3A is a photomicrograph of the uterus of female mice received 8.5 days (8.5dpc) after mating (coitum), which was fixed with Buan fixative, dehydrated with benzyl benzoate and cleaned. All uterine implantation sites failed to develop, except one (at the arrow), where the embryo (fig. 3B) appeared normal and at approximately 12 somites.

FIG. 4 shows the DNA typing of donors and offspring in series C (see below and Table 2), which confirms the genetic identity between cloned offspring and cumulus cell donors and the heterogeneity between oocyte donors and host surrogate females. Placental DNA from 6 cloned series C progeny (lanes 10-15) was compared to DNA from three cumulus cell donor females (lanes 1-3), three oocyte recipient females (lanes 4-6) and three host females (lanes 7-9). Control DNA was from C57BL/6 (lane 16), C3H (lane 17), DBA/2 (lane 18), B6C3F1 (lane 19) and B6D2F1 (lane 20). The left side of FIGS. 4A and 4B shows a ladder of 100 base pair (bp) DNA size markers. FIG. 4A depicts PCR typing with the strain-specific marker D1Mit 46. FIG. 4B depicts PCR typing with the strain-specific marker D2Mitl 02. The PCR-amplified DNA of F1 hybrid mice (FIGS. 4A and 4B) showed additional gel bands not seen in the DNA of the inbred parental line (lanes 16-20). This additional band corresponds to a heteroduplex derived from the two parental products, the configuration of which results in abnormal gel migration. FIG. 4C depicts Southern blot typing of strain-specific Emv loci (Emv1, Emv2 and Emv 3).

FIG. 5 is a schematic diagram of the cloning procedure of the present invention.

Detailed Description

The mitotic cell cycle ensures that each cell that divides gives equal genetic material to both daughter cells. DNA synthesis does not extend through the entire cell division cycle, but is restricted to a portion of the cycle, namely the pre-mitotic synthesis phase (or "S" phase). A time interval after DNA synthesis and before cell division (G2); another interval (G1) occurs after the division and before the next S phase. Thus, the cell cycle includes an M (mitosis) phase, a G1 phase (interval 1), an S phase, a G2 phase (interval 2), and finally a return to the M phase. The cycle of many non-dividing cells (e.g., all dormant fibroblasts) in a tissue is terminated post-mitotically to pre-S phase. Such "quiescent" cells that exit the cell cycle prior to S phase are said to be in G0 phase. Cells entering the G0 phase may be left in this state for a brief or long time. For example, podocytes and neurons are characterized by not dividing in adult animals, but remaining in stage G0. More than 90% of cumulus cells surrounding newly ovulated (mouse) oocytes are in stage G0 or G1. The nuclei of cells at stage G0 or G1 have a double (2n) DNA content, i.e.they differ morphologically (n-1 times autosomal) with two copies each. The nucleus of the cell at the G2 phase has a 4-fold DNA content, i.e. during the S phase, two copies of each different chromosome have been replicated each time.

The present invention describes methods for generating vertebrate clones. In this method, each clone develops from an enucleated oocyte that has received the nucleus (or a portion thereof, including at least the chromosome) of an adult somatic cell. In one embodiment of the invention, cloned mice are generated following microinjection of cumulus cell (i.e., ovulated follicle cell) nuclei into enucleated oocytes using the methods of the invention. In another embodiment of the invention, cloned mice are generated following injection of nuclei of adult tail fibroblasts into enucleated oocytes using the methods of the invention. In embodiments of the invention employing fibroblasts, some fibroblasts are cultured in vitro in a serum-free medium; thus, these fibroblasts are "starved" in order to induce them to be in the G0 or G1 phase of the cell cycle (as is known in the art), and are assumed to contain chromosomes. Culturing other fibroblasts in vitro in a serum-containing medium; thus, these fibroblasts continue the cell cycle by dividing, assuming they are 2C to 4C. In another embodiment of the invention, thymocytes, splenocytes, macrophages are used as adult somatic cell nuclear donors.

Other animals, such as, but not limited to, primates, cows, pigs, cats, dogs, horses, etc., can also be cloned using the methods of the invention. The methods of the invention shown herein provide a high rate of successful development of embryos to the morula/blastocyst stage, a high rate of transfer of embryos to surrogate recipients, and a success rate of producing newborn mammals of greater than 2%. The magnitude of these efficiencies means that the method of the present invention is easily reproducible.

The steps and substeps of one embodiment of the method of cloning an animal of the present invention are described in the example of FIG. 5. Briefly, (1) oocytes are harvested from oocyte donor animals. (2) Removal of the MetII plate results in enucleated oocytes (chromosome-free eggs). (3) Harvesting somatic cells from adult donors, (4) selecting cells that are healthy in appearance, (5) obtaining their nuclei (or nuclear composition including chromosomes). (6) A single nucleus is injected into the cytoplasm of an enucleated oocyte. (7) The nuclei were allowed to stay in the cytoplasm of the enucleated oocytes for 6 hours, during which time chromosome condensation was observed. (8) Activating the oocyte in the presence of an inhibitor of microtubule and/or microfilament assembly, (9) to inhibit the formation of polar bodies. During this activation, the formation of false pronuclei is observed. (10) Eggs forming pseudopronuclei are selected and placed in embryo culture. (11) The embryos are then transferred to surrogate mothers and (12) live offspring are born.

Accordingly, one embodiment of the mammalian cloning method of the present invention comprises the steps of: (a) collecting somatic cell nuclei or at least partial nuclei containing chromosomes from somatic cells of an adult animal; (b) inserting at least a portion of the somatic cell nucleus into the enucleated oocyte to form a nucleus-implanted oocyte; (c) allowing the nucleus-implanted oocyte to develop into an embryo; and (d) allowing the embryo to develop into a viable offspring. Each of these steps will be described in detail below. Somatic cell nuclei (or nuclear compositions containing chromosomes) can be collected from somatic cells with more than two-fold chromosomes (e.g., cells with one or two-fold normal diploid genomic material). Preferably, somatic cell nuclei are collected from somatic cells having a diploid chromosome. Preferably, the somatic cell nucleus is inserted into the cytoplasm of the enucleated oocyte. The insertion of the nucleus is preferably achieved by microinjection, more preferably by piezoelectrically driven microinjection.

Activation of the oocyte may occur before, during or after insertion into the nucleus of a somatic cell. In one embodiment, the activation step occurs 0 to 6 hours after insertion into the somatic cell nucleus, such that the nucleus is in contact with the oocyte cytoplasm for a period of time prior to oocyte activation. Activation can be by various means, including but not limited to, electrical activation, or contact with ethanol,Sperm cytoplasmic factor, oocyte receptor ligand peptide mimetics, Ca²⁺Released drug stimulant (such as caffeine), Ca²⁺Ionophores (e.g., a2318, ionomycin), phosphoprotein signaling modulators, protein synthesis inhibitors, and the like, or combinations thereof. In one embodiment of the invention, the cells are contacted with strontium ions (Sr)²⁺) Activation is achieved.

Preferably, the nucleus-implanted oocyte injected with the activated diploid chromosomes is contacted with a microtubule and/or microfilament assembly disruptor (described below) to prevent polar body formation, such that all chromosomes of the donor nucleus remain in the nucleus-implanted host oocyte. Whereas activated nuclear-implanted oocytes injected with 2C to 4C nuclei are preferably not exposed to such agents in order to form polar bodies to reduce chromosome number to 2 n.

The step of developing the embryo may include the substep of transferring the embryo to a female mammalian surrogate recipient in which the embryo develops into a viable fetus. As will be appreciated by those skilled in the art, any stage of embryo transfer (from two cells to morula/blastocyst) can be performed.

Embodiments of the invention may have one or more of the following advantages as well as other advantages not listed. First, delivery of nuclei (or delivery of nuclear compositions including chromosomes) by microinjection is suitable for a variety of cell types, whether the cells are grown in vitro or in vivo, regardless of donor size, morphology, developmental stage, etc. Second, delivery of a nucleus by microinjection allows careful control (e.g., minimization) of the cytoplasmic and nuclear cytoplasmic volume of the nuclear donor cell introduced into an enucleated oocyte at the time of nuclear injection, since foreign materials may be toxic to developmental potential. Third, delivery of the nucleus by microinjection allows careful control of the injection of additional agents (along with the donor nucleus) into the oocyte at the time of injection of the nucleus. These reagents are exemplified below. Fourth, delivery of the nucleus by microinjection allows the donor nucleus to be contacted with the cytoplasm of the enucleated oocyte for a period of time prior to activation. This contact may cause chromatin remodeling/reprogrammingThis facilitates subsequent embryo development. Fifth, delivery of nuclei by microinjection allows multiple options for subsequent activation regimens (in one embodiment Sr is used)²⁺). Different activation schemes may exert different effects on developmental potential. Sixth, activation may be performed in the presence of a microtubule and/or microfilament disrupting agent (cytochalasin B in one embodiment to prevent chromosome extrusion) exit and a modifying agent for cell differentiation (dimethyl sulfoxide in one embodiment to promote a favorable developmental outcome). Seventh, in one embodiment, piezoelectric-driven microinjection is used to deliver nuclei, which allows for rapid and efficient processing of the sample, thereby reducing damage to the manipulated cells. This is due, in part, to the fact that the preparation of somatic cell nuclei and the introduction of enucleated oocytes can be carried out using the same injection needle (as opposed to conventional microinjection methods which require at least one needle change between zona pellucida granulation and penetration of the plasma membrane of the oocyte). In addition, oocytes from some species of animals (e.g., mice) are not amenable to microinjection using conventional needles, and piezoelectric-driven microinjection offers high success rates. Finally, not only the individual steps of the invention, but also the relationship of these steps to each other as a whole, lead to high cloning efficiency. The various steps will be described in more detail below to show how the steps may be arranged relative to each other in the present invention.

Oocyte acceptor

It has been reported that the in vivo maturation stage of oocytes upon enucleation and nuclear transport are important for the success of nuclear delivery methods. Generally, reports of mammalian nuclear delivery describe the use of MetII oocytes as recipients. MetII oocytes are the cell type that is normally activated by the fertilization sperm. It is known that the chemical composition of the oocyte cytoplasm changes throughout the maturation process. For example, cytoplasmic activity associated with the metaphase factor of maturation ("MPF") is highest when immature oocytes are in the metaphase of the first meiosis ("Met i"), decreases with the formation and expulsion of the first polar body ("Pb 1"), and again reaches a maximum level at Met ii. MPF activity remains high in oocytes that are arrested at MetII and decreases rapidly following oocyte activation. When somatic cell nuclei are injected into the cytoplasm of MetII oocytes (i.e., oocytes with high MPF activity), the nuclear membrane is disrupted and chromatin is condensed, resulting in the formation of metaphase chromosomes.

Oocytes that may be used in the methods of the invention include immature (e.g., stage GV) and mature (i.e., stage MetII) oocytes. Mature oocytes may be obtained, for example, by inducing superovulation in an animal by injection of gonadotropin or other hormones (e.g., sequential administration of equine and human chorionic gonadotropin), followed by surgical harvesting of the oocytes after ovulation (e.g., 80-84 hours after onset of estrus in domestic cats, 72-96 hours after onset of estrus in cows, and 13-15 hours after onset of estrus in mice). Where only immature oocytes are available, they are cultured in a maturation-promoting medium until they progress to MetII; this is called in vitro maturation ("IVM"). IVM Methods for immature bovine oocytes are described in WO98/07841 and for immature mouse oocytes in Eppig & Telfer (Methods in Enzymology 225,77-84, Academic Press, 1993).

Enucleation of oocyte

Preferably, the oocyte is contacted with a medium containing microtubule and/or microwire disrupting agents prior to and during enucleation. Disruption of the microfilaments and/or microtubules imparts relative fluidity to the cell membrane and underlying cortical cytoplasm, thereby allowing the portion of the oocyte enclosed within the membrane to be readily aspirated into the pipette with minimal disruption to the cell structure. One microtubule disrupting agent of choice is cytochalasin B (5 μ g/ml). Other suitable microtubule disrupting agents, such as thiaurethane pyridazinol, 6-dimethylaminopurine, and colchicine, are also known to those skilled in the art. Microfilament disruptors are also known and include, but are not limited to, cytochalasin D, methoprene (jasplakinolide), lasagnolin A (1atrunculin A), and the like.

In a preferred embodiment of the invention, enucleation of MetII oocytes is accomplished by aspiration with a piezo-electrically driven micropipette. Throughout the enucleation microsurgery, MetII oocytes were fixed with a conventional holding pipette with the flat tip of a piezoelectrically driven enucleation pipette (internal diameter of about 7 microns) in contact with a transparent tape. A suitable piezoelectric Drive unit is sold under the name piezoelectric Micromanipulator/piezoelectric Impact Drive unit (Piezo Micromanipulator/Piezo Impact Drive unit) by Prime Tech Ltd. The unit uses the piezoelectric effect to advance the (injection) pipette holder a very small distance (about 0.5 microns) in a highly controlled rapid manner. The intensity and spacing between each pulse can be varied and adjusted by a control unit. A piezoelectric pulse (e.g., intensity =1-5, speed =4-16) is applied to advance (or bore) the pipette through the zona pellucida while maintaining a small negative pressure within the pipette. In this way, the pipette tip quickly passes through the zona pellucida and thereby advances to a position adjacent to the Met ii plate (a discernable translucent region in the cytoplasm of Met ii oocytes from several animals, usually near the first polar body). The oocyte cytoplasm containing the metaphase plate, which contains the chromosome-spindle complex, is then gently aspirated into the injection pipette in minimal amounts, and the injection pipette (now containing the MetII chromosome) is gently withdrawn. The effect of this procedure is to pinch off that part of the oocyte cytoplasm which contains the MetII chromosome. The injection pipette is then pulled out of the zona pellucida, discharging the chromosome into the adjacent medium, and preparing the next oocyte for a chromosome removal microsurgical operation. Where appropriate, a batch of oocytes may be screened to confirm complete enucleation. For oocytes containing granular cytoplasm (e.g., porcine, ovine, feline oocytes), it is advantageous to stain with DAN-specific fluorochromes (e.g., Hoeschst 33342) and simply examine with low UV radiation (enhanced with image-enhanced video monitors) in determining enucleation efficiency.

Enucleation of MetII oocytes may be accomplished by other methods, such as those described in U.S. Pat. No. 4,994,384. For example, in contrast to piezo-electrically driven micropipettes, coring can be accomplished microscopically with conventional micropipettes. This was achieved by tearing the zona pellucida of the oocyte with a glass needle along the zona pellucida about 10-20% of the MetII chromosome position (spindle in the cortex of the oocyte is shown by differential interference microscopy). The oocyte is placed in a small drop of medium containing cytochalasin B in a micromanipulation chamber. Chromosomes were removed with an enucleated pipette having an untapered beveled mouth.

After enucleation, the oocytes are readily reconstituted with adult somatic cell nuclei. Preferably, the enucleated oocyte is prepared within about 2 hours of insertion into the donor nucleus.

Preparation of adult somatic cell nuclei

Cells derived from in vivo or in vitro grown cell populations containing cells bearing chromosomes that are diploid (i.e., cells at G0 or G1) or more than diploid (e.g., cells at G2, usually quadruplicate) may be suitable nuclear donors. In one embodiment of the invention, the cell is a follicle (cumulus cell) cell obtained from an adult mammal and dispersed by mechanical and/or enzymatic means (e.g., with hyaluronidase). The resulting dispersed cell suspension can be placed in a micro-chamber to facilitate detailed examination, selection, and manipulation of individual cells to avoid those cells having certain characteristics, such as exhibiting a progressive stage of apoptosis, necrosis, or division. The cells selected in this way were gently and repeatedly aspirated to rupture the plasma membrane and harvest the corresponding nuclei. The individually selected nuclei are then aspirated into an injection pipette described below for insertion into the enucleated oocyte.

In another embodiment of the invention, the donor of the adult cell nucleus is a fibroblast. Fibroblasts can be obtained from animals by methods well known to those skilled in the art. For example, fibroblasts can be obtained from adult mouse tails by subjecting minced tail tissue to short-term culture (e.g., 5% CO)₂At 37.5 deg.c for 5-7 days) during which the fibroblasts in the culture become the predominant cell type. In yet another embodiment of the invention, thymocytes, splenocytes, macrophages are employedAs donors to adult somatic nuclei. Methods for obtaining thymocyte or splenocyte cell suspensions are well known to those skilled in the art. Macrophages can be obtained, for example, by irrigating the peritoneal cavity or lungs by methods known to those skilled in the art.

Other somatic cells that may be used as a nuclear source include, but are not limited to, epithelial cells, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes, monocytes, nucleated red blood cells, podocytes, cardiomyocytes, skeletal muscle cells, smooth muscle cells, and other cells from organs including, but not limited to, skin, lung, pancreas, liver, kidney, bladder, stomach, intestine, etc. (and their suitable progenitor cells), obtained directly from in vivo sources, or obtained after in vitro culture.

Insertion of donor nuclei into enucleated oocytes

The nucleus (or nuclear composition comprising a chromosome) may be injected directly into the cytoplasm of an enucleated oocyte using microinjection techniques. In one preferred method of injecting somatic cell nuclei into enucleated oocytes, a piezo-electrically driven micropipette is employed, wherein apparatus and techniques substantially as described above (with respect to enucleation of oocytes) may be employed, with the improvements described in detail herein.

For example, an injection pipette is prepared as described above with a flat mouth having an inner diameter of about 5 microns. The injection needle contained mercury near the mouth and was placed in a piezo-electrically driven unit according to the manufacturer's instructions. The presence of a mercury droplet near the injection pipette tip increases momentum and thus improves the penetration capability. The injection pipette tip containing the single selected nucleus is brought into close contact with the zona pellucida of the enucleated oocyte and several piezoelectric pulses (intensity level 1-5, speed 4-6 set by the controller) are applied to advance the pipette while maintaining a slight negative pressure in the pipette. When the pipette tip passes through the zona pellucida, the contained zona pellucida plug is squeezed into the perivitelline space and the nucleus is pushed forward close to the pipette tip. The pipette tip was then juxtaposed to the plasma membrane and advanced toward the opposite side of the oocyte until the holding pipette almost reached the opposite side of the oocyte cortex. The oocyte membrane is now deeply nested around the nozzle of the injection needle. After applying 1 to 2 electrical piezoelectric pulses (typically 1-2 intensity at a speed of 1), the egg membrane is punctured at the pipette tip, showing a sharp relaxation of the egg membrane, which is clearly visible. The nuclei and accompanying minimal (about 6pL) medium were then drained into the plasma. The pipette is then gently withdrawn, leaving the newly introduced nucleus in the cytoplasm of the oocyte. The method is carried out very rapidly, usually with a batch of 10-15 enucleated oocytes, which are maintained in culture at other times.

Other microinjection protocols can be used to insert donor nuclei, using conventional injection pipettes. Examples of suitable microinjection methods for inserting sperm nuclei into hamster oocytes using conventional pipettes are described in Yanagida, K., Yanagimachi, R., Perreault, S.D., and R.G.Kleinfeld, Biology of Reproduction 44,440-447(1991), the disclosure of which is incorporated herein by reference.

Activation of host oocytes

In one embodiment of the invention, the nucleus-implanted oocyte is returned to culture conditions for 0-6 hours prior to activation. Thus, in embodiments of the invention, oocytes may be activated at any time up to about 6 hours (latency period) after nuclear implantation by electrical activation, injection of one or more oocyte-activating substances, or transfer of oocytes into a medium containing one or more oocyte-activating substances.

Agents that provide an activation stimulus (or a combined activation stimulus) include, but are not limited to, sperm cytoplasmic activating factor and certain pharmaceutical compounds (e.g., Ca)²⁺And other signal transduction modulators) that can be introduced by microinjection after or simultaneously with nuclear implantation. Certain stimuli can be provided following transfer (immediately or after incubation) of nucleus-implanted oocytes into a medium containing one or more members of the activating subgroup of compoundsActivating stimuli, these activating compounds including Ca²⁺Releasing stimulants (e.g. caffeine, Ca)²⁺Ionophores such as A23187 and ionomycin, and ethanol), phosphoprotein signaling modulators (e.g., 2-aminopurine, staurosporine, and sphingosine), protein synthesis inhibitors (e.g., A23187, cyclohexamide), 6-dimethylaminopurine, or combinations of the foregoing (e.g., 6-dimethylaminopurine and ionomycin). In one embodiment of the invention, the strontium titanate powder is prepared by adding 2-10 millimoles of Sr²⁺And does not contain Ca²⁺The mouse oocytes were activated by incubation in CZB medium for 1-6 hours.

In embodiments of the invention where an activation stimulus is applied simultaneously with or subsequent to nuclear implantation, the nucleus-implanted oocyte is transferred to a culture medium containing one or more microtubule and/or microfilament inhibitors (e.g., 5 micrograms/ml cytochalasin B) or formulations as described above to inhibit chromosome extrusion (through the "polar body") at or shortly after the application of the activation stimulus.

In one embodiment of the invention, the enucleated oocyte may be activated prior to nuclear implantation. The activation method is as described above. Following contact activation stimulation, the oocytes may be incubated for up to about 6 hours and then injected into the nucleus of a diploid cell as described above. In this embodiment, somatic cell-derived chromosomes are transformed directly into the pronucleus-like structures of nucleus-implanted oocytes without inhibiting "polar body" extrusion by incubation with cytokinesis-preventing agents (such as cytochalasin B).

Embryonic development to produce live fetuses and offspring

After the formation of the pronuclei, the embryos can be developed by culturing in a medium that does not contain a microtubule disrupting agent. The culture may be continued to the 2-8 cell stage or morula/blastocyst stage, at which time the embryo may be transferred to the oviduct or uterus of the surrogate mother.

In addition, to increase yield, embryos can be divided and cells clonally expanded. Alternatively, or in addition, the yield of a live embryo may be increased by the methods of the invention, by clonal expansion of the donor, and/or if continuous (nuclear) transfer methods are used, the nuclear composition of the resulting embryo may be transferred back into the enucleated oocyte according to the methods described above in the present invention to produce a new embryo. In another embodiment of the invention, the pre-nuclear embryos are cultured in vivo after direct transfer into a suitable recipient.

Modulation of cell division or embryonic development

In one embodiment of the invention, nuclear implantation of an oocyte allows for the introduction of one or more agents, which have the potential to alter the outcome of embryonic development, before, during or after the nucleus is associated with the enucleated oocyte. Alternatively, the agent may be introduced before or after nuclear implantation. For example, the nucleus may be co-injected with antibodies which are directed against proteins which have a putative regulatory role, possibly affecting the outcome of the method of the invention. Such molecules may include, but are not limited to, proteins involved in vesicle trafficking (e.g., synaptotagmin), molecules that mediate chromatin-ovalbumin communication (e.g., DNA damaging cell cycle checkpoint molecules such as chk1), molecules that have a putative role in oocyte signaling (e.g., STAT3) or molecules that modify DNA (e.g., DNA methyltransferase). Members of this class of molecules may also be (indirect) target molecules of regulatory pharmaceutical preparations introduced by microinjection, which have a similar effect as antibodies. Both antibodies and pharmaceutical agents act by binding to their respective target molecules. This binding reduces the function of the target molecule when the target has an inhibitory effect on the developmental outcome, and promotes this function when the target has a positive effect on the developmental outcome. In addition, modulation of important functions in the cloning process can be achieved directly by injection of a protein (such as the type of protein described above) rather than by injection of a formulation that binds the protein.

In another embodiment of the invention, exogenous ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) may be introduced into the oocyte by microinjection, either before or after nuclear implantation. For example, injection of recombinant DNA carrying the desired cis-activity signal can result in transcription of sequences on the recombinant DNA by resident or co-injected transcription factors, followed by expression of the encoded protein that antagonizes the development inhibitory factor or has a positive effect on embryonic development. In addition, the transcript may have antisense activity against mRNA encoding a developmental inhibitor protein. Alternatively, antisense modulation can be achieved by injection of nucleic acids (or their derivatives) that can exert an inhibitory effect by interacting directly with their nucleic acid targets that are not previously transcribed in the oocyte.

Recombinant DNA (linear or otherwise) introduced by the methods of the invention may contain a functional replicon comprising one or more functional genes expressed under the control of a promoter capable of exhibiting a developmental expression profile ranging from narrow to broad. For example, when a promoter is active only in the early zygote, the promoter may direct immediate and brief expression. The introduced DNA may be lost at some point during embryonic development, or integrated into one or more genomic loci, and stably replicated throughout the life of the resulting transgenic individual. In one embodiment, a DNA construct encoding a putative "anti-aging" protein (e.g., telomerase or superoxide dismutase) may be introduced into the oocyte by microinjection. Alternatively, these proteins can be injected directly.

Examples

The following examples describe the methods of the invention and the development of viable offspring from oocytes injected with adult somatic cell nuclei. Specifically, the examples describe the cloning of mice from enucleated oocytes injected with nuclei isolated from adult mice cumulus cells, podocytes, neurons, fibroblasts, splenocytes, thymocytes, and macrophages. The examples described herein are intended to be merely illustrative of animal oocytes, adult somatic cells, and media that can be used in the methods of the invention and are not intended to be limiting, as other methods of the invention will be readily apparent to those of skill in the art

Examples of embodiments.

Reagent

All inorganic and organic compounds were purchased from Sigma Chemical Co, (st. louis, MO), unless otherwise stated.

The medium used for post-microsurgical culturing of oocytes is CZB medium (Chatot, et al 1989.J. reprod. fert.86,679-688) to which 5.56mM D-glucose was added. The CZB medium contains 81.6 mM NaCl, 4.8 mM KCl, and 1.7 mM CaCl₂1.2 mmole MgSO₄、1.8mM KH₂PO₄25.1 mmole NaHCO₃0.1 mmol of Na₂EDTA, 31 mmol sodium lactate, 0.3 mmol sodium pyruvate, 7 units/ml penicillin G, 5 units/ml streptomycin sulfate and 4 mg/ml bovine serum albumin.

The medium from which the oocytes were collected from the oviducts, subsequently processed and micromanipulated was modified CZB, which contained 20 millimolar Hepes, reduced amounts of NaHCO₃(5 mmol) and 3 mg/ml bovine serum albumin. This medium is referred to herein as Hepes-CZB. The pH of the CZB and Hepes-CZB media was about 7.5. For microinjection purposes, 0.1 mg/ml polyvinyl alcohol (PVA, cold water soluble, average molecular weight 10X 10) is preferably used³) In place of BSA in Hepes-CZB, PVA is beneficial when repeated use of one pipette for multiple nuclear/oocyte transfers because it keeps the injection pipette wall less viscous than BSA for a longer period of time.

The medium used to activate the reconstituted oocytes is Ca-free²⁺CZB of (1), which contains 10 mmol of SrCl₂And 5 μ g/ml cytochalasin B. Sr is²⁺The stock solution (100 mmol in distilled water) was stored at room temperature. A stock solution of cytochalasin B (500. mu.g/ml in DMSO) was stored at-20 ℃. Before use, the composition is Ca-free²⁺CZB 1: 10 diluted Sr²⁺Storage of the solution to thereby Sr²⁺Is 10 millimolar. With no Ca content²⁺CZB diluted the cytochalasin B stock solution so that the final concentration of cytochalasin in the final 1% DMSO concentration was 5 μ g/ml.

The medium used to isolate brain cells was Nuclear Isolation Medium (NIM) consisting of 123.0mM KCl, 2.6mM NaCl, 7.8mM NaH₂PO₄、1.4mM KH₂PO₄、3mM Na₂EDTA. The pH was adjusted to 7.2 by adding a small amount of 1 MHCl. Before injection, PVP (average molecular weight 3X 10) is added³Icnbiochemics, Costa Mesa, CA) in NIM suspension of brain cells.

Other media used in the examples are disclosed as appropriate.

Animal(s) production

5-10 weeks old B6D2F1(C57BL/6 xDBA/2), B6C3F1(C57BL/6 xC 3H/He) and DBA/2 female mice were used as oocyte donors. 5-10 week old female mice, C57BL/6, C3H/He, DBA/2, B6D2F1, and B6C3F1, were used as donors of cumulus cell nuclei. Male mice, 10-12 weeks old, B6C3F1 were used as donors of fibroblast nuclei. Male and female mice, 5-10 weeks old, of B6D2F1 were used as donors of nuclei of other adult cells. The surrogate mother mouse is a CD-1 female mouse, and is mated with a vasectomized male mouse of the same strain.

All Animals used in these examples were maintained according to the guidelines established by the Laboratory animal Care and Use Committee of the institute of Laboratory National Research Committee, University of Hawaii (revised DHEW publication [ NIH ]80-23,1985), Laboratory animal Service at the University of Hawaii (Laboratory animal Service at the University of Hawaii) and the Committee of Laboratory animal Research Council of Laboratory Resources National Research Council of Laboratory origin (Committee on Care and Use of Laboratory Animals of the institute of Laboratory Resources Council). Methods of animal handling were reviewed and approved by the animal care and use committee of the university of hawaii.

Example 1

Preparation of somatic cells

In this example, cumulus cells were isolated from a mouse oviduct as a source of adult somatic cell nuclei for injection into enucleated mouse oocytes. The methods for obtaining cloned mice generated in series A-D, Table 2 and described below, are also described in Wakayama, et al 1998, Nature394, 369-374.

Superovulation of female mice, B6D2F1(C57BL/6 xDBA/2, used in series A and B), B6C3F1(C57BL/6 xC 3H/He, used in series C) or B6C3FI cloned mice generated in series D was induced by sequential intravenous injections of 5-7.5 units of equine chorionic gonadotropin (eCG) and 5-7.5 units of human chorionic gonadotropin (hCG). At 13 hours after hCG injection, cumulus-oocyte complexes (see fig. 1A) were collected from the oviduct and treated in Hepes-CZB medium supplemented with bovine testicular hyaluronidase (0.1% [ w/v ],300 units/ml, ICN Biochemicals, Costa Mesa, CA) to disperse cumulus cells. Intermediate sized cumulus cells (10-12 microns in diameter) are the most common (> 70%), and were selected for injection. After dispersion, the cells were transferred to Hepes-CZB containing 10% (w/v) polyvinylpyrrolidone (average molecular weight 360000 daltons) and left for up to 3 hours at the pre-injection room temperature.

Example 2

Preparation of somatic cells

In this example, podocytes and brain cells (neurons) were isolated from adult mice. These cells are characterized by not dividing in adult animals and permanently residing in the G0 phase of the cell cycle.

The seminiferous tubules were isolated from the testis and exposed to a solution of 1 mg/ml collagenase in Hepes-CZB at 30 ℃ for 20 minutes. The vials were then cut with razor blades and placed in Hepes-CZB with 1 mg/ml trypsin and occasionally agitated. The resulting suspension was then allowed to stand. The podocyte-rich fraction settles first. The suspended cells were aspirated and the remainder was resuspended in fresh medium. Podocytes with characteristic morphological features are easily identified under low power microscopy. The manipulation of individual podocytes was performed with a large syringe pipette (approximately 10 microns inner diameter).

Neural cells were isolated from the cerebral cortex of adult B6D2F1 female mice. Brain tissue was removed with sterile scissors, washed rapidly in red blood cell lysis buffer, and gently homogenized by hand in Nuclear Isolation Medium (NIM) for several seconds at room temperature. Nuclei carrying prominent nucleoli were separately collected from the resulting suspensions using a syringe pipette and then delivered to the recipient enucleated oocytes.

EXAMPLE 3 preparation of somatic cells

Fibroblasts were prepared from the tail of adult B6C3F1 mice. The tails of the resulting mice were isolated, the skin removed, cut into small pieces, and then placed in 5 ml Dulbecco's modified Eagle's medium (DMEM, Sigma) supplemented with 10% fetal calf serum (FCS, Hyclone, Logan, UT) in tissue culture dishes. In the presence of 5% CO₂After incubation at 37.5 ℃ for 5-7 days in air, many fibroblasts were seen to spread along the inner surface of the plate. In some experiments, the medium in the dishes was replaced with DMEM without FCS and incubated for an additional 3 to 5 days. To detach the fibroblasts from the plate, Ca-free solutions containing 0.25% trypsin and 0.75 mM ethylenediaminetetraacetic acid (EDTA, Specialty Media, Lavallette, N.J.) were used²⁺Free of Mg²⁺Phosphate Buffered Saline (PBS) in place of the medium. After 10 minutes, the medium is agitated by pipetting for several minutes to release the cells from the plate surface. The medium was collected and centrifuged (150 Xg, 10 min) to pellet the cells. The cells were then washed three times by centrifugation in DMEM medium.

EXAMPLE 4 preparation of somatic cells

Spleens were removed from adult B6D2F1 male and female mice. After washing in CZB medium to remove blood adhering to the surface, each spleen was placed in 5 ml of Hepes-CZB medium and torn into small pieces to disperse the cells in the medium.

EXAMPLE 5 preparation of somatic cells

The thymus was removed from adult B6D2F1 male and female mice. After washing in CZB medium to remove blood adhering to the surface, each thymus was placed in 5 ml of Hepes-CZB medium and torn into small pieces to disperse the cells in the medium.

EXAMPLE 6 preparation of somatic cells

Immediately after the sacrifice of the female or male mice (B6D2F1), 5 ml of 0.9% NaCl or CZB medium was injected into their peritoneal cavity through a hypodermic needle. The abdomen was then massaged and the medium was recovered through the needle. The recovered medium containing peritoneal macrophages was centrifuged to pellet the cells. The cells were then resuspended in Hepes-CZB medium.

EXAMPLE 7 enucleation of mature unfertilized oocytes

In this example, murine Met ii oocytes were harvested, enucleated, and then microinjected into nuclei isolated from the cells of examples 1 to 6 using a piezo-electrically driven micropipette. All manipulation of oocytes, culturing, and insertion of the nucleus are performed under a layer of mineral oil, which preferably contains vitamin E (e.g. from e.r. squibband Sons, Princeton, NJ) as an antioxidant.

Enucleation of oocytes was performed by aspiration with a piezo-driven micropipette using a piezo-micromanipulator model MB-U of Prime Tech Ltd. (Tsukuba, Ibaraki-ken, Japan). The device uses the piezoelectric effect to advance the pipette holder forward a very short distance (about 0.5 microns) at a very high speed at a time. The intensity and speed of the pulses are regulated by a controller.

Oocytes (obtained 13 hours after hCG injection from eCG-exposed females) were separated from cumulus at about 5% (v/v) CO₂Was placed in CZB medium at 37.5 ℃ under air until needed. A set of oocytes (usually 15-20 in number) was transferred to 1 drop (about 10. mu.l) of Hepes-CZB containing 5. mu.g/ml cytochalasin B, which was previously placed in the operating chamber of the microscope stage. After an oocyte is held by the oocyte holding pipette, its zona pellucida is "drilled out" by applying several piezoelectric pulses to the enucleated pipette tip (approximately 10 microns in inside diameter). The Met II chromosome-spindle complex (as a translucent spot in the egg mass) and a small amount of accompanying egg mass were aspirated into a pipette and then gently aspirated from the oocyte until the elongated cytoplasmic bridge was pinched off. In one groupAfter all oocytes (typically 20 oocytes) have been enucleated (approximately 10 minutes spent), they are transferred to CZB without cytochalasin B and left for up to 2 hours at 37.5 ℃. Oocytes were fixed and stained as described above for evaluation, and the enucleation efficiency was 100%.

Example 8

Insertion of adult somatic cell nuclei into enucleated oocytes

To inject donor nuclei into enucleated oocytes prepared as described above, microinjection chambers were prepared with lids from plastic plates (100 mm. times.150 mm; Falcon Plastics, Oxnard, Calif., catalog number 1001). A row of two circular drops and one elongated drop was placed along the center line of the dish. The first drop (about 2. mu.l; 2 mm in diameter) was used to wash the pipette (Hepes-CZB, containing 12% [ w/v ] PVP, average molecular weight 360000 daltons). The second drop (about 2. mu.l; 2 mm in diameter) contained Hepes-CZB-matched donor cell suspension. The third elongated drop (6. mu.l; 2 mm wide and 6mm long) was Hepes-CZB medium for the oocyte. Each droplet was covered with mineral oil. The plate was placed on the stage of an inverted microscope with a Hoffman modulated contrast lens.

The donor cell nuclei were microinjected into the oocytes using the piezoelectric microinjection method described above. The nuclei were removed from the respective somatic cells and gently aspirated into and out of the injection pipette (approximately 7 microns inner diameter) until their nuclei were "bare" or nearly bare (i.e., essentially no visible cytoplasmic material). For cells with "tough" plasma membranes (i.e., tail fibroblasts), several piezoelectric pulses are applied to disrupt the plasma membrane. After "naked" nuclei are drawn deep into the pipette, (nuclei of) the next cell are drawn into the same pipette. These nuclei are injected one after the other into enucleated oocytes.

To inject the nuclei, a small amount (about 0.5 microliter) of mercury is placed near the proximal end of the injection pipette and then connected to the piezoelectric drive unit described above. After mercury is pushed towards the pipette tip, a small amount of medium (about 10pL) is aspirated into the pipette.

The enucleated oocyte was placed in a drop of CZB medium containing 5. mu.g/ml cytochalasin B on a microscope stand. The oocyte is held with a holding pipette while the injection pipette tip is brought into close contact with the zona pellucida. Several piezoelectric pulses (e.g., intensity 1-2, velocity 1-2) are given to advance the pipette while slightly applying a negative pressure therein. The zona pellucida pellet in the pipette is discharged into the perivitelline space as the pipette tip passes through the zona pellucida. As the donor nucleus advances forward until it is close to the injection pipette tip, the pipette is mechanically advanced until its mouth almost reaches the opposite side of the oocyte cortex. After applying 1 to 2 electric pulses of pressure (typically 1-2 intensity at a rate of 1), the membrane of the egg is pierced, squeezing the nucleus and a minimal amount (about 6pL) of accompanying medium into the egg mass. Sometimes, the medium can be withdrawn. The pipette is then gently withdrawn, leaving the nucleus in the egg mass. One nucleus was injected per oocyte. Using this method, about 5-20 oocytes can be microinjected in 10-15 minutes. All injections were usually performed at room temperature of 24-28 ℃. All manipulations were carried out at room temperature (24-26 ℃). Each nucleus was injected into a separate enucleated oocyte within 10 minutes after denudation.

FIG. 1B depicts cumulus cell nuclei in enucleated oocytes within 10 minutes after injection.

Nuclei of the podocytes and brain cells prepared as described in example 2 were injected into enucleated oocytes by piezoelectric microinjection using the above-described method of injecting cumulus cells.

Nuclei of tail fibroblasts, thymocytes and macrophages prepared in examples 3, 4, 5 and 6, respectively, were injected into enucleated oocytes by piezoelectric microinjection using the above-described method of injecting cumulus cells.

Some oocytes containing injected nuclei were then immediately activated as described in example 9. Other similar oocytes were incubated for up to about 6 hours prior to activation.

Example 9 oocyte activation

After injection of somatic cell nuclei, some groups of oocytes were immediately placed in a medium containing 10 mM Sr²⁺And 5. mu.g/ml cytochalasin B, Ca-free²⁺The CZB was left for 6 hours. Other groups of enucleated oocytes injected with cumulus cell nuclei were treated at 5% (v/v) CO₂Is left in air at 37.5 ℃ for about 1 to 6 hours, preferably about 1 to 3 hours, in CZB, during which time the nuclei in the oocytes disagglomerate and are converted into visible chromosomes { is the description correct? }. Then in the presence of 10 mmol of Sr²⁺And 5. mu.g/ml cytochalasin B, Ca-free²⁺The oocytes are incubated in CZB for about 6-7 hours for activation. Sr²⁺The treatment activates the oocyte, whereas cytochalasin B prevents the subsequent formation of polar bodies and thus prevents chromosome expulsion, thereby ensuring that all chromosomes of the adult somatic cell nucleus remain within the cytoplasm of the activated oocyte. Examination of enucleated oocytes injected with cumulus cell nuclei revealed that the chromosomes aggregated within 1 hour after injection (see FIG. 1C). In the absence of Sr²⁺After 1-6 hours of subsequent incubation in a medium containing Sr²⁺And cytochalasin B, their cumulus cell-derived chromosomes separate (see figure 1D) to form a structure similar to a pronucleus formed after normal fertilization (referred to herein as a pseudo-pronucleus). Examination of 47 fixed and stained oocytes revealed that 64% had two pseudopronuclei (see FIGS. 1E and 1E'), and 36% had three or more pseudopronuclei. Oocytes with at least one clear false pronucleus are considered to be normally activated. Chromosome analysis was performed on 13 such oocytes fixed prior to the first cut (data not shown) and revealed that 85% had a normal diploid chromosome number (2n = 40).

With no Sr content²⁺And cytochalasin B washed in CZB medium at 5% (v/v) CO₂Until they reach the two-to eight-cell stage or morula/blastocyst stage.

FIG. 1F depicts a viable blastocyst produced upon injection of a cumulus cell nucleus into an enucleated oocyte.

Example 10 embryo transfer

Two to eight-cell embryos (24 or 48 hours after the start of activation) were transferred to the oviduct or uterus of surrogate mother mice (CD-1, albino) that had been mated 1 day before with vas-ligated CD-1 males, respectively. Morula/blastocyst (72 hours after activation) was transferred into the uterus of a surrogate mother rat that mated with vas deferens ligated male rat 3 days ago. When cumulus cells or fibroblasts were used as nuclear donors, the female mice received were sacrificed at 19.5 days post-mating (dpc) and examined for the presence of a fetus in their uterus and the implantation site. If a live fetus is present, it is fed to another lactating surrogate mother mouse (CD-1). All the female mice received were sacrificed 8.5 to 12.5 days post-mating when nuclei of other somatic cells (i.e., splenocytes, thymocytes, and macrophages) were taken, and examined for the presence of fetuses in their uterus and implantation sites.

Example 11DNA typing

The DNA of control lines C57BL/6J (B6), C3H/HeJ (C3), DBA/2J (D2), B6C3F1 and B6D2F1, as well as hybrids, were obtained from spleen tissue. DNA from tail tip biopsies were obtained from three cumulus cell donor females (B6C3F1), three oocyte recipient females (B6D2F1) and three generation pregnant females (CD-1). Total DNA of 6B 6C3F 1-derived clonal progeny was prepared from their attached placenta.

For the microsatellite markers D1Mit46, DS2Mit102 and D3Mit49, primer pairs (map pairs ) were purchased from Research Genetics (Huntsville, AL) and typed as described in Dietrich, W. et AL Genetics 131, 423-.

Endogenous ecotropic murine leukemia proviral DNA sequence (Emv locus) was identified after hybridizing PvuII digested genomic DNA to diagnostic probe pEc-B4 according to the method described by Taylor, B.A. and L.Rowe, Genomics 5,221-232 (1989). Probe labeling, Southern blotting and hybridization procedures were performed as described in Johnson, K.R. et al, Genomics 12,503-509 (1992).

Example 12 examination of placenta

After a term fetus (19.5dpc) was found in utero, the placenta was isolated, weighed, and fixed with a cloth fixative to facilitate later examination of histological details. Typically, only one or two implanted clonal mouse offspring in each host surrogate mother mouse reach the term of childbirth (term). During the course of this study, it was noted that the placenta of the cloned fetuses was significantly larger than that of the normal fetuses (see table 7). To investigate the possibility of large placentas due to a low number of tubes per uterus (during normal pregnancy, the uterus of each mouse carries several or up to 10) the number of normal pregnant litters was deliberately reduced by the following method: c57BL/6 female mice were mated with C3H/He male mice. The next day, eggs containing pronucleus were collected from the oviduct, and 2-3 eggs were transferred into the oviduct of each pseudopregnant female mouse (CD-1) to implant only 1-2 embryos. Embryos and placentas were weighed 19.5 days post-mating. Results

And cloning of cumulus cell nuclei. Pre-implantation development of host enucleated oocytes injected with cumulus cell nuclei is described in Table 1. Of the 182 oocytes that were subjected to activation stimulation immediately after injection, 153 (84.1%) were successfully activated and survived. Of these 153 oocytes, 61 developed extracorporeally to the morula/blastocyst stage. However, 474 of 508 injected oocytes activated 1-3 hours after injection (93.3%) were successfully activated and survived, and 151 of 182 injected oocytes activated 3-6 hours after injection (83.0%) were successfully activated and survived. 277 (58.4%) and 101 (66.9%) of these developed in vitro to the morula/blastocyst stage, respectively. Thus, the proportion of oocytes that developed to morula/blastocyst in vitro was significantly higher (p < 0.005) when they were activated 1-6 hours after injection of the nucleus compared to oocytes activated immediately after injection, and the time interval between nuclear injection and oocyte activation appeared to affect the proportion of oocytes developed in these experiments.

The development of host enucleated oocytes injected with cumulus cell nuclei is described in table 2. In the first series of experiments (series a), a total of 142 developing embryos (at the two-cell to morula/blastocyst stage) were transferred to 16 recipient females. These female mice were examined 8.5 days and 11.5 days post-mating (dpc) and found 5 live and 5 dead fetuses in utero. In the second series of experiments (series B), a total of 800 embryos were transferred to 54 surrogate mother mice. When a Caesarean section was performed at 18.5-19.5dpc, 17 live tires were found. Of these, 6 fetuses died soon after birth, 1 fetuses died about 7 days after birth, but the remaining 10 fetuses survived and appeared healthy. All of these mice, including the first fetus (designated "Cumulina", on the anterior side in fig. 2A, together with their albino somatic pregnant females), have been mated, born and fed normal offspring. FIG. 2B is a photograph of Cumulina together with mice generated by its mating with CD-1 (albino) male mice at 2.5 months. Some of these offspring have developed into fertile adult mice.

In a third series of experiments (series C in table 2), B6C3F1 cumulus cell nuclei were injected into enucleated B6D2F1 oocytes. Although B6D2F1 mice were black, B6C3F1 mice carried copies of the agouti a (type a wild gray) gene and were therefore wild gray. Therefore, the offspring of this experiment should have a wild grey coat colour, rather than the black colour of the B6D2F1 oocyte donor. A total of 298 embryos derived from B6C3F1 cumulus cell nuclei were transferred to 18 surrogate mother mice. Caesarean sections were carried out at 19.5dpc and showed 6 live fetuses, the placentas of which were subjected to DNA typing analysis (see example 6 above). Although 1 fetus died one day after birth, the 5 female mice present were all healthy and had a wild gray coat phenotype. Figure 2C shows two such cloned wild gray pups and their albino offspring pregnant females (in the center of the photograph). On the left side of the photograph are corresponding wild gray B6C3F1 cumulus cell donor mice. Cloned young mice (central) resemble the same "twin" sisters of cumulus cell donor mice (i.e. they are clones). The B6D2F1 oocyte donor (black) is shown on the right side of the photograph.

Additional experiments (series D in table 2) were performed to investigate whether clones could be cloned efficiently in subsequent recloning rounds. In this experiment, cumulus cells were harvested from B6C3F1 (wild gray) clones produced in series C, and their nuclei were injected into enucleated B6D2F1 oocytes to produce embryos transferred as described in series A-C. A total of 287 embryos derived from cloned B6C2F1 cumulus cell nuclei were transferred to 18 surrogate mother mice. Caesarean section was performed 19.5 days after mating to obtain 8 live fetuses. Although 1 litter died soon after birth, 7 surviving females were healthy and had the expected wild gray fur phenotype. These results suggest that clones (series B and C) and cloned clone (series D) produced similar efficiencies. Subsequently, the process can be repeated with series D animals as cumulus cell chromosome donors (data not shown), resulting in cloning of the birth clone (third generation cloning). Thus, it appears that the continuous production of clones did not undergo changes (positive or negative changes) that affect the outcome of the cloning process.

Confirmation of the genetic identity of the clones to the cumulus cell donor. As shown in FIGS. 4A, 4B and 4C, DNA typing of the donor and series C progeny confirmed that the cloned progeny were genetically identical to cumulus cell donors and not identical to oocyte donors and host breeding females. PCR typing of DNA was performed with diagnostic highly variable alleles (strain-specific markers) of C57BL/6, C3H, DBA/2 and CD-1 mouse strains. These lines or their F1 hybrids were used in this work, so they all constitute all the genotypes present. In all figures, the placental DNA of 6 cloned series C progeny (lanes 10-15) was compared to the DNA of three cumulus cell donor females (B6C3F1, lanes 1-3), three oocyte donor females (B6D2F1, lanes 4-6) and three host females (CD-1, lanes 7-9). Control DNA was from C57BL/6 (lane 16), C3H (lane 17), DBA/2 (lane 18), B6C3F1 (lane 19) and B6D2F1 (lane 20). FIGS. 4A and 4B depict the results of DNA typing using agarose gel and strain-specific markers D1Mit46 and D2Mit102, and FIG. 4C depicts the results of DNA typing using Southern blot analysis and strain-specific Emv loci (Emv1, Emv2 and Emv 3).

The data in these figures show genetic overlap between cumulus cell nuclear donors and putative clones, and genetic differences from oocyte donors or surrogate mothers. Thus, the genome of each of the 6 cloned mice was derived from the nucleus of cumulus cells.

The following aspects demonstrate that all live offspring in the series B-D reported herein represent clones derived solely from cumulus cell chromosomes. (1) Oocytes/eggs have not been exposed to sperm in vitro. (2) Surrogate mother mice (CD-1, albino) were mated with vasectomized, infertile male mice (CD-1, albino) that demonstrated no fertility. When such vasectomized males have an impossible fertilization behavior, the offspring should be albino. (3) 2-8 cell embryos or blastocysts are transferred into the oviduct/uterus of surrogate mother rats. It is well known that 2-8 cell mouse embryos/blastocysts are generally difficult to fertilize by sperm. (4) The term animals born had black eyes. The 10 surviving litters in series B had black coat and the 5 surviving litters in series C had wild grey coat. This coat colour is genetically identical to the result predicted by the nuclear donor genotype in each case. Because B6D2F1 mice lacked the agouti a gene, the wild gray mice in series C must inherit their wild gray coat color from nuclei other than B6D2F 1. (5) DNA typing of the diagnostic highly variable alleles of the B6, C3, D2 and CD-1 strains used herein (fig. 4) demonstrated that, needless to say, the 6 cloned offspring in series C, including the one that died shortly after birth, were isogenic to the three cumulus cell donor females (B6C3F1) used and did not contain DNA derived from either oocyte donors (B6D2F1) or host surrogate mothers (CD-1). (6) After enucleation, chromosome extrusion polar bodies were inhibited by using cytochalasin B. Thus, if enucleation of an oocyte is not successful at all or only partially successful, all embryos will be hyperploid and will not develop into normal offspring. (7) In the simulation, 204 oocytes were enucleated and examined after staining after fixation, and no chromosomes were found, suggesting that the efficiency of chromosome removal was over 99.99%.

In example 1, the cell type used was identified with high certainty as a cumulus cell. Cells were not cultured in vitro. Sufficient time is provided for the transformation of cumulus cell nuclei into condensed chromosomes in the cytoplasm of enucleated MetII oocytes. The rate of embryo development into morula/blastocyst and implantation is very high. Extending the time between nuclear injection and oocyte activation is advantageous for both pre-and post-implantation development (see tables 1 and 2) and may increase the chances that the cumulus cell genes undergo embryonic development reprogramming.

It is believed that piezoelectric micromanipulators also give a higher proportion of embryo development. The device enables the manipulation of oocytes and donor cells (e.g., drilling of zona pellucida to enucleate oocytes and injection into donor cell nuclei) to be performed very quickly and efficiently. The method of introducing the donor nucleus into the oocyte using a piezo-electrically driven pipette appears to cause less damage to the oocyte than methods using electrical pulses, sendai virus or polyethylene glycol, which also allow direct introduction of the somatic nucleus into the cytoplasm of the oocyte. In addition, microinjection minimizes the amount of somatic cytoplasm that is introduced into an enucleated oocyte. This may also allow for better development of the embryo of the present invention prior to implantation.

Cloning with podocyte nuclei and brain cell nuclei. Enucleated oocytes injected with podocyte nuclei and brain cell nuclei developed individually in vitro into morula/blastocysts of approximately 63 (40%) and 50 (22%), respectively, of which 59 and 46 were transferred into the uterus of recipient surrogate mothers. FIG. 3 depicts embryonic development following transfer of a podocyte nucleus injected into an enucleated oocyte. FIG. 3A is a photograph of the uterus of mice received 8.5 days post-mating (dpc). In the uterus of a surrogate pregnant mother sacrificed at 8.5dpc, it was found that all implantation sites of the uterus could not develop except for one live fetus (fig. 3B) (table 3). Enucleated oocytes injected with brain cell nuclei did not develop more than 6-7dpc (Table 3). Thus, the methods of the invention provide embryonic and fetal development of oocytes injected with podocytes or brain cell nuclei.

Adult fibroblast nuclei were used for cloning.

Table 5 describes the results of an experiment in which nuclei of adult male (wild gray) tail fibroblasts from B6C3F1 mice were injected into enucleated oocytes from female (non-wild gray) B6D2F1 mice. As shown in the table, the activated oocytes injected with fibroblast (cultured in serum-containing medium) nuclei developed about 50% into the morula/blastocyst stage. Of these, 177 two-cell or morula/blastocyst-stage embryos were transferred to recipient surrogate mothers, with 1.1% reaching term (i.e., two live offspring were born). Activated oocytes injected with fibroblast nuclei cultured in serum-free medium developed approximately 58% to the morula/blastocyst stage. Of these, 97 two-cell or morula/blastocyst-stage embryos were transferred to recipient surrogate mother mice, and 1.0% of the embryos reached term (i.e., 1 live offspring was born). All live offspring were male, with black eye and wild grey coat colour, as did fibroblast nuclear donors. All of the above offspring were confirmed to be viable at mating. Whether fibroblasts are cultured in serum-free media or serum-containing media, there appears to be little or no effect on the number of viable progeny obtained.

Clones were cloned with adult splenocytes, thymocytes, and macrophage nuclei.

Table 4 also describes the development of enucleated oocytes that received adult splenocytes, thymocytes, or macrophage nuclei. In these studies, thymocytes provided 3.1% of activated oocyte development to morula/blastocyst, but none developed beyond this stage.

The splenocyte nucleus provides 21% -22% of the activated oocyte embryonic development to the morula/blastocyst stage. Although many were implanted after metastasis, they appeared to be resorbed after 6-7 dpc.

Macrophage nuclei provide 23% -31% of the activated oocyte embryos to develop to morula/blastocyst, but the embryos are absorbed or their development is stopped before 6-7 dpc.

Thus, the methods of the invention provide embryonic and fetal development of oocytes injected with thymocytes, splenocytes, or macrophage nuclei. Since the nuclei of thymocytes, splenocytes, and macrophages in these studies have been shown to be more limited in support of embryonic development than cumulus cell nuclei or fibroblast nuclei, it appears that the nuclei of these cells may support the development of viable offspring, but less efficiently than nuclei of other adult cells.

Cumulus cell nuclear cloning with inbred and hybrid strains of mice

Experiments were performed in which cumulus cell nuclei from three different inbred and two hybrid mice were injected into enucleated oocytes. The results of the experiment are described in table 6. When cumulus cell nuclei of inbred mice (C57BL/6, C3H/He and DBA/2) were injected into the oocytes of the hybrid (B6D2F1), some of the oocytes developed into normal-looking blastocysts, and one (DBA/2 XB 6D2F1) developed into full-term live offspring. In contrast, when cumulus cell nuclei of the crossed B6D2F1 and B6C3F1 mice were injected into enucleated oocytes of mice of the same cross line, respectively, a total of 41 viable offspring (2% -4% of the transferred embryos) were obtained. These offspring are all females. They have black eyes and the same coat color as the cumulus cell nucleus donor.

Differences in placenta weight of cloned and normal mouse pregnancies

During our study, a significant difference in placenta weight was noted between the cloned and normal mice during pregnancy. As shown in Table 7, the cloned mice had placenta weights ranging from 0.25 to 0.33 grams on average, whereas the control (normal) placenta, having the same number of fetuses, weighed from about 0.12 to 0.15 grams, which was about half the weight of the cloned mouse placenta.

We believe that all live progeny reported herein represent clones derived from adult somatic nuclei, especially cumulus cells and fibroblasts, without genetic contamination. The reason is as follows: (1) the oocytes/eggs did not contact sperm in vitro during the experiment. In mammals, intact oocytes are unable to develop to the stage of labor without sperm. (2) The surrogate mother mouse (CD-1) was mated with a ligated vas deferens, a male mouse (CD-1) that was confirmed to be unable to grow. Even though vasectomized males ejaculate sperm and fertilize CD-1 oocytes, all of their offspring should be albinism. The reconstituted two-to eight-cell embryo or blastocyst is transferred into the oviduct/uterus of a surrogate mother mouse. Even if vasectomized males ejaculate sperm, these developing embryos are never fertilized by sperm. (3) All term animals born had dark eyes (non-albino), and the coat color was inherited in a pattern that was completely consistent with the predicted outcome of the nuclear donor genotype in each case. B6D2F1 mice used as oocyte recipients lacked the agouti gene. Thus, the only way to obtain wild-gray offspring is through the donor cell nuclei (e.g., tail fibroblasts and some cumulus cells) of the B6C3F1 mouse. (4) The sex of the cloned mice matched the sex of the donor mice. Clones obtained from female cumulus cells were all female. Clones obtained from male tail fibroblasts were all male. (5) Chromosome extrusion into the polar body is inhibited by the use of cytochalasin B. Thus, even if enucleation of oocytes is completely unsuccessful or only partially successful, all zygotes will be hyperploid; such embryos cannot develop into normal offspring.

It has been demonstrated herein that the methods of the present invention can be used to obtain viable cloned mouse progeny from adult cumulus cells and adult fibroblast nuclei. The success rate is as high as 3%. This method is by far the most successful method with cumulus cell nuclei. The reason for this is not clear. Each mouse oocyte was surrounded by approximately 5000 cumulus cells (data not shown). Cumulus cells are known to communicate with each other through gap junctions throughout follicular development. Those cumulus cells that are closest to the oocyte (corona radiata cells) are in contact with the oocyte by gap junctions. Without wishing to be bound by theory, it is believed that significant ion and small molecule (< 2,000Mr) exchange between the oocyte and the surrounding cumulus cells occurs with confidence. This may affect cumulus cell genes, making the genome more "programmable" within the enucleated oocyte cytoplasm.

It was found that the method of the present invention gives the best cloning results when cumulus cell nuclei of hybrid mice are injected into enucleated oocytes of corresponding cross-bred mice. The only exception was the example of dBA/2 cumulus cell nuclei injected into the oocytes of the hybrid line (B6D2F 1). It is not known at present why cumulus cell nuclei of inbred mice are generally unable to support development following embryo implantation. Mann and Stewart (Development 113,1325-1333(1991)) reported that the developmental potential of the androgenetic condensation chimera was somewhat dependent on the mouse strain. In addition, it is well known that embryos from mouse hybrids are much easier to culture in vitro than embryos from selfed mice (Suzuki, et al (1996) reprod.Fertil.Dev.8, 975-980). It appears that heterosis promotes the development of the cloned embryo up to the stage of labor.

Three live cloned mice were generated by the method of the invention using fibroblasts from adult male mice. It has been previously proposed that the key success in cloning sheep is to bring the donor cells to the G0 phase of the cell cycle. For example, Wilmut et al do this by culturing the cells in serum-free media to "starve" them. In this experiment, culturing adult fibroblasts in serum-free medium did not appear to have a significant beneficial effect on increasing cloning success. It has also been reported that cloned pups can be obtained from foetal cells cultured with serum (Cibeli et al, Science 280,1256-1258 (1998)). Actively dividing cell populations appear to support post-nuclear transfer development to the stage of labor, while serum starvation is not a necessary treatment at least in mouse models.

In these experiments, it was noted that all cloned embryos had large placentas, almost twice as large as normal placentas. Occasionally, large placenta was found but no identifiable embryos (data not shown). The large placenta is also noted by Kono et al in diploid parthenogenetic embryos developed from the fusion of mature oocytes with very young, small oocytes (Nature Genet.13,91-94 (1996)). These parthenogenetic embryos had too large a placenta at 13.5 days of gestation. Kono et al suggested that gene expression lacking maternal alleles might explain why embryonic and placental development is greater than in normal mice. Other genes may be of particular importance for placental development, such as maternally expressed Mash2(Guillemot et al, (1995). Nature Genet.9,235-242), and paternally expressed (maternally expressed) genes required for proliferation of polar trophectodermal cells (Barton et al (1985), J.Embryol.Exp.Morphol.90, 267-285). In addition, some cloned mice that died soon after birth weighed more than others. In addition, dead clonal larvae derived from somatic cells tend to be larger than live larvae as reported by Kato et al (Science 282,2095-2098 (1998)). This suggests that some genes do not completely complete programming or reprogram to function properly during nuclear reprogramming of somatic cells after nuclear transfer. Without wishing to be bound by theory, these findings are consistent with possible changes in characteristic (imprinted) gene expression.

Although the present invention has been described herein with reference to the preferred embodiments, it should be understood that the invention is not intended to be limited to the particular forms disclosed. On the contrary, it is intended to cover all modifications and alternatives falling within the spirit and scope of the invention.

TABLE 1 enucleated oocytes injected with cumulus cell nuclei Pre-implantation developmental oocytes survival of oocytes after activation of oocytes activated 72 hours after activation of embryos number of activated oocytes from activation of oocyte development embryo number (mean% + -SD) activation time Total cell mother cell number Single cell and abnormal two cells to eight cell morula/blastocyst and injection simultaneously 233230182153 (84.1) 177561 (39.9 + -16.6) 1-3 hours after injection of a 573565508474 (93.3) 20177277 (58.4 + -12.6) b 3-6 hours after injection of 195191182151 (83.0) 941101 (66.9 + -14.4) b

The superscript a or b in the same column indicates a significant difference between the two (P < 0.005). Post-implantation development of enucleated oocytes injected with cumulus cell nuclei (Chi-square assay analysis data. Table 2

Series a, caesarean section at 8.5dpc or 11.5 dpc; series B and C, performed with caesarean section at 18.5-19.5 dpc. In series a and B, each donor nucleus is from a B6D2F1 cumulus cell. In series C, each donor nucleus was from a B6C3F1 cumulus cell. In series D, each donor nucleus was from B6C3Fl cloned mice of series C.

Superscripts a and b within the same column indicate a significant difference between a and b: implanting (p < 0.005); fetal development (p < 0.05). The data were analyzed using the Chi-square test.

Death occurs 6-7 days after mating;death occurs 7-8 days after mating;10 days after mating: death was caused by death

TABLE 3 development of enucleated eggs injected with podocyte or brain cell nuclei

All recipients were sacrificed at 8.5 dpc. Superscripts a and b in the same column indicate a significant difference between a and b (P < 0.005).

Death occurred 6 to 7 days after mating.

Survived 12.5 days post-mating.

TABLE 4 development of enucleated mouse eggs injected with different types of adult somatic nuclei 1

Adult cell class	Cell donor	Nuclear transfer postdeposition	Number of oocytes (%)		Transferred embryos	Number (%)
								Model (III)	Sex	Live oocytes	Activated	Until mulberry fruit develops	Number (recipient)	Implant site	Tire
		Number of cells		Embryo/blastocyst
								Thymocyte splenocyte macrophage	Male, female, male	176968052308205	168(95.5)58(60.4)49(61.3)38(73.1)187(60.7)109(22.9)	5(3.1)011(22.4)8(21.1)58(31.0)25(22.9)	0011(2)8(1)52(5)25(3)	--10(90.9)6(75)26(50.0)19(76.0)	--2(18.2)*04(7.7)*0

¹The number of colonized sites and fetuses was measured 8.5 to 12.5 days post-coital.

^*Death or growth arrest after 6 to 7 days post-mating.

Table 5 full term development of enucleated oocytes of mice injected with adult male mouse tail fibroblast nuclei:

the fibroblast is cultured in serum-containing culture medium for 5-7 days

Comparison of Effect after culturing in serum-free Medium for 3-5 days

^*The two-cell to blastocyst-stage embryo is transferred into the oviduct or uterus of the recipient.There was no significant difference in the success rate of obtaining live offspring between female and male clones. The data were analyzed using the Chi-square test.

Table 6 full term development of enucleated mouse oocytes following injection of cumulus cell nuclei from various strains and hybrids of mice:^*these data were calculated at the blastocyst stage of embryo culture.All two-cell embryos were transferred to recipient females.These data include the data in table 2, B6D2F1 mice for series a and B, and table 2, B6C3F1 mice for series C.

Superscript indicates a statistically significant difference between a and b (p < 0.005). The data were analyzed using the Chi-square test. TABLE 7 placental weight of cloned mice at 19.5 days post-coitus

Adult somatic cells for cloning	Sex of fetal rat	Placenta hominis
				Number of checks	Weight (grams)*) Mean ± standard deviation (range)
		Cumulus cell	Female			23	0.33a±0.08(0.21-0.61)
Fibroblast cell	Male sex			3	0.34a±0.07(0.29-0.39)
		-	Female (No clone)			10	0.12b±0.02(0.10-0.16)
-	Male (No clone)			11	O.15b±0.03(0.10-0.18)

Superscript indicates statistically significant differences between a and b (p < 0.001). Data were analyzed using the schradenn t test.

Claims (46)

1. A method of cloning an animal, the method comprising the steps of:

(a) collecting somatic cell nuclei from somatic cells of an adult animal;

(b) inserting at least a portion of a somatic cell nucleus, including a chromosome, into an enucleated oocyte to form a nucleus-implanted oocyte;

(c) allowing the nucleus-implanted oocyte to develop into an embryo; and

(d) allowing the embryo to develop into a viable offspring.

2. The method of claim 1, wherein the adult somatic cell is a cumulus cell.

3. The method of claim 1, wherein the adult somatic cell is a fibroblast.

4. The method of claim 3, wherein the fibroblast cells are cultured cells.

5. The method of claim 3, wherein the fibroblast cells are from an adult male animal.

6. The method of claim 3, wherein the fibroblast cells are from an adult female animal.

7. The method of claim 1, wherein the adult somatic cell nucleus has a diploid chromosome.

8. The method of claim 1, wherein the adult somatic cell nucleus is 2C to 4C.

9. The method of claim 1, wherein the adult somatic cell nucleus is inserted into the cytoplasm of an enucleated oocyte.

10. The method of claim 1, wherein the inserting step is accomplished by microinjection.

11. The method of claim 10, wherein the microinjection is piezo-electrically driven microinjection.

12. The method of claim 1, wherein the enucleated oocyte is arrested in the metaphase of the second meiosis.

13. The method of claim 1, further comprising the step of activating the oocyte prior to, simultaneously with, or after insertion into the nucleus of an adult somatic cell.

14. The method of claim 13, wherein the activating step occurs within 0 to 6 hours after insertion into the nucleus of the adult somatic cell.

15. The method of claim 13, wherein the activating step occurs within 1 to 3 hours after insertion into the nucleus of the adult somatic cell.

16. The method of claim 13, wherein the activating step comprises electrical activation, or exposure to a chemical activator.

17. The method of claim 16, wherein the chemical activator is selected from the group consisting of ethanol, sperm cytoplasmic factor, oocyte receptor ligand peptide mimetics, Ca²⁺Released drug stimulant, Ca²⁺Ionophores, strontium ions, phosphoprotein signaling modulators, inhibitors of protein synthesis, and combinations thereof.

18. The method of claim 16, wherein the chemical activator is selected from the group consisting of caffeine, Ca²⁺Ionophore A23187, ethanol, 2-aminopurine, staurosporine, sphingosine, cyclohexylamide, ionomycin, 6-dimethylaminopurine, and combinations thereof.

19. The method of claim 17, wherein the activator comprises Sr²⁺。

20. The method of claim 1, further comprising the step of disrupting microtubule and/or microfilament assembly in the oocyte for a period of time before or after insertion into the nucleus of an adult somatic cell.

21. The method of claim 20, wherein the time interval is 0 to 6 hours.

22. The method of claim 20, wherein microtubule and/or microfilament assembly is disrupted with an agent selected from cytochalasin B, thiabendazole, colchicine, and combinations thereof.

23. The method of claim 22, wherein the microtubule and/or microfilament assembly is disrupted with cytochalasin B.

24. The method of claim 1, further comprising the step of disrupting the microfilament in the oocyte for a period of time before or after insertion into the nucleus of an adult somatic cell.

25. The method of claim 24, wherein the time interval is 0 to 6 hours.

26. The method of claim 24, wherein the microfilament is disrupted with cytochalasin D, methoprene, lasikalin a, or a combination thereof.

27. The method of claim 1, wherein the step of developing an embryo into a viable offspring further comprises the substep of transferring the embryo to a female replacement recipient, wherein the embryo develops into a viable fetus.

28. The method of claim 1, wherein the inserting step further comprises adding an agent to the cytoplasm of the oocyte.

29. The method of claim 28, wherein the agent is selected from the group consisting of a foreign protein, a derivative of a foreign protein, an antibody, a pharmaceutical agent, and a combination thereof.

30. The method of claim 28, wherein the inserting step further comprises inserting an exogenous nucleic acid or a derivative of an exogenous nucleic acid into the cytoplasm of the oocyte.

31. The method of claim 1, wherein the animal is selected from the group consisting of mammals, amphibians, fish, and birds.

32. The method of claim 31, wherein the mammal is selected from the group consisting of primates, ovines, bovines, porcines, bears, felines, canines, equines, rodents.

33. The method of claim 32, wherein the mammal is a mouse.

34. An animal whose somatic and germ line cells contain only chromosomes derived from adult somatic nuclei of an adult animal.

35. The animal of claim 34, wherein the animal is selected from the group consisting of mammals, amphibians, fish, and birds.

36. The animal of claim 35, wherein the mammal is selected from the group consisting of primates, ovines, bovines, porcines, bears, felines, canines, equines, rodents.

37. The animal of claim 36, wherein the mammal is a mouse.

38. The animal of claim 34, wherein the adult somatic cell is a cumulus cell.

39. The method of claim 34, wherein the adult somatic cell is a fibroblast.

40. The method of claim 39, wherein the fibroblast cells are cultured cells.

41. The method of claim 40, wherein the fibroblast cells are from an adult male animal.

42. The method of claim 40, wherein the fibroblast cells are from an adult female animal.

43. A method of modulating embryonic development, the method comprising the steps of:

(a) combining the nucleus of the adult somatic cell with the enucleated oocyte to form a nucleus-implanted oocyte;

(b) inserting an agent into the cytoplasm of the oocyte before, simultaneously with or after the combining step; and

(c) allowing the agent-treated nucleus-implanted oocyte to develop into an embryo.

44. The method of claim 43, wherein the agent is selected from the group consisting of a foreign protein, a derivative of a foreign protein, an antibody, a pharmaceutical agent, a foreign nucleic acid, a derivative of a foreign nucleic acid, and combinations thereof.

45. The method of claim 43, wherein the inserting step comprises microinjection.

46. The method of claim 45, wherein the microinjection is piezo-electrically driven microinjection.