WO2006062507A1 - Procede pour preselectionner des cellules pour des procedures de transfert nucleaire - Google Patents

Procede pour preselectionner des cellules pour des procedures de transfert nucleaire Download PDF

Info

Publication number
WO2006062507A1
WO2006062507A1 PCT/US2004/040816 US2004040816W WO2006062507A1 WO 2006062507 A1 WO2006062507 A1 WO 2006062507A1 US 2004040816 W US2004040816 W US 2004040816W WO 2006062507 A1 WO2006062507 A1 WO 2006062507A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
donor
transgenic
cells
nuclear transfer
Prior art date
Application number
PCT/US2004/040816
Other languages
English (en)
Inventor
Yann Echelard
Li How Chen
Original Assignee
Gtc Biotherapeutics, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Gtc Biotherapeutics, Inc. filed Critical Gtc Biotherapeutics, Inc.
Priority to PCT/US2004/040816 priority Critical patent/WO2006062507A1/fr
Priority to US11/081,945 priority patent/US20060123500A1/en
Publication of WO2006062507A1 publication Critical patent/WO2006062507A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0273—Cloned vertebrates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
    • A23C9/158—Milk preparations; Milk powder or milk powder preparations containing additives containing vitamins or antibiotics
    • A23C9/1585—Antibiotics; Bacteriocins; Fungicides from microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/20—Dietetic milk products not covered by groups A23C9/12 - A23C9/18
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • C12N15/877—Techniques for producing new mammalian cloned embryos
    • C12N15/8772—Caprine embryos
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/102—Caprine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C2230/00—Aspects relating to animal feed or genotype
    • A23C2230/15—Animal milk with modified composition due to manipulation of the animal, e.g. animal milk comprising antibodies, selection of animals having specific genotypes

Definitions

  • the present invention relates to the development of methods that increase the efficiency of cell-line screening for use in the production of transgenic animals.
  • the current invention provides a method for improving cell lines through pre-selection methods such that downstream nuclear transfer procedures are improved.
  • the present invention relates generally to the field of somatic cell nuclear transfer (SCNT) and to the creation of desirable transgenic animals. More particularly, it concerns improved methods for selecting, generating, and propagating superior somatic cell-derived cell lines, and using these transfected cells and cell lines to generate transgenic non-human mammalian animal species, especially for the production of ungulates. Typically these transgenic animals will be used for the production of molecules of interest, including biopharmaceuticals, antibodies and recombinant proteins that are the subject of the transgene(s) of interest.
  • SCNT somatic cell nuclear transfer
  • transgenic animals are animals that carry the gene(s) of interest that has been deliberately introduced into existing somatic cells and/or germline cells at an early stage of development. As the animals develop and grow the protein product or specific developmental change engineered into the animal becomes apparent, and is present in their genetic complement and that of their offspring.
  • transgenic production of recombinant monoclonal antibodies in milk where often several transgenes have to be expressed in the same secretory cells of the mammary epithelium at equivalent levels.
  • Co-integration of the transgenes in the same chromosomal locus to avoid segregation of heavy chain and light chain genes during herd propagation, is also desirable.
  • transgenic animals have been produced by various methods in several different species, methods to readily and reproducibly produce transgenic animals capable of expressing a desired protein or biopharmaceutical in high quantity or demonstrating the genetic alteration or enhancement caused by the insertion of the transgene(s) at reasonable costs are still lacking.
  • the methods of the invention are typically applied to primary somatic cells, in the context of nuclear transfer, for the accelerated generation of a herd of transgenic animals useful in the production of recombinant proteins in milk.
  • the current invention provides a method for the accelerated production of transgenic animals.
  • the method involves prescreening cells destined for transgenic procedures for abnormalities. By eliminating problem cell lines the resulting transgenic animal technologies are improved in efficiency.
  • the methods of the invention include transfecting a selected non-human mammalian cell-line with a given transgene construct containing at least one DNA encoding a desired gene; selecting a cell line(s) in which the desired gene has been inserted into the genome of that cell or cell-line; and, performing a nuclear transfer procedure to generate a transgenic animal heterzygous for the desired gene.
  • An additional step that may performed according to the invention is to expand the biopsied cell-line obtained from the heterozygous animal in cell and/or cell- line in culture.
  • An additional step that may performed according to the invention is to biopsy the heterozygous transgenic animal.
  • a nuclear transfer procedure can be conducted to generate a mass of transgenic cells useful for research, serial cloning, or in vitro use.
  • surviving cells are characterized by one of several known molecular biology methods including without limitation FISH, Southern Blot, PCR. The methods provided above will allow for the accelerated production of herd homozygous for desired transgene(s) and thereby the more efficient production of a desired biopharmaceutical.
  • the current invention allows for the production of genetically desirable livestock or non-human mammals.
  • multiple proteins can be integrated into the genome of a transgenic cell line that has been pre-screened to remove the possibility of cell line abnormalities, and/or screened after selected procedures to remove cell lines that become abnormal after the integration of a genetic sequence of interest. Successive rounds of transfection with another the DNA for an additional gene/molecule of interest (e.g., molecules that could be so produced, without limitation, include antibodies, and desired biopharmaceuticals).
  • an additional gene/molecule of interest e.g., molecules that could be so produced, without limitation, include antibodies, and desired biopharmaceuticals.
  • beta casein promoter is one such promoter turned on during lactation in mammary epithelial cells, while other promoters could be turned on under different conditions in other cellular tissues.
  • the methods of the current invention will allow the accelerated development of one or more homozygous animals that carry a particularly beneficial or valuable gene, enabling herd scale-up and potentially increasing herd yield of a desired protein much more quickly than previous methods.
  • the methods of the current invention will also provide for the replacement of specific transgenic animals lost through disease or their own mortality. It will also facilitate and accelerate the production of transgenic animals constructed with a variety of DNA constructs so as to optimize the production and lower the cost of a desirable biopharmaceutical.
  • homozygous transgenic animals are more quickly developed for xenotransplantation purposes or developed with humanized Ig loci.
  • FIG. 1 Shows a flowchart of the methods involved in practicing the invention.
  • FIG. 2 Shows A Generalized Diagram of the Process of Creating Cloned Animals through Nuclear Transfer.
  • SOF Synthetic Oviductal Fluid
  • FBS Fetal Bovine Serum
  • BSA Bovine Serum Albumin
  • Cytocholasin-B A metabolic product of certain fungi that selectively and reversibly blocks cytokinesis while not effecting karyokinesis.
  • Cytoplast - The cytoplasmic substance of eukaryotic cells.
  • Fusion Slide A glass slide for parallel electrodes that are placed a fixed distance apart. Cell couplets are placed between the electrodes to receive an electrical current for fusion and activation.
  • Karyoplast - A cell nucleus, obtained from the cell by enucleation, surrounded by a narrow rim of cytoplasm and a plasma membrane.
  • Nuclear Transfer - or "nuclear transplantation” refers to a method of cloning wherein the nucleus from a donor cell is transplanted into an enucleated oocyte.
  • Parthenogenic The development of an embryo from an oocyte without the penetrance of sperm Porcine - of, relating to or resembling swine or pigs
  • Reconstructed Embryo - A reconstructed embryo is an oocyte that has had its genetic material removed through an enucleation procedure. It has been "reconstructed” through the placement of genetic material of an adult or fetal somatic cell into the oocyte following a fusion event.
  • Selective Agent Compounds, compositions, or molecules that can act as selection markers for cells in that they are capable of killing and/or preventing the growth of a living organism or cell not containing a suitable resistance gene.
  • such agents include, without limitation, Neomycin, puromycin, zeocin, hygromycin, G418, gancyclovir and FIAU.
  • increasing the dosage of the selective agent will kill all cell lines that only contain one integration site (e.g., heterozygous animals and/or cells).
  • Somatic Cell Any cell of the body of an organism except the germ cells.
  • Somatic Cell Nuclear Transfer Also called therapeutic cloning, is the process by which a somatic cell is fused with an enucleated oocyte.
  • the nucleus of the somatic cell provides the genetic information, while the oocyte provides the nutrients and other energy-producing materials that are necessary for development of an embryo. Once fusion has occurred, the cell is totipotent, and eventually develops into a blastocyst, at which point the inner cell mass is isolated.
  • Transgenic Organism An organism into which genetic material from another organism has been experimentally transferred, so that the host acquires the genetic information of the transferred genes in its chromosomes in addition to that already in its genetic complement.
  • Ungulate of or relating to a hoofed typically herbivorous quadraped mammal, including, without limitation, sheep, swine, goats, cattle and horses.
  • Xenotransplantation any procedure that involves the use of live cells, tissues, and organs from one animal source, transplanted or implanted into another animal species (typically humans) or used for clinical ex-vivo perfusion
  • the accelerated development of superior transgenic genotypes of mammals with improved efficiencies, characteristics, or enhanced biopharmaceutical production, including caprines and bovines, are provided.
  • This invention relates to the genetic characterization of transfected somatic cells prior to use as karyoplasts donors in nuclear transfer prior to engage in the nuclear transfer procedure.
  • Analysis of several murine, caprine and bovine transgenic lines has shown that in the cases of transgenes incorporating the chicken globin (Chung et al., 1993) insulator sequence there is a correlation between the number of copies of trangenes at the integration sites and the expression level of the transgene. In both murine and caprine cases, it was observed that if the copy number of the integrated transgene is very high (>20 copies) this can lead to over expression of the transgene, affecting the health of the animal.
  • transgene-encoded protein in milk >20 gll
  • low-copy number transgene integrations (1 to 2 copies), or transgene integrations located on the X chromosome will often lead to lower expression level of the transgene ( ⁇ 1 gll), often incompatible with successful commercialization of these transgenic lines.
  • transfected somatic cells for nuclear transfer opens the possibility to prescreen the cell line prior to their use in the generation of transgenic animals.
  • Several methods can be employed for genotyping screening of the transgected somatic cells:
  • FISH is preferred for use with the current invention since qualitative information is obtained: number and chromosomal location of integration sites, percentage mosaicism of the donor cell line; as well as semiquantitative information. By comparing the intensity of the signal given by the transgene integration sites with either endogenous signals or control lines, one can evaluate the copy number. Southern blotting helps evaluate in the transgene is rearranged. FISH can also be used to identify the specific chromosome onto which the transgene(s) is (are) integrated. There is an obvious value in screening out Y- chromosome specific integrations (only males are transgenic), and it is also a good idea to reject X-chromosome integration, since sometimes the transgene is associated with X inactivation.
  • prescreening is particularly useful in the generation of cell lines containing multiple transgenes (as for full antibody production). In this case it is particularly important to determine if all the transgenes are present in the cell lines, and if they are present in the same locus.
  • Pre-screening methods also allow the elimination of cell lines with chromosomal abnormalities. Such cell lines inevitably arise in culture, and if employed will give rise to non-viable or poorly viable animals.
  • abnormalities that can be looked for are: chromosomal complement (using Giemsa staining), evidence of chromosome breakage and translocation (using Giemsa staining or several banding procedures); abnormal sex chromosome complements (XO, XXY, XYY etc.), evidence of chromosomal instability (sister chromatin exchange).
  • Cell lines are first transfected with the transgene of interest using standard procedures (Ex: electroporation, lipofection). Recombinant clones are then isolated using standard methods (for example drug resistance), giving rise to isolated colonies. An aliquot (a few thousand cells) for each colony is frozen, to stop cell division and prevent the onset of senescence. For each colony, the remaining cells are kept in culture, expanded and genotyped. Clonal cell lines that have low-copy integration ( ⁇ 2 copies) or very high-copy integration (>20 copies) are then identified and discarded; only cell lines that have preferred copy number (for example 3-20 copies) are retained and used in nuclear transfer procedures aiming at creating transgenic animals.
  • standard procedures for example drug resistance
  • Donor cells are primary cells, they have to be used before as nuclear transfer karyoplasts before the growth arrest brought on by the onset of senescence (with goat fibroblasts, typically 30 to 50 cell divisions).
  • methods have been developed that will allow the rapid identification of promising transfected candidates, freeze an aliquot, and pursue genotyping on the remainder of the cells.
  • the ability to freeze early small aliquots of cells is important, since it maximizes the number of generations that we can use to perform somatic cell nuclear transfer. Possibly (although the data on this is probably not very strong) the use of a "younger" cell line could also lead to a healthier offspring.
  • the inventors have successfully applied the preferred methods of the current invention to the cloning of goats and are working on transitioning this technology to other species.
  • transgenic cell-lines can be characterized using standard molecular biology methods (PCR, Southern blotting, FISH).
  • PCR Southern blotting
  • FISH FISH-labeled immunoglobulinous hybridization
  • Cell lines carrying a transgene(s) of the appropriate copy number generally with a single integration site (although the same technique could be used with multiple integration sites) can then be used as karyoplast donors in a somatic cell nuclear transfer protocol. Following nuclear transfer, and embryo transfer to a recipient animal, and gestation, live transgenic offspring are obtained.
  • this transgenic offspring carries only one transgene integration on a specific chromosome, the other homologous chromosome not carrying an integration in the same site. Hence the transgenic offspring is heterozygous for the transgene.
  • Protocol Using G418 selection I. Plate primary cells at 2 x 10 /10 cm petri dish.
  • the cells following the initial transfection, and isolation of the cell line, the cells be subjected immediately to increased selection to generate the homozygous cell line prior to generate an offspring.
  • the present invention relates to cloning procedures in which cell nuclei derived from somatic or differentiated fetal or adult mammalian cell lines are utilized. These cell lines include the use of serum starved differentiated fetal or adult caprine or bovine (as the case may be) cell populations and cell lines later reintroduced to serum as mentioned infra, these cells are transplanted into enucleated oocytes of the same species as the donor nuclei. The nuclei are reprogrammed to direct the development of cloned embryos, which can then be transferred to recipient females to produce fetuses and offspring, or used to produce cultured inner cell mass cells (CICM).
  • CICM cultured inner cell mass cells
  • the cloned embryos can also be combined with fertilized embryos to produce transfer. However, these methods do not generate Ca +2 oscillations patterns similar to sperm in a typical in vivo fertilization pattern. [0036] Significant advances in nuclear transfer have occurred since the initial report of success in the sheep utilizing somatic cells (Wilmut et al, 1997). Many other species have since been cloned from somatic cells (Baguisi et al, 1999 and Cibelli et al, 1998) with varying degrees of success.
  • Primary somatic cells are differentiated non-germ cells that were obtained from animal tissues transfected with a gene of interest using a standard lipid-based transfection protocol. The transfected cells were tested and were transgene-positive cells that were cultured and prepared as described in Baguisi et al, 1999 for use as donor cells for nuclear transfer.
  • the enucleation and reconstruction procedures can be performed with or without staining the oocytes with the DNA staining dye Hoechst 33342 or other fluorescent light sensitive composition for visualizing nucleic acids.
  • the Hoechst 33342 is used at approximately 0.1 - 5.0 ⁇ g/ml for illumination of the genetic material at the metaphase plate.
  • Enucleation and reconstruction was performed with, but may also be performed without, staining the oocytes with Hoechst 3342 at approximately 0.1-5.0 ug/ml and ultraviolet illumination of the genetic material/metaphase plate.
  • the karyoplast/cytoplast couplets were incubated in equilibrated Synthetic Oviductal Fluid medium supplemented with fetal bovine serum (1% to 15%) plus 100 U/ml penicillin and 100 ⁇ g/ml streptomycin (SOF/FBS).
  • the couplets were incubated at 37-39°C in a humidified gas chamber containing approximately 5% CO 2 in air at least 30 minutes prior to fusion.
  • Fusion was performed using a fusion slide constructed of two electrodes.
  • the fusion slide was placed inside a fusion dish, and the dish was flooded with a sufficient amount of fusion buffer to cover the electrodes of the fusion slide.
  • Cell couplets were removed from the culture incubator and washed through fusion buffer.
  • a stereomicroscope cell couplets were placed equidistant between the electrodes, with the karyoplast/cytoplast junction parallel to the electrodes.
  • an initial single simultaneous fusion and activation electrical pulse of approximately 2.0 to 3.0 kV/cm for 20 (can be 20-60) ⁇ sec was applied to the cell couplets using a BTX ECM 2001 Electrocell Manipulator.
  • the fusion treated cell couplets were transferred to a drop of fresh fusion buffer. Fusion treated couplets were washed through equilibrated SOF/FBS, then transferred to equilibrated SOF/ FBS with (1 to 10 ⁇ g/ml) or without cytochalasin-B. The cell couplets were incubated at 37- 39°C in a humidified gas chamber containing approximately 5% CO 2 in air.
  • Fused couplets received an additional single electrical pulse (double pulse) of approximately 2.0 kV/cm for 20 (20-60) ⁇ sec starting at 1 hour (15 min-1 hour) following the initial fusion and activation treatment to facilitate additional activation.
  • another group of fused cell couplets received three additional single electrical pulses (quad pulse) of approximately 2.0 kV/cm for 20 ⁇ sec, at fifteen- minute intervals, starting at 1 hour (15 min to 1 hour) following the initial fusion and activation treatment to facilitate additional activation.
  • Non-fused cell couplets were re- fused with a single electrical pulse of approximately 2.6 to 3.2 kV/cm for 20 (20-60) ⁇ sec starting at 1 hours following the initial fusion and activation treatment to facilitate fusion. All fused and fusion treated cell couplets were returned to SOF/FBS with (1 to 10 ⁇ g/ml) or without cytochalasin-B. The cell couplets were incubated at least 30 minutes at 37-39°C in a humidified gas chamber containing approximately 5% CO 2 in air.
  • Primary caprine fetal fibroblast cell lines to be used as karyoplast donors were derived from 35- and 40-day fetuses. Fetuses were surgically removed and placed in equilibrated phosphate-buffered saline (PBS, Ca ++ /Mg ++ -free). Single cell suspensions were prepared by mincing fetal tissue exposed to 0.025 % trypsin, 0.5 mM EDTA at 38°C for 10 minutes.
  • PBS phosphate-buffered saline
  • fetal cell medium fetal cell medium [equilibrated Medium-199 (M199, Gibco) with 10% fetal bovine serum (FBS) supplemented with nucleosides, 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine and 1%
  • Transfected fetal somatic cells were seeded in 4-well plates with fetal cell medium and maintained in culture (5% CO2, 39 0 C). After 48 hours, the medium was replaced with fresh low serum (0.5 % FBS) fetal cell medium. The culture medium was replaced with low serum fetal cell medium every 48 to 72 hours over the next 2 - 7 days following low serum medium, somatic cells (to be used as karyoplast donors) were harvested by trypsinization. The cells were re-suspended in equilibrated M199 with 10% FBS supplemented with 2 mM L-glutamine, 1% penicillin/streptomycin (10,000 I. U. each/ml) for at least 6 hours prior to fusion to the enucleated oocytes.
  • Oocyte donor does were synchronized and superovulated as previously described (Gavin W.G., 1996), and were mated to vasectomized males over a 48-hour interval. After collection, oocytes were cultured in equilibrated Ml 99 with 10% FBS supplemented with 2 mM L-glutamine and 1 % penicillin/streptomycin (10,000 LU. each/ml).
  • oocytes were treated with cytochalasin-B (Sigma, 5 ⁇ g/ml in SOF with 10% FBS) 15 to 30 minutes prior to enucleation.
  • Metaphase-II stage oocytes were enucleated with a 25 to 30 ⁇ m glass pipette by aspirating the first polar body and adjacent cytoplasm surrounding the polar body ( ⁇ 30 % of the cytoplasm) to remove the metaphase plate. After enucleation, all oocytes were immediately reconstructed.
  • Donor cell injection was conducted in the same medium used for oocyte enucleation.
  • One donor cell was placed between the zona pellucida and the ooplasmic membrane using a glass pipet.
  • the cell-oocyte couplets were incubated in SOF for 30 to 60 minutes before electrofusion and activation procedures.
  • Reconstructed oocytes were equilibrated in fusion buffer (300 mM mannitol, 0.05 mM CaCl 2 , 0.1 mM MgSO 4 , 1 mM K 2 HPO 4 , 0.1 mM glutathione, 0.1 mg/ml BSA) for 2 minutes.
  • Electrofusion and activation were conducted at room temperature, in a fusion chamber with 2 stainless steel electrodes fashioned into a "fusion slide" (500 ⁇ m gap; BTX- Genetronics, San Diego, CA) filled with fusion medium.
  • Fusion was performed using a fusion slide.
  • the fusion slide was placed inside a fusion dish, and the dish was flooded with a sufficient amount of fusion buffer to cover the electrodes of the fusion slide. Couplets were removed from the culture incubator and washed through fusion buffer. Using a stereomicroscope, couplets were placed equidistant between the electrodes, with the karyoplast/cytoplast junction parallel to the electrodes. It should be noted that the voltage range applied to the couplets to promote activation and fusion can be from 1.0 kV/cm to 10.0 kV/cm.
  • the initial single simultaneous fusion and activation electrical pulse has a voltage range of 2.0 to 3.0 kV/cm, most preferably at 2.5 kV/cm, preferably for at least 20 ⁇ sec duration.
  • This is applied to the cell couplet using a BTX ECM 2001 Electrocell Manipulator.
  • the duration of the micropulse can vary from 10 to 80 ⁇ sec.
  • the treated couplet is typically transferred to a drop of fresh fusion buffer. Fusion treated couplets were washed through equilibrated SOF/FBS, then transferred to equilibrated SOF/ FBS with or without cytochalasin-B.
  • cytocholasin-B its concentration can vary from 1 to 15 ⁇ g/ml, most preferably at 5 ⁇ g/ml.
  • the couplets were incubated at 37-39 0 C in a humidified gas chamber containing approximately 5% CO 2 in air.
  • mannitol may be used in the place of cytocholasin-B throughout any of the protocols provided in the current disclosure (HEPES -buffered mannitol (0.3 mm) based medium with Ca +2 and BSA).
  • each sample may be first analyzed by PCR using primers for a specific transgenic target protein, and then subjected to Southern blot analysis using the cDNA for that specific target protein.
  • genomic DNA was digested with EcoRl (New England Biolabs, Beverly, MA), electrophoreses in 0.7 % agarose gels (SeaKem®, ME) and immobilized on nylon membranes (MagnaGraph, MSI, Westboro, MA) by capillary transfer following Standard procedures known in the art.
  • Membranes were probed with the 1.5 kb Xho I to Sal I hAT cDNA fragment labeled with 32 P dCTP using the Prime-It® kit (Stratagene, La Jolla, CA). Hybridization was executed at 65 0 C overnight. The blot was washed with 0.2 X SSC, 0.1 % SDS and exposed to X-OMATTM AR film for 48 hours.
  • the present invention allows for increased efficiency of transgenic procedures by increasing the number of potentially useful transgenic lines. Since it allows the rapid generation of transgenic animals with a substantial yield of recombinant protein production.
  • the present invention also includes a method of cloning a genetically engineered or transgenic mammal, by which a desired gene is inserted, removed or modified in the differentiated mammalian cell or cell nucleus prior to insertion of the differentiated mammalian cell or cell nucleus into the enucleated oocyte.
  • the present invention is preferably used for cloning caprines or bovines but could be used with any mammalian species.
  • the present invention further provides for the use of nuclear transfer fetuses and nuclear transfer and chimeric offspring in the area of cell, tissue and organ transplantation.
  • Suitable mammalian sources for oocytes include goats, sheep, cows, pigs, rabbits, guinea pigs, mice, hamsters, rats, primates, etc.
  • the oocytes will be obtained from ungulates, and most preferably goats or cattle. Methods for isolation of oocytes are well known in the art. Essentially, this will comprise isolating oocytes from the ovaries or reproductive tract of a mammal, e.g., a goat.
  • a readily available source of ungulate oocytes is from hormonally induced female animals.
  • oocytes may preferably be matured in vivo before these cells may be used as recipient cells for nuclear transfer, and before they can be fertilized by the sperm cell to develop into an embryo.
  • Metaphase II stage oocytes which have been matured in vivo have been successfully used in nuclear transfer techniques. Essentially, mature metaphase II oocytes are collected surgically from either non-superovulated or superovulated animals several hours past the onset of estrus or past the injection of human chorionic gonadotropin (hCG) or similar hormone.
  • hCG human chorionic gonadotropin
  • the ability to modify animal genomes through transgenic technology offers new alternatives for the manufacture of recombinant proteins.
  • the production of human recombinant pharmaceuticals in the milk of transgenic farm animals solves many of the problems associated with microbial bioreactors (e.g., lack of post-translational modifications, improper protein folding, high purification costs) or animal cell bioreactors (e.g., high capital costs, expensive culture media, low yields).
  • the current invention enables the use of transgenic production of biopharmaceuticals, hormones, plasma proteins, and other molecules of interest in the milk or other bodily fluid (i.e., urine or blood) of transgenic animals homozygous for a desired gene.
  • Proteins capable of being produced in through the method of the invention include: antithrombin III, lactoferrin, urokinase, PF4, alpha- fetoprotein, alpha- 1 -antitrypsin, C-I esterase inhibitor, decorin, interferon, ferritin, prolactin, CFTR, blood Factor X, blood Factor VIII, as well as monoclonal antibodies.
  • antithrombin III lactoferrin, urokinase, PF4, alpha- fetoprotein, alpha- 1 -antitrypsin, C-I esterase inhibitor, decorin, interferon, ferritin, prolactin, CFTR, blood Factor X, blood Factor VIII, as well as monoclonal antibodies.
  • Telomerase would be among such compounds.
  • a 5' element of the chicken beta- globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila. Cell 74, 505-514.

Abstract

L'invention concerne la production d'animaux transgéniques grâce à des procédés de présélection conçus pour améliorer l'efficacité du transfert nucléaire et par conséquent la production de caractéristiques de mammifères transgéniques par rapport à des protéines d'intérêt. L'invention est utilisée dans la production d'animaux transgéniques ongulés capables de produire des produits biopharmaceutiques souhaités dans leur lait à un rendement plus élevé qu'un hétérozygote comparable ou de produire d'autres animaux présentant des attributs physiologiques améliorés.
PCT/US2004/040816 2004-12-07 2004-12-07 Procede pour preselectionner des cellules pour des procedures de transfert nucleaire WO2006062507A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/US2004/040816 WO2006062507A1 (fr) 2004-12-07 2004-12-07 Procede pour preselectionner des cellules pour des procedures de transfert nucleaire
US11/081,945 US20060123500A1 (en) 2004-12-07 2005-03-16 Methods of prescreening cells for nuclear transfer procedures

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2004/040816 WO2006062507A1 (fr) 2004-12-07 2004-12-07 Procede pour preselectionner des cellules pour des procedures de transfert nucleaire

Publications (1)

Publication Number Publication Date
WO2006062507A1 true WO2006062507A1 (fr) 2006-06-15

Family

ID=36578207

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2004/040816 WO2006062507A1 (fr) 2004-12-07 2004-12-07 Procede pour preselectionner des cellules pour des procedures de transfert nucleaire

Country Status (1)

Country Link
WO (1) WO2006062507A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6580017B1 (en) * 1998-11-02 2003-06-17 Genzyme Transgenics Corporation Methods of reconstructed goat embryo transfer

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6580017B1 (en) * 1998-11-02 2003-06-17 Genzyme Transgenics Corporation Methods of reconstructed goat embryo transfer

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BAUGISI A. ET AL: "Production of Goats by Nuclear Transfer", NATURE GENETICS, vol. 17, May 1999 (1999-05-01), pages 456 - 461 *
KEEFER C.L ET AL: "Generation of Dwarf Goat (Capra hircus) Clones Following Nuclear Transfer with Transfected and Nontransfected Fetal Fibroblasts and In Vitro-Matured Oocytes", BIOLOGY OF REPRODUCTION, vol. 64, 2001, pages 849 - 856 *

Similar Documents

Publication Publication Date Title
US20060191025A1 (en) Method for the rapid selection of homozygous primary cell lines for the production of transgenic animals by somatic cell nuclear transfer
Niemann et al. Application of transgenesis in livestock for agriculture and biomedicine
US5994619A (en) Production of chimeric bovine or porcine animals using cultured inner cell mass cells
Niemann et al. Transgenic livestock: premises and promises
US20060123500A1 (en) Methods of prescreening cells for nuclear transfer procedures
CA2350233A1 (fr) Mammiferes transgeniques et clones
US7652192B2 (en) Cloning of transgenic unglulates comprising artificial chromosomes
US11647737B2 (en) Genetically modified rabbit expressing an exogenous protein in milk
US20060168671A1 (en) Injection of caprine sperm factor (cSF), phospholipase C zeta (PLCzeta) and adenophostin A as alternative methods of activation during nuclear transfer in the caprine species
AU2002252076A1 (en) Cloning of transgenic animals comprising artificial chromosomes
US20060174359A1 (en) Method for selecting cell lines to be used for nuclear transfer in mammalian species
Skrzyszowska et al. Generation of transgenic rabbits by the novel technique of chimeric somatic cell cloning
Wolf et al. Nuclear transfer technology in mammalian cloning
WO1998037183A1 (fr) Production de cellules donneuses transgeniques pour le transfert nucleaire
JP2007510437A (ja) 相同的組み換えを用いる牛β−カゼイン遺伝子ターゲッティングベクター
Yang et al. Transgenic farm animals: applications in agriculture and biomedicine
WO2003064633A1 (fr) Procede et systeme de fusion et d'activation suite au transfert nucleaire dans des embryons reconstitues
Niemann et al. Production of biopharmaceuticals in transgenic animals
WO2006062507A1 (fr) Procede pour preselectionner des cellules pour des procedures de transfert nucleaire
Tian et al. Generation of transgenic mice for cardiovascular research
US20060021070A1 (en) Production of chimeric bovine or porcine animals using cultured inner cell mass
DEYKIN et al. RESEARCH RESULTS IN PHARMACOLOGY
Duszewska et al. The use of green fluorescent protein (GFP) to select bovine embryos
Wang et al. Transgenic technologies for animals as bioreactors
Takahashi et al. Strategy for selective acquisition of transgenic marmosets using the piggyBac transposon system

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 11081945

Country of ref document: US

AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 04813173

Country of ref document: EP

Kind code of ref document: A1