Patent Application
20080044392
Before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified cell culture techniques, serum, media or methods and may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting which will be limited only by the appended claims.
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. However, publications mentioned herein are cited for the purpose of describing and disclosing the protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a cell” includes a plurality of such cells, and a reference to “an oocyte” is a reference to one or more oocytes, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred materials and methods are now described.
The present invention relates to methods of producing primary cell cultures. The term “primary cell culture” denotes a mixed cell population of cells that permits interaction of many different cell types isolated from a tissue. The word “primary” takes its usual meaning in the art of tissue culture. For example, a primary culture of epidermal tissue may allow the interaction between mesenchymal and epithelial cells.
The primary cell culture is produced from tissue biopsy material. The term “tissue” refers to a group or layer of similarly specialised cells, which together perform certain special functions. Accordingly, the term “tissue biopsy” as used herein refers to a specimen obtained by removing a group or layer of similarly specialised cells from animals for use in primary cell culture. The term includes aspiration biopsies; brush biopsies; chorionic villus biopsies; endoscopic biopsies; excision biopsies; needle biopsies (specimens obtained by removal by aspiration through an appropriate needle or trocar that pierces the skin, or the external surface of an organ, and into the underlying tissue to be examined); open biopsies; punch biopsies (trephine); shave biopsies; sponge biopsies; and wedge biopsies.
The tissue biopsy may be taken from any animal, for which the study of tissue-specific progenitor cells or stem cell-like cells is required. Suitable mammalian animals include members of the Orders Primates, Rodentia, Lagomorpha, Cetacea, Carnivora, Perissodactyla and Artiodactyla. Members of the Orders Perissodactyla and Artiodactyla are particularly preferred because of their similar biology and economic importance.
For example, Artiodactyla comprise approximately 150 living species distributed through nine families: pigs (Suidae), peccaries (Tayassuidae), hippopotamuses (Hippopotamidae), camels (Camelidae), chevrotains (Tragulidae), giraffes and okapi (Giraffidae), deer (Cervidae), pronghorri (Antilocapridae), and cattle, sheep, goats and antelope (Bovidae). Many of these animals are used as feed animals in various countries. More importantly, with respect to the present invention, many of the economically important animals such as goats, sheep, cattle and pigs have very similar biology and share high degrees of genomic homology.
The Order Perissodactyla comprises horses and donkeys, which are both economically important and closely related. Indeed, it is well known that horses and donkeys interbreed.
In one embodiment, the tissue biopsies will be obtained from ungulates, and in particular, bovids, ovids, cervids, suids, equids and camelids. Examples of such representatives are cows or bulls, bison, buffalo, sheep, big-horn sheep, horses, ponies, donkeys, mule, deer, elk, caribou, goat, water buffalo, camels, llama, alpaca, and pigs. Especially preferred bovine species are Bos taurus, Bos indicus, and Bos buffaloes cows or bulls.
In another embodiment, the tissue biopsies will be obtained from primates, especially humans.
The general purpose of the primary cell culture is to “isolate,” “proliferate” or “selectively expand” tissue-specific progenitor cells or stem cell-like cells present in a tissue biopsy. The terms “isolate,” “proliferate” or “selectively expand” as used herein refers to the culturing process by which the tissue-specific progenitor cells or stem cell-like cells are increased in number relative to the other cells present in the tissue biopsy.
The term “progenitor cell” is used synonymously with “stem cell”. Both terms refer to an undifferentiated cell which is capable of proliferation and giving rise to more progenitor cells having the ability to generate a large number of mother cells that can in turn give rise to differentiated, or differentiable daughter cells. In a preferred embodiment, the term progenitor or stem cell refers to mesenchymal connective tissue derived stem cell-like cells (MCTs). The characteristics of MCTs are reminiscent of pluripotent stem cells. The MCTs are characterised by loss of contact inhibition, anchorage independent growth, de novo expression of alkaline phosphatase and activation of the germ line specific Oct4 promoter. The proliferative potential of these cells is significantly increased compared to primary fibroblasts.
In one embodiment the MCT is the MCT deposited under the Budapest Treaty at the Deutsche Sammlung Von Mikroorganismen und Zellkulturen GmbH (DSMZ), Germany on September 2004, under accession number #12345.
After the tissue biopsy has been obtained, the initial step in the isolation, proliferation or selective expansion of the tissue-specific progenitor cells, stem cell-like cell or MCT present in a tissue biopsy involves the culturing of the tissue biopsy. The terms “culture,” “cultured” and “culturing” are used herein interchangeably, to refer to the process by which the tissue biopsy is grown in vitro.
The tissue biopsy is preferably subjected to physical and/or chemical dissociating means capable of dissociating cellular stratum in the tissue sample. Methods for dissociating cellular layers within the tissues are well known in the field. For example, the dissociating means may be either a physical or a chemical disruption means. Physical dissociation means might include, for example, scraping the tissue biopsy with a scalpel, mincing the tissue, physically cutting the layers apart, or perfusing the tissue with enzymes. Chemical dissociation means might include, for example, digestion with enzymes such as trypsin, dispase, collagenase, trypsin-EDTA, thermolysin, pronase, hyaluronidase, elastase, papain and pancreatin. Non-enzymatic solutions for the dissociation of tissue can also be used.
In one embodiment, dissociation of the tissue biopsy is achieved by placing the tissue biopsy in a pre-warmed enzyme solution containing an amount of trypsin sufficient to dissociate the cellular stratum in the tissue biopsy. Preferably, the enzyme solution used in the method is calcium and magnesium free.
Where the tissue biopsy is derived from an animals skin (comprising epithelial and dermal cells) the amount of trypsin that might be used in the method is preferably between about 5 and 0.1% trypsin per volume of solution. Desirable the trypsin concentration of the solution is about 2.5 to 0.25%, with about 0.5% trypsin being most preferred.
The time period over which the tissue biopsy is subjected to the trypsin solution may vary depending on the size of the tissue biopsy taken. Preferably the tissue biopsy is placed in the presence of the trypsin solution for sufficient time to weaken the cohesive bonding between the tissue stratum. For example, where the tissue sample is taken from an animal's skin the tissue biopsy might be placed in trypsin for between 5 to 60 minutes. In one embodiment, the tissue biopsy is immersed in the trypsin solution for between 10 and 30 minutes with 15 to 20 minutes being optimal for most tissue biopsies.
After the tissue biopsy has been immersed in the trypsin solution for an appropriate amount of time, the dissociated cells are removed and suspended in tissue culture medium. The terms “culture media,” “tissue culture media” or “tissue culture medium” are recognised in the art, and refers generally to any substance or preparation used for the cultivation of living cells. There are a large number of tissue culture media that exist for culturing tissue from animals. Some of these are complex and some are simple. Examples of media that would be useful in the present invention include Modified Eagle's Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), RPMI 1640, F-12, IMDM, Alpha Medium and McCoy's Medium. Most preferably, the culture medium is DMEM.
In one embodiment, enzymatically dissociated and eviscerated fetuses or mesenchymal explant (<1 mm3) cultures of connective tissue are suspended in DMEM supplemented with 1 mM glutamine, 1% non-essential amino acids, 1% vitamin solution, 0.1 mM mercaptoethanol, 100 U/ml penicillin, and 100 mg/ml streptomycin (all from Sigma, Deisenhofen, Germany).
In order to encourage the tissue-specific progenitor cells or stem cell-like cells to proliferate, serum is added to the tissue culture medium. The serum in the culture medium may be allogeneic serum (ie., from the same animal species, but not the same animal), autologous serum (ie., from the same animal) or xenogeneic serum (ie., from a different animal species). In one embodiment, heat-inactivated autologous serum is used.
When the dissociated tissue biopsy is initially cultured the amount of serum used is typically about 10%. The term “about” as used herein to describe the amount of serum used in the culture medium indicates that in certain circumstances the amount of serum used will be slightly more (approximately 10% more) or slightly less (approximately 10% less), than the stated amount. For example, about 10% serum would mean that as little as 9% serum might be used or up to a maximum of 11% serum. About 30% serum would mean that as little as 27% serum might be used serum (i.e. within 10% of the stated volume) or as much as 33% serum (i.e. within 10% of the stated volume).
The dissociated tissue biopsy cells, including the tissue-specific progenitor cells or stem cell-like cells are incubated in a humidified 95% air/5% C02 atmosphere at 37° C.
After the second passage of the cells after setting up the culture, the serum concentration is adjusted to about 30%. The precise timing of this stage is difficult to predict as this will vary depending upon the type of tissue used and the age of the material. For example, fetal tissue is typically faster growing than adult tissue. The presence of the increased serum concentration enables the tissue-specific progenitor cells or stem cell-like cells to proliferate, while the other cells present such as keratinocytes, basal cells, Langerhans cells, fibroblasts and melanocytes, have depressed growth. Approximately, every 48 hours or so, 50% of the culture medium is preferably replaced with fresh medium.
As the tissue-specific progenitor cells or stem cell-like cells proliferate they generally take on a 3D appearance. Once the 3D-colonies reach approximately 200-300 μm in diameter they are isolated and trypsinised to obtain single cells suspensions. Subsequently, 104 cells are seeded into bacteriological culture dishes to prevent attachment. Supplementation of the culture medium (DMEM/30% FCS) with dexamethasone results in aggregations of small multicellular spheroids usually within 24 hours, which continue to grow up to a diameter of >400 μm after 10-15 days.
The maximal replicative limit can be determined by serially subpassaging the cells as 12.5×103 cell aliquots seeded per cm2 in 6-well-dishes, trysinised after 5-7 days, counted and reseeded.
In one embodiment, the tissue-specific progenitor cells or stem cell-like cells are mesenchymal connective tissue derived stem cell-like cells (MCTs). The MCTs show several characteristics not found in fibroblasts, e.g. they have a significantly extended proliferative capacity of >100 cell doublings in vitro. This allows an extended amplification of clonal cell strains or mass cultures and could simplify genetic modifications and potentially enables two rounds of genetic modifications and selection. Also enough cells for grafting procedures can be obtained, as MCTs might be suitable for directed differentiation into several cell types.
Once the tissue-specific progenitor cells, stem cell-like cells or MCTs have been isolated or proliferated they can then be used, for example, for direct transplantation or to produce differentiated cells in vitro for transplantation or in nuclear transfer techniques. The invention accordingly provides, for example, stem cells that may serve as a source for many other, more differentiated cell types.
One embodiment pertains to the progeny of the tissue-specific progenitor cells, stem cell-like cells or MCTs, e.g. those cells which have been derived from the cells of the initial tissue biopsy. Such progeny can include subsequent generations of tissue-specific progenitor cells, stem cell-like cells or MCTs, as well as lineage committed cells generated by inducing differentiation of the tissue-specific progenitor cells, stem cell-like cells or MCTs after their isolation from the tissue biopsy, e.g., induced in vitro.
Another embodiment relates to cellular compositions enriched for tissue-specific progenitor cells, stem cell-like cells or MCTs, or the progeny thereof. In certain embodiments, the cells will be provided as part of a pharmaceutical preparation, e.g., a sterile, free of the presence of unwanted virus, bacteria and other pathogens, as well as pyrogen-free preparation. That is, for animal administration, the tissue-specific progenitor cells, stem cell-like cells or MCTs should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.
In certain embodiments, such cellular compositions can be used for transplantation into animals, preferably mammals, and even more preferably humans. The tissue-specific progenitor cells, stem cell-like cells or MCTs can be autologous, allogeneic or xenogeneic with respect to the transplantation host.
Yet another aspect of the present invention concerns cellular compositions, which include as a cellular component, substantially pure preparations of the tissue-specific progenitor cells, stem cell-like cells or MCTs, or the progeny thereof. Cellular compositions of the present invention include not only substantially pure populations of the tissue-specific progenitor cells, stem cell-like cells or MCTs, but can also include cell culture components, e.g., culture media including amino acids, metals, coenzyme factors, as well as small populations of non-tissue-specific progenitor cells, stem cell-like cells or MCTs cells, e.g., some of which may arise by subsequent differentiation of isolated tissue-specific progenitor cells, stem cell-like cells or MCTs of the invention. Furthermore, other non-cellular components include those which render the cellular component suitable for support under particular circumstances, eg., implantation, eg., continuous culture.
As common methods of administering the tissue-specific progenitor cells, stem cell-like cells or MCTs of the present invention to animals, particularly humans, which are described in detail herein, include injection or implantation of the tissue-specific progenitor cells, stem cell-like cells or MCTs into target sites in the animals, the cells of the invention can be inserted into a delivery device which facilitates introduction by, injection or implantation, of the cells into the animals. Such delivery devices include tubes, eg., catheters, for injecting cells and fluids into the body of a recipient animal. In a preferred embodiment, the tubes additionally have a needle, eg., a syringe, through which the cells of the invention can be introduced into the animal at a desired location. The tissue-specific progenitor cells, stem cell-like cells or MCTs of the invention can be inserted into such a delivery device, eg., a syringe, in different forms. For example, the cells can be suspended in a solution or embedded in a support matrix when contained in such a delivery device. As used herein, the term “solution” includes a pharmaceutically acceptable carrier or diluent in which the cells of the invention remain viable. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. The solution is preferably sterile and fluid to the extent that easy syringability exists. Preferably, the solution is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Solutions of the invention can be prepared by incorporating tissue-specific progenitor cells, stem cell-like cells or MCTs as described herein in a pharmaceutically acceptable carrier or diluent and, as required, other ingredients enumerated above, followed by filtered sterilisation.
Support matrices in which the tissue-specific progenitor cells, stem cell-like cells or MCTS can be incorporated or embedded include matrices which are recipient-compatible and which degrade into products which are not harmful to the recipient. Natural and/or synthetic biodegradable matrices are examples of such matrices. Natural biodegradable matrices include plasma clots, eg., derived from a mammal, and collagen matrices. Synthetic biodegradable matrices include synthetic polymers such as polyanhydrides, polyorthoesters, and polylactic acid. Other examples of synthetic polymers and methods of incorporating or embedding cells into these matrices are known in the art. See eg., U.S. Pat. Nos. 4,298,002 and 5,308,701. These matrices provide support and protection for the fragile progenitor cells in vivo and are, therefore, the preferred form in which the tissue-specific progenitor cells, stem cell-like cells or MCTs are introduced into the recipient animals.
The present invention also provides substantially pure tissue-specific progenitor cells, stem cell-like cells or MCTs cells which can be used therapeutically for treatment of various disorders.
To illustrate, the tissue-specific progenitor cells, stem cell-like cells or MCTs of the invention can be used in the treatment or prophylaxis of a variety of disorders. For instance, the tissue-specific progenitor cells, stem cell-like cells or MCTs can be used to produce populations of differentiated cells for repair of damaged tissue eg pancreatic tissue, cardiac tissue, nerves and the like. Likewise, such cell populations can be used to regenerate or replace pancreatic tissue, cardiac tissue or nerves lost due to, pancreatolysis, eg., destruction of pancreatic tissue, such as pancreatitis, heart disease or neuropathy.
Yet another embodiment provides methods for screening various compounds for their ability to modulate growth, proliferation or differentiation of tissue-specific progenitor cells, stem cell-like cells or MCTs. In an illustrative embodiment, the subject tissue-specific progenitor cells, stem cell-like cells or MCTs, and their progeny, can be used to screen various compounds or natural products. Such explants can be maintained in minimal culture media for extended periods of time (eg., for 7-21 days or longer) and can be contacted with any compound, eg., small molecule or natural product, eg., growth factor, to determine the effect of such compound on one of cellular growth, proliferation or differentiation of the tissue-specific progenitor cells, stem cell-like cells or MCTs. Detection and quantification of growth, proliferation or differentiation of these cells in response to a given compound provides a means for determining the compound's efficacy at inducing one of the growth, proliferation or differentiation. Methods of measuring cell proliferation are well known in the art and most commonly include determining DNA synthesis characteristic of cell replication. There are numerous methods in the art for measuring DNA synthesis, any of which may be used according to the invention. In an embodiment of the invention, DNA synthesis has been determined using a radioactive label (3H-thymidine) or labelled nucleotide analogues (BrdU) for detection by immunofluorescence. The efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the compound. A control assay can also be performed to provide a baseline for comparison. Identification of the progenitor cell population(s) amplified in response to a given test agent can be carried out according to such phenotyping as described above.
In one embodiment, the tissue-specific progenitor cells, stem cell-like cells or MCTs are used for cloning mammals by nuclear transfer or nuclear transplantation. In the subject application, the terms “nuclear transfer” or “nuclear transplantation” are used interchangeably; however, these terms as used herein refers to introducing a full complement of nuclear DNA from one cell to an enucleated cell.
The first step in the preferred methods involves the isolation of a recipient oocyte from a suitable animal. In this regard, the oocyte may be obtained from any animal source and at any stage of maturation. Methods for isolation of oocytes are well known in the art. For example, oocytes can be isolated from either oviducts and/or ovaries of live animals by oviductal recovery procedures or transvaginal oocyte recovery procedures well known in the art. See, eg., Pieterse et al., 1988, “Aspiration of bovine oocytes during transvaginal ultrasound scanning of the ovaries,” Theriogenology 30: 751-762. Furthermore, oocytes can be isolated from ovaries or oviducts of deceased animals. For example, ovaries can be obtained from abattoirs and the oocytes aspirated from these ovaries. The oocytes can also be isolated from the ovaries of a recently sacrificed animal or when the ovary has been frozen and/or thawed.
Briefly, in one preferred embodiment, immature (prophase I) oocytes from mammalian ovaries are harvested by aspiration. For the successful use of techniques such as genetic engineering, nuclear transfer and cloning, once these oocytes have been harvested they must generally be matured in vitro before these cells may be used as recipient cells for nuclear transfer.
The stage of maturation of the oocyte at enucleation and nuclear transfer has been reported to be significant to the success of nuclear transfer methods. (See eg., Prather et al., Differentiation, 48, 1-8, 1991). In general, successful mammalian embryo cloning practices use the metaphase II stage oocyte as the recipient oocyte because at this stage it is believed that the oocyte can be or is sufficiently activated to treat the introduced nucleus as it does a fertilising sperm.
The in vitro maturation of oocytes usually takes place in a maturation medium until the oocyte has extruded the first polar body, or until the oocyte has attained the metaphase II stage. In domestic animals, and especially cattle, the oocyte maturation period generally ranges from about 16-52 hours, preferably about 28-42 hours and more preferably about 18-24 hours post-aspiration. For purposes of the present invention, this period of time is known as the “maturation period.”
Oocytes can be matured in a variety ways and using a variety of media well known to a person of ordinary skill in the art. See, eg., U.S. Pat. No. 5,057,420; Saito et al., 1992, Roux's Arch. Dev. Biol. 201: 134-141 for bovine organisms and Wells et al., 1997, Biol. Repr. 57: 385-393 for ovine organisms and WO97/07668, entitled “Unactivated Oocytes as Cytoplast Recipients for Nuclear Transfer,” all hereby incorporated herein by reference in the entirety, including all figures, tables, and drawings.
One of the most common media used for the collection and maturation of oocytes is TCM-199, and 1 to 20% serum supplement including fetal calf serum (FCS), newborn serum, estrual cow serum, lamb serum or steer serum. Example 1 shows one example of a preferred maintenance medium: TCM-199 with Earl salts supplemented with 15% cow serum and including 10IU/ml pregnant mare serum gonadotropin and 5IU/ml human chorionic gonadotropin (SuigonR Vet, Intervet, Australia). Oocytes can be successfully matured in this type of medium within an environment comprising 5% CO2 at 39° C.
While it will be appreciated by those skilled in the art that freshly isolated and matured oocytes are preferred, it will also be appreciated that it is possible to cryopreserve the oocytes after harvesting or after maturation. Accordingly, the term “cryopreserving” as used herein can refer to freezing an oocyte, a cell, embryo, or animal of the invention. The oocytes, cells, embryos, or portions of animals of the invention are frozen at temperatures preferably lower than 0° C., more preferably lower than −80° C., and most preferably at temperatures lower than −196° C. Oocytes, cells and embryos in the invention can be cryopreserved for an indefinite amount of time. It is known that biological materials can be cryopreserved for more than fifty years. For example, semen that is cryopreserved for more than fifty years can be utilised to artificially inseminate a female bovine animal. Methods and tools for cryopreservation are well known to those skilled in the art. See, eg., U.S. Pat. No. 5,160,312, entitled “Cryopreservation Process for Direct Transfer of Embryos”.
If cyropreserved oocytes are utilised then these must be initially thawed before placing the oocytes in maturation medium. Methods of thawing cryopreserved materials such that they are active after the thawing process are well-known to those of ordinary skill in the art.
In a further preferred embodiment, mature (metaphase II) oocytes, which have been matured in vivo, are harvested and used in the nuclear transfer methods disclosed herein. Essentially, mature metaphase II oocytes are collected surgically from either non-superovulated or superovulated cows or heifers 35 to 48 hours past the onset of estrus or past the injection of human chorionic gonadotropin (hCG) or similar hormone.
Where oocytes have been cultured in vitro cumulus cells that may have accumulated may be removed to provide oocytes that are at a more suitable stage of maturation for enucleation. Cumulus cells may be removed by pipetting or vortexing, for example, in the presence of 0.5% hyaluronidase.
After the maturation period as described above the zona pellucida may be removed from the oocytes if desired. The advantages of zona pellucida removal are described in PCT/AU02/00491, which is incorporated in its entirety herein by reference. The removal of the zona pellucida from the oocyte may be carried out by any method known in the art including physical manipulation (mechanical opening), chemical treatment or enzymatic digestion (Wells and Powell, 2000). Physical manipulation may involve the use of a micropipette or a microsurgical blade. Preferably, enzymatic digestion is used.
In one particularly preferred embodiment, the zona pellucida is removed by enzymatic digestion in the presence of a protease or pronase. Briefly, mature oocytes are placed into a solution comprising a protease, pronase or combination of each at a total concentration in the range of 0.1% - 5%, more preferably 0.25% -2% and most preferably about 0.5%. The mature oocyte is then allowed to incubate at between 30° C. to about 45° C., preferably about 39° C. for a period of 1 to 30 minutes. Preferably the oocytes are exposed to the enzyme for about 5 minutes. Although pronase may be harmful to the membranes of oocytes, this effect may be minimised by addition of serum such as FCS or cow serum. The unique advantage of zona removal with pronase is that no individual treatment is required, and the procedure can be performed in quantities of 100's of oocytes. Once the zona pellucida has been removed the zona pellucida-free mature oocyte are rinsed in 4 ml Hepes buffered TCM-199 medium supplemented with 20% FCS and 10 μg/ml cytochalasin B and then enucleated.
The terms “enucleation”, “enucleated” and “enucleated oocyte” are used interchangeably herein and refers to an oocyte which has had part of its contents removed.
Enucleation of the oocyte may be achieved physically, by actual removal of the nucleus, pronuclei or metaphase plate (depending on the oocyte), or functionally, such as by the application of ultraviolet radiation or another enucleating influence. All of these methods are well known to those of ordinary skill in the art. For example, physical means includes aspiration (Smith & Wilmut, Biol. Reprod., 40: 1027-1035 (1989)); functional means include use of DNA-specific fluorochromes (See, for example, Tsunoda et al., J. Reprod. Fertil. 82: 173 (1988)), and irradiation with ultraviolet light (See, for example, Gurdon, Q. J. Microsc. Soc., 101: 299-311 (1960)). Enucleation may also be effected by other methods known in the art. See, for example, U.S. Pat. No. 4,994,384; U.S. Pat. No. 5,057,420; and Willadsen, 1986, Nature 320:63-65, herein incorporated by reference.
Preferably, the oocyte is enucleated by means of manual bisection. Oocyte bisection may be carried out by any method known to those skilled in the art. In one preferred embodiment, the bisection is carried out using a microsurgical blade as described in International Patent Application No. WO98/29532 which is incorporated by reference herein. Briefly, oocytes are split asymmetrically into fragments representing approximately 30% and 70% of the total oocyte volume using an ultra sharp splitting blade (AB Technology, Pullman, W A, USA). The oocytes may then be screened to identify those of which have been successfully enucleated. This screening may be effected by staining the oocytes with 1 microgram per millilitre of the Hoechst fluorochrome 33342 dissolved in TCM-199 media supplemented with 20% FCS, and then viewing the oocytes under ultraviolet irradiation with an inverted microscope for less than 10 seconds. The oocytes that have been successfully enucleated (demi-oocytes) can then be placed in a suitable culture medium, eg., TCM-199 media supplemented with 20% FCS.
In the present invention, the recipient oocytes will preferably be enucleated at a time ranging from about 10 hours to about 40 hours after the initiation of in vitro maturation, more preferably from about 16 hours to about 24 hours after initiation of in vitro maturation, and most preferably about 16-18 hours after initiation of in vitro maturation.
The bisection technique described herein requires much less time and skill than other methods of enucleation and the subsequent selection by staining results in high accuracy. Consequently, for large-scale application of cloning technology the present bisection technique can be more efficient than other techniques.
A single tissue-specific progenitor cell, stem cell-like cell or MCTs of the present invention of the same species as the enucleated oocyte can then be transferred by fusion into the enucleated oocyte thereby producing a reconstituted cell.
Analysis of cell cycle stage may be performed as described in Kubota et al., PNAS 97: 990-995 (2000). Briefly, cell cultures at different passages are grown to confluency. After trypsinisation, cells are washed with TCM-199 plus 10% FCS and re-suspended to a concentration of 5×105 cells/ml in 1 ml PBS with glucose (6.1 mM) at 4° C. Cells are fixed overnight by adding 3 ml of ice-cold ethanol. For nuclear staining, cells are then pelleted, washed with PBS and re-suspended in PBS containing 30 μg/ml propidium iodide and 0.3 mg/ml RNase A. Cells are allowed to incubate for 1 h at room temperature in the dark before filtered through a 30 μm mesh. Cells are then analyzed.
To examine the ploidy of the tissue-specific progenitor cells, stem cell-like cells or MCTs at various passages, chromosome counts may be determined at different passages of culture using standard preparation of metaphase spreads (See, for example, Kubota et al., PNAS 97: 990-995 (2000)).
Cultured tissue-specific progenitor cells, stem cell-like cells or MCTs may also be genetically altered by transgenic methods well-known to those of ordinary skill in the art. See, for example, Molecular cloning a Laboratory Manual, 2nd Ed., 1989, Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory Press; U.S. Pat. No. 5,612,205; U.S. Pat. No. 5,633,067; EPO 264 166, entitled “Transgenic Animals Secreting Desired Proteins Into Milk”; WO94/19935, entitled “Isolation of Components of Interest From Milk”; WO93/22432, entitled “Method for Identifying Transgenic Pre-implantation Embryos”; and WO95/175085, entitled “Transgenic Production of Antibodies in Milk,” all of which are incorporated by reference herein in their entirety including all figures, drawings and tables. Any known method for inserting, deleting or modifying a desired gene from a mammalian cell may be used for altering the tissue-specific progenitor cells, stem cell-like cells or MCTs to be used as the nuclear donor. These procedures may remove all or part of a gene, and the gene may be heterologous. Included is the technique of homologous recombination, which allows the insertion, deletion or modification of a gene or genes at a specific site or sites in the cell genome.
Examples for modifying a target DNA genome by deletion, insertion, and/or mutation are retroviral insertion, artificial chromosome techniques, gene insertion, random insertion with tissue specific promoters, gene targeting, transposable elements and/or any other method for introducing foreign DNA or producing modified DNA/modified nuclear DNA. Other modification techniques include deleting DNA sequences from a genome and/or altering nuclear DNA sequences. Nuclear DNA sequences, for example, may be altered by site-directed mutagenesis.
The present invention can thus be used to provide adult mammals with desired genotypes. Multiplication of adult ungulates with proven genetic superiority or other desirable traits is particularly useful, including transgenic or genetically engineered animals, and chimeric animals. Furthermore, cell and tissues from the nuclear transfer fetus, including transgenic and/or chimeric fetuses, can be used in cell, tissue and organ transplantation.
Methods for generating transgenic cells typically include the steps of (1) assembling a suitable DNA construct useful for inserting a specific DNA sequence into the nuclear genome of tissue-specific progenitor cells, stem cell-like cells or MCTs; (2) transfecting the DNA construct into the tissue-specific progenitor cells, stem cell-like cells or MCTs; (3) allowing random insertion and/or homologous recombination to occur. The modification resulting from this process may be the insertion of a suitable DNA construct(s) into the target genome; deletion of DNA from the target genome; and/or mutation of the target genome.
DNA constructs can comprise a gene of interest as well as a variety of elements including regulatory promoters, insulators, enhancers, and repressors as well as elements for ribosomal binding to the RNA transcribed from the DNA construct.
DNA constructs can also encode ribozymes and anti-sense DNA and/or PNA, identified previously herein. These examples are well known to a person of ordinary skill in the art and are not meant to be limiting.
Due to the effective recombinant DNA techniques available in conjunction with DNA sequences for regulatory elements and genes readily available in data bases and the commercial sector, a person of ordinary skill in the art can readily generate a DNA construct appropriate for establishing transgenic cells using the materials and methods described herein.
Transfection techniques are well known to a person of ordinary skill in the art and materials and methods for carrying out transfection of DNA constructs into cells are commercially available. Materials typically used to transfect cells with DNA constructs are lipophilic compounds, such as Lipofectin™ for example. Particular lipophilic compounds can be induced to form liposomes for mediating transfection of the DNA construct into the cells.
Target sequences from the DNA construct can be inserted into specific regions of the nuclear genome by rational design of the DNA construct. These design techniques and methods are well known to a person of ordinary skill in the art. See, for example, U.S. Pat. No. 5,633,067; U.S. Pat. No. 5,612,205 and PCT publication WO93/22432, all of which are incorporated by reference herein in their entirety. Once the desired DNA sequence is inserted into the nuclear genome, the location of the insertion region as well as the frequency with which the desired DNA sequence has inserted into the nuclear genome can be identified by methods well known to those skilled in the art.
Once the transgene is inserted into the nuclear genome of the donor tissue-specific progenitor cells, stem cell-like cells or MCTs, that cell, like other donor tissue-specific progenitor cells, stem cell-like cells or MCTs of the invention, can be used as a nuclear donor in nuclear transfer methods. The means of transferring the nucleus of a tissue-specific progenitor cells, stem cell-like cells or MCTs into the enucleated oocyte preferably involves cell fusion to form a reconstituted cell.
Fusion is typically induced by application of a DC electrical pulse across the contact/fusion plane, but additional AC current may be used to assist alignment of donor and recipient cells. Electrofusion produces a pulse of electricity that is sufficient to cause a transient breakdown of the plasma membrane and which is short enough that the membrane reforms rapidly. Thus, if two adjacent membranes are induced to breakdown and upon reformation the lipid bilayers intermingle, small channels will open between the two cells. Due to the thermodynamic instability of such a small opening, it enlarges until the two cells become one. Reference is made to U.S. Pat. No. 4,997,384 by Prather et al., (incorporated by reference in its entirety herein) for a further discussion of this process. A variety of electrofusion media can be used including eg., sucrose, mannitol, sorbitol and phosphate buffered solution.
Fusion can also be accomplished using Sendai virus as a fusogenic agent (Graham, Wister Inot. Symp. Monogr., 9, 19, 1969). Fusion may also be induced by exposure of the cells to fusion-promoting chemicals, such as polyethylene glycol.
Preferably, the donor tissue-specific progenitor cells, stem cell-like cells or MCTs and enucleated oocyte are placed in a 500 μm fusion chamber and covered with 4 ml of 26° C.-27° C. fusion medium (0.3M mannitol, 0.1 mM MgSO4, 0.05 mM CaCl2). The cells are then electrofused by application of a double direct current (DC) electrical pulse of 70-100V for about 15 μs, approximately 1 s apart. After fusion, the resultant fused reconstituted cells are then placed in a suitable medium until activation, eg., TCM-199 medium.
In a preferred method of cell fusion the donor tissue-specific progenitor cell, stem cell-like cell or MCT is firstly attached to the enucleated oocyte. For example, a compound is selected to attach the progenitor cell, stem cell-like cell or MCT to the enucleated oocyte to enable fusing of the donor cell and enucleated oocyte membranes. The compound may be any compound capable of agglutinating cells. The compound may be a protein or glycoprotein capable of binding or agglutinating carbohydrate. More preferably the compound is a lectin. The lectin may be selected from the group including Concanavalin A, Canavalin A, Ricin, soybean lectin, lotus seed lectin and phytohemaglutinin (PHA). Preferably the compound is PHA.
In one preferred embodiment, the method of electrofusion described above also comprises a further fusion step, or the fusion step comprises described above comprises one donor progenitor cell, stem cell-like cell or MCT and two or more enucleated oocytes. The double fusion method has the advantageous effect of increasing the cytoplasmic volume of the reconstituted cell.
A reconstituted cell is typically activated by electrical and/or non-electrical means before, during, and/or after fusion of the nuclear donor and recipient oocyte (See, for example, Susko-Parrish et al., U.S. Pat. No. 5,496,720). Activation methods include:
The activated reconstituted cells, or embryos, are typically cultured in medium well known to those of ordinary skill in the art, and include, without limitation, TCM-199 plus 10% FSC, Tyrodes-Albumin-Lactate-Pyruvate (TALP), Ham's F-10 plus 10% FCS, synthetic oviductal fluid (“SOF”), B2, CR1aa, medium and high potassium simplex medium (“KSOM”).
The reconstituted cell may also be activated by known methods. Such methods include, eg., culturing the reconstituted cell at sub-physiological temperature, in essence by applying a cold, or actually cool temperature shock to the reconstituted cell. This may be most conveniently done by culturing the reconstituted cell at room temperature, which is cold relative to the physiological temperature conditions to which embryos are normally exposed. Suitable oocyte activation methods are the subject of U.S. Pat. No. 5,496,720, to Susko-Parrish et al., herein incorporated by reference in its entirety.
The activated reconstituted cells may then be cultured in a suitable in vitro culture medium until the generation of cells and cell colonies. Culture media suitable for culturing and maturation of embryos are well known in the art. Examples of known media, which may be used for bovine embryo culture and maintenance, include Ham's F-10 plus 10% FCS, TCM-199 plus 10% FCS, Tyrodes-Albumin-Lactate-Pyruvate (TALP), Dulbecco's Phosphate Buffered Saline (PBS), Eagle's and Whitten's media. One of the most common media used for the collection and maturation of oocytes is TCM-199, and 1 to 20% serum supplement including fetal calf serum, newborn serum, estrual cow serum, lamb serum or steer serum. A preferred maintenance medium includes TCM-199 with Earl salts, 10% FSC, 0.2 mM Na pyruvate and 50 μg/ml gentamicin sulphate. Any of the above may also involve co-culture with a variety of cell types such as granulosa cells, oviduct cells, BRL cells and uterine cells and STO cells.
Afterward, the cultured reconstituted cell or embryos are preferably washed and then placed in a suitable media, eg., TCM-199 medium containing 10% FCS contained in well plates which preferably contain a suitable confluent feeder layer. Suitable feeder layers include, by way of example, fibroblasts and epithelial cells, e.g., fibroblasts and uterine epithelial cells derived from ungulates, chicken fibroblasts, murine (e.g., mouse or rat) fibroblasts, STO and SI-m220 feeder cell lines, and BRL cells.
In one embodiment, the feeder cells comprise mouse embryonic fibroblasts. Preparation of a suitable fibroblast feeder layers are well known in the art.
The reconstituted cells are cultured on the feeder layer until the reconstituted cells reach a size suitable for transferring to a recipient female, or for obtaining cells which may be used to produce cells or cell colonies. Preferably, these reconstituted cells will be cultured until at least about 2 to 400 cells, more preferably about 4 to 128 cells, and most preferably at least about 50 cells. The culturing will be effected under suitable conditions, i.e., about 39° C. and 5% CO2, with the culture medium changed in order to optimise growth typically about every 2-5 days, preferably about every 3 days.
The methods for embryo transfer and recipient animal management in the present invention are standard procedures used in the embryo transfer industry. Synchronous transfers are important for success of the present invention, i.e., the stage of the nuclear transfer embryo is in synchrony with the estrus cycle of the recipient female. This advantage and how to maintain recipients are reviewed in Siedel, G. E., Jr. (“Critical review of embryo transfer procedures with cattle” in Fertilization and Embryonic Development in Vitro (1981) L. Mastroianni, Jr. and J. D. Biggers, ed., Plenum Press, New York, N.Y., page 323), the contents of which are hereby incorporated by reference.
Briefly, blastocysts may be transferred non-surgically or surgically into the uterus of a synchronized recipient. Other medium may also be employed using techniques and media well-known to those of ordinary skill in the art. In one procedure, cloned embryos are washed three times with fresh KSOM and cultured in KSOM with 0.1% BSA for 4 days and subsequently with 1% BSA for an additional 3 days, under 5% CO2, 5% O2 and 90% N2 at 39° C. Embryo development is examined and graded by standard procedures known in the art. Cleavage rates are recorded on day 2 and cleaved embryos are cultured further for 7 days. On day seven, blastocyst development is recorded and one or two embryos, pending availability of embryos and/or animals, is transferred non-surgically into the uterus of each synchronized foster mother.
Foster mothers preferably are examined for pregnancy by rectal palpation or ultrasonography periodically, such as on days 40, 60, 90 and 120 of gestation. Careful observations and continuous ultrasound monitoring (monthly) preferably is made throughout pregnancy to evaluate embryonic loss at various stages of gestation. Any aborted fetuses should be harvested, if possible, for DNA typing to confirm clone status as well as routine pathological examinations.
The reconstituted cell, activated reconstituted cell, fetus and animal produced during the steps of such method, and cells, nuclei, and other cellular components which may be harvested therefrom, are also asserted as embodiments of the present invention. It is particularly preferred that the term animal produced be a viable animal.
The present invention can also be used to produce embryos, fetuses or offspring which can be used, for example, in cell, tissue and organ transplantation. By taking a fetal or adult cell from an animal and using it in the cloning procedure a variety of cells, tissues and possibly organs can be obtained from cloned fetuses as they develop through organogenesis. Cells, tissues, and organs can be isolated from cloned offspring as well. This process can provide a source of materials for many medical and veterinary therapies including cell and gene therapy. If the cells are transferred back into the animal in which the cells were derived, then immunological rejection is averted. Also, because many cell types can be isolated from these clones, other methodologies such as hematopoietic chimerism can be used to avoid immunological rejection among animals of the. same species.
Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises”, is not intended to excluded other additives, components, integers or steps.
The invention will now be further described by way of reference only to the following non-limiting examples. It should be understood, however, that the examples following are illustrative only, and should not be taken in any way as a restriction on the generality of the invention described above. In particular, while the invention is described in detail in relation to the use of mouse and porcine cells, it will be clearly understood that the findings herein are not limited to these-types of cells, but would be useful growing any type of cell from any animal.
OG2-transgenic mice carrying the GFP reporter gene under transcriptional control of the exclusively germline-specific Oct-4 promoter, were employed for fetal explant cultures. Mesenchymal explants with an average size of <1 mm3 were isolated from fetuses of days 11.5, 13.5 and 14.5 p.c., pasted with recalcified microdrops of bovine plasma to cell culture dishes and cultured individually in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 2 mM glutamine and 10% FCS as described infra. Specific care was taken to isolate explants from connective tissue of the neck and shoulder regions. Fluorescence microscopy using Zeiss Axiomat LSM and excitation wavelength of 488 nm was used to detect GFP. No GFP positive-cells were revealed in the initial explants and GFP expression could not be detected in the first outgrowths after 2 days (
The expression of the endogenous Oct4 gene was confirmed by RT-PCR detection of the corresponding mRNA in subpassages of the mesenchymal outgrowths (
The isolation of Oct4 expressing cells from murine somatic explants raised the question whether similar cells could be obtained from livestock species. Mesenchymal explants of porcine fetuses (day 25 p.c.) were established and subpassaged once using standard culture protocols yielding morphologically homogenous cell cultures. Immunostaining showed uniform labelling with a vimentin specific and no labelling with a cytokeratin-specific antibody (data not shown), indicative for fibroblasts. RT-PCR with porcine Oct4 specific primers prove that cultures maintained in DMEM/30% FCS activated the germ line specific Oct4 gene (not shown).
Upon change of the culture medium to high serum concentrations, ie. DMEM containing 30% FCS, the cultures did no longer show contact inhibition. After confluency was reached the growth of 3D-colonies became apparent (
Staining. for endogenous alkaline phosphatase (AP) activity revealed a massive induction of AP-positive cells, which were nearly exclusively accumulated within the 3D-colonies (
Adult porcine fibroblasts (3 different origins, 0.5-1.5 y old donors) derived from subdermal tissue explants did not display 3D-colony growth (
Apparently the altered phenotype of porcine fetal cultures was reversible. When high serum cultures were split and one part of the population was returned to standard medium, colony-growth ceased and AP-positive cells disappeared nearly completely within two passages, suggesting that induction and proliferation of MCTs are dependent upon high serum levels in culture (
Culture medium supplemented with high-serum resulted in a dramatically altered growth curve (
To investigate the growth potential of the colony forming fetal cells, 3D-colonies of 200-300 μm diameter were isolated and trypsinised to obtain single cells suspensions. Subsequently, 104 cells were seeded into bacteriological culture dishes to prevent attachment. Supplementation of the culture medium (DMEM/30% FCS) with dexamethasone resulted in aggregation of small multicellular spheroids within 24 hours, which continued to grow up to a diameter of >400 μm after 10-15 days and contained nearly exclusively AP positive cells (
To determine the developmental potential, MCTs were injected into murine blastocysts, which were subsequently transferred to pseudopregnant recipients. MCTs of both sexes were isolated from double transgenic fetuses of OG2 and Rosa26 mouse strains. These cells carried the germline specific Oct-4 GFP and the ubiquitously active lacZ reporter gene constructs and thus allowed to distinguish them from the cells of the recipient blastocysts.
Day 13.5-15.5 fetuses derived from the injected blastocysts were isolated and analyzed for chimerism either by staining for lacZ activity or by fluorescence microscopy to identify GFP positive cells. Of a total of 19 analyzed fetuses, 7 contained progeny cells from the injected MCTs (Table 2). Chimerism was detected in mesenchymal organs, such as liver, muscle and tongue, but also in the genital ridges.
The present invention demonstrates the presence of tissue-specific progenitor cells or stem cell-like cells (MCTS) in fetal mesenchymal tissue cultures of rodents and livestock species that can be specifically enriched by the methods disclosed herein. MCT cells are characterised by extended proliferative capacity, altered morphology, de novo expression of the stem cell markers Oct4, Stat3 and AP, as well as contact- and anchorage-independent growth.
The explant culture technique employing higher than normal serum levels seems to be essential for an initially stimulation of the MCT proliferation. Standard culture using low serum levels of 10% or less are associated with a progressive loss of MCTS.
Transcriptional activity of the Oct4 promoter in MCTs indicates that these cells have characteristics of stem cells. Oct4 controls the expression of several genes including Fgf4, Rex-1, Sox-2, OPN, hCG, Utf-1 and INFt. Variation in the level of Oct-4 expression by as little as 30% has been shown to maintain cells either in the totipotent state or to drive embryonic stem cells into differentiation.
Chimeric fetuses, obtained by injection of murine MCTs into recipient blastocysts, showed that the MCTs were able to contribute to various mesenchymal organs and in particular the genital ridges. Genital ridges showed contribution of MCTs to the primordial germ cells, as some albeit few cells expressed GFP fluorescence driven by the germ line specific Oct-4 promoter, indicating that germ line transmission might be possible. The finding that GFP positive cells were not found outside of the genital ridges indicates that the Oct-4 marker was correctly activated in cells committed to the germ line. It also suggests that at least some of the MCTs descendants were capable to migrate into the genital ridge. The relatively low percentage of chimerism might be due to the fact, that the cells used for blastocyst injection were not preselected for Oct-4-GFP expression.
Preferentially, chimerism was found in liver, muscle and tongue. No chimerism was detected in heart and brain, two organs, which showed a high rate of spontaneous cell fusions in a recent study. However, we cannot fully exclude the possibility that fusion with differentiated cells might have contributed to the observed chimerism.
Two remarkable characteristics of MCTs are 3D-colony growth and the ability to grow in suspension. Our data provides convincing evidence that unlike many cell lines derived from tumours or cells transformed by oncogenic agents, the MCT subpopulation does not result from spontaneous immortalisation or transformation. MCTs do not exhibit a crisis followed by clonal outgrowth and chromosomal abnormalities or aneuplodies, and show reversibility of the altered growth characteristics after exposure to standard cell culture conditions.
Primary fibroblasts were prepared by enzymatic isolation of eviscerated fetuses or by mesenchymal explant (<1 mm3) cultures of connective tissue pasted to the dish surface by employing recalcified microdrops of bovine plasma and maintained in Dulbecco's Modified Eagles Medium (DMEM) medium supplemented with 1 mM glutamine, 1%.non-essential amino acids 1% vitamin solution, 0.1 mM mercaptoethanol, 100 U/ml penicillin, 100 mg/ml streptomycin (all from Sigma, Deisenhofen, Germany), containing 10% FCS from selected batches (Gibco, Karlsruhe, Germany, batch numbers 40G321K, 40G2810K) and incubated in a humidified 95% air/5% C02 atmosphere at 37° C. (Keus et al., 2000, Biol. Reprod., 62: 412-419; Keus et al., 2002, Cloning Stem Cells, 4: 147-165). Outgrowing cells were trypsinised and subpassaged once prior to cryoconservation. For high serum culture the serum content of the standard medium was increased to 30% FCS. For suspension culture, colonies were selectively isolated and completely dissociated in a trypsin solution, then 104 cells were seeded into bacteriological dishes (35 mm). Every second day 50% of the medium was replaced with new medium. To determine the maximal replicative limit, cultures were serially subpassaged and 12.5×103 cells were seeded per cm2 in 6-well-dishes, trysinised after 5-7 days, counted and reseeded. The number of accumulated population doublings per passage was determined using the equation, PD =log (A/B)/log2, in which A is the number of collected cells and B is the number of plated cells. Murine fibroblasts were obtained from day 11.5-14.5 fetuses or adult animals of OG2 mice (Chang et al., 2002, Proc. Natl. Acad. Sci. USA; 99:12877-12882) (homozygous for a Oct4-GFP transgene) or from double transgenic fetuses of crosses of OG2 with Rosa26 mice. Confocal microscopy was applied to detect GFP using a Zeiss Axiomat LSM and an excitation wavelength of 488 nm. ES cells (wild type GS1 129/Sv) were cultured as described previously (Gotz et al., 1998, Proc Natl Acad Sci USA; 95:12370-12375).
In brief, total RNA was isolated from cells grown in 6-well dishes and reverse transcribed into cDNA using random hexamers as primers. Murine Oct4 and GFP cDNAs were amplified by PCR with the following primers and conditions:
(35 cycles, annealing temperature 57° C.) for the murine Oct4:
(35 cycles) for GFP. Porcine Oct4 was amplified with:
primers (AJ251914) and 36 cycles. The PCR reactions were performed in 20 μl volumes, consisting of 20 mM Tris.HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 μM dNTPs, 1 μM of specific primer pairs and 0.5 units of Taq DNA polymerase (Gibco).
DNA synthesis was measured by 5-bromo-2′deoxy-uridine (BrdU) incorporation as described in Keus et al. (2002, Cloning Stem Cells, 4:231-243). Incorporated BrdU was detected by a chromogenic immunoassay employing an anti-BrdU antibody conjugated with alkaline phosphatase.
Cells grown on gelatinised coverslips, were fixed in cold 80% methanol. The following monoclonal antibody dilutions were used: anti-vimentin (AMF-17b, 1:200) (Developmental Studies Hybridoma Bank, Iowa) and anti-cytokeratin (peptide 17, 1:100, Sigma). A rhodamine-labelled secondary anti-mouse antibody (1:2000, Molecular Probes, N L) was used. In some cases the nuclei were counterstained with 1 mM Hoechst 33342 (Keus et al, 1995, J. Cell Biol., 130: 949-957). The samples were examined with an Olympus BX60 microscope equipped with phase-contrast and epifluorescence optics, using band-pass rhodamine and Hoechst filter sets.
Cell cultures were washed with PBS, fixed in 3.7% paraformaldehyde for 15 minutes, washed in PBS and then incubated in a solution containing 25 mM TrisHC pH 9.0, 4 mM MgCl2, 0.4 mg/ Na-α-naphtylphosphate, 1 mg/ml Fast Red TR (Sigma) and 0.05% Triton X-100 for 60 minutes.
Rosa26 homozygous mice were obtained from Jackson Laboratory (NY) and mated with homozygous OG2 animals to generate double-transgenic fetuses carrying both marker genes, which were used to isolate MCTs. Between day 11.5 and day 15.5 fetuses were isolated and employed for fetal cell cultures using the explant method described supra.
For blastocyst injections, 6-10 week old female CD2F1 mice were superovulated with 10 U PMSG at noon on day -2, followed by 10 U hCG on day 0 and were then mated with CD2F1 males. The next day females were checked for plug formation. At day 3.5 females were sacrificed, and the uterine tracts were isolated and flushed with PBS containing 1% albumin. Blastocysts were isolated and incubated in 1% albumin at 37° C. Single blastocysts were transferred into a micromanipulation unit (Zeiss) and fixed with a holding pipette. On average 2-15 double transgenic cells (OG2/Rosa26) were injected into the blastocoel by the aid of a microcapillary. In total, 8-10 blastocysts were transferred into the uteri of day 2.5 or day 3.5 pseudopregnant NMRI females that had been obtained by matings of NMRI females with vasectomised males. Fetuses were recovered at day 10.5-15.5 and either stained for lacZ positive cells (Friedrich & Soriano, 1991, Genes Dev., 5, 1513-1523) as whole mounts, or dissected and screened for GFP expression in genital ridges and other organs.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2003905692 | Oct 2003 | AU | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/AU04/01408 | 10/15/2004 | WO | 00 | 3/16/2007 |
