WO2001032855A1 - Transgenic animal production using lopu-derived oocytes - Google Patents
Transgenic animal production using lopu-derived oocytes Download PDFInfo
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- WO2001032855A1 WO2001032855A1 PCT/US2000/029751 US0029751W WO0132855A1 WO 2001032855 A1 WO2001032855 A1 WO 2001032855A1 US 0029751 W US0029751 W US 0029751W WO 0132855 A1 WO0132855 A1 WO 0132855A1
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- 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
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- 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
- C12N2510/00—Genetically modified cells
Definitions
- the field of the invention is methods for the development and propagation of transgenic animals.
- transgenic animals carrying a gene fused to a tissue-specific promoter such as milk
- tissue-specific promoter such as milk
- the use of transgenic animals carrying such transgenes makes it possible to produce desired polypeptides in the animals. These polypeptides can be produced in larger quantities and with less expense than those produced using more traditional methods of protein production in microorganisms or animal cells. Once transgenic animals are generated, their offspring can be used in efficient, tissue-specific production of desired polypeptides.
- the invention features methods for the generation of a transgenic animal using oocytes recovered through laparoscopic aspiration of follicles.
- This oocyte recovery procedure is also known as laparoscopic ovum pick up (LOPU), laparoscopic follicle aspiration, or laparoscopic oocyte aspiration.
- LOPU laparoscopic ovum pick up
- a transgene may be introduced into LOPU-derived oocytes, for example, by microinjection of a DNA construct or retroviral particles, or by retroviral infection (Wall, supra).
- the oocytes are then fertilized, and the formed zygote, or morulae or blastocyst grown from the zygote is transferred into a recipient.
- a transgene is introduced into a zygote, or a cleaved embryo, morulae, or blastocyst formed from by culturing the zygotes by microinjection or infection, followed by transfer into a recipient.
- a transgenic animal may be generated by recovering oocytes from a transgenic founder, using the LOPU procedure, followed by fertilization and transfer to a recipient.
- non-transgenic oocytes may be recovered, using the LOPU procedure, and fertilized in vitro with transgenic semen to produce a transgenic animal.
- the invention features a method for generating a non-human transgenic animal, comprised of the following steps:
- the invention features a method for generating a non-human transgenic animal, involving:
- the invention features a method for generating a non-human transgenic animal, involving:
- the transgene is introduced into the oocyte, zygote, or a cleaved embryo, morulae, or blastocyst formed from culturing the zygote by microinjection.
- the transgene is introduced into the oocyte, zygote, or a cleaved embryo, morulae, or blastocyst formed from culturing the zygote by retroviral infection.
- fertilization of the oocyte is done using transgenic semen.
- the invention features a method for generating a non-human transgenic animal, said method comprising: (a) recovering an oocyte from a transgenic donor animal by laparoscopic ovum pick up;
- the animal is a ruminant, such as a goat or a sheep.
- the donor and/or recipient animal is prepubertal.
- transgenic animal is meant a non-human animal containing a transgene.
- transgene is meant a DNA sequence that is introduced into the germline of a non-human animal by way of human intervention using any of the methods described herein, or methods known to one skilled in the art.
- tissue-specific expression is meant the expression within a specifically desired tissue, or a product released from a specific tissue.
- tissue-specific expression refers to the production of a protein in the milk, urine, or blood specifically.
- prepubertal is meant less than 5 months of age.
- microinjection is meant a procedure used for the introduction of material into a cell, involving injecting the material into the cell.
- microinjection refers to DNA or retroviral particles introduced into an oocyte, zygote, cleaved embryo, morulae, or blastocyst.
- retroviral infection is meant a procedure used for the introduction of a retrovirus into a cell.
- the retrovirus contains a transgene, and the result of retroviral infection is the introduction of the transgene into the cell.
- the invention provides a number of advantages. The LOPU procedure can be repeated on the same animal for several months without a reduction in the number of oocytes recovered.
- FIG. 1 Brief Description of the Drawing Figure 1 is a schematic representation of steps involved in the generation of transgenic animals using oocytes recovered by the LOPU procedure.
- Example 1 Synchronization and Gonadotrophin Stimulation of Goats to be Used as Donors of Oocytes Recovered by LOPU
- Adult Goats Adult goats may be subjected to LOPU without any hormonal stimulation. However, higher numbers of oocytes are obtained if donor goats are synchronized and stimulated with gonadotrophins. Synchronization of donor goats may be achieved using established protocols known to those skilled in the art. The following is an example of a synchronization protocol which may be used.
- Intravaginal sponges containing 60 mg of medroxyprogesterone acetate were inserted into the vagina of donor goats and left in place for 7 to 10 days, with an injection of 125 ⁇ g cloprostenol given 48 hours before sponge removal.
- the sponge was left in place until the time of oocyte collection, while for the recovery of oocytes which were more mature, the sponge was removed up to 48 hours before oocyte collection.
- the priming of the ovaries was achieved using gonadotrophin preparations including follicle stimulating hormone (FSH), equine chorionic gonadotrophin (eCG), and human menopausal gonadotrophin (hMG). Any established regime for superovulation known by those skilled in the art may be used. The following hormonal regimes are examples of methods which may be used.
- a total dose equivalent to 120 mg of NIH-FSH-P1 was given twice daily in decreasing doses (35 mg/dose on the first day, 25 mg/dose on the second day) beginning 48 hours before sponge removal.
- 70 mg of NIH- FSH-P1 was given together with 400 IU of eCG 36 to 48 hours before LOPU.
- the recovered oocytes were then matured in vitro as described in Example 3.
- An alternative strategy for the recovery of oocytes is to aspirate oocytes that have been matured in vivo. For this purpose it is essential to control the number of hours between the luteinizing hormone (LH) peak and the time at which the oocytes are collected. This may be achieved by drug- induced depletion of the endogenous LH peak.
- LH luteinizing hormone
- the FSH/LH contents of the hypophysis may be depleted using gonadotrophin releasing hormone (GnRH) agonists such as buserelin or deslorelin.
- GnRH gonadotrophin releasing hormone
- the hypophysis may be made refractory to hypothalamic GnRH using a GnRH antagonist such as cetrorelix.
- the desired GnRH agonist/antagonist may be administered by means of repeated injections, or more appropriately, by means of drug release devices such as subcutaneous implants or pumps.
- the GnRH agonist/antagonist is administered to the donor goats for at least 7 days prior to the start of gonadotrophin stimulation, and the treatment is continued until the LOPU procedure occurs. Follicular development is then stimulated by means of administration of gonadotrophins using a similar protocol as described above.
- Prepubertal Goats To recover oocytes from prepubertal goats, synchronization is not required. However, for recovering high numbers of oocytes, donor goats may need to be stimulated with gonadotrophin. This may be achieved by applying the same regimes used for superovulation of adult goats, as described above.
- Oocytes from donor goats were recovered by aspiration of follicle contents (puncture or folliculocentesis) under laparoscopic observation.
- the laparoscopy equipment used (Richard Wolf, Germany) was composed of a 7 mm telescope, light cable, light source, 7 mm trocar for the laparoscope, atraumatic grasping forceps, and two 5 mm "second puncture" trocars.
- the follicle puncture set was composed of a puncture pipette, tubing, a collection tube, and a vacuum pump.
- the puncture pipette was made using a PVC pipette (5 mm external diameter, 2 mm internal diameter), and a 20G short bevel hypodermic needle, which was cut to a length of 5 mm and fixed into the tip of the pipette with instant glue.
- the connection tubing was made of silicon with an internal diameter of 5 mm, and connected the puncture pipette to the collection tube.
- the collection tube was a 50 ml centrifuge tube with an inlet and an outlet available in the cap. The inlet was connected to the pipette, and the outlet was connected to a vacuum line. Vacuum was provided by a vacuum pump connected to the collection tube by means of PVC 8 mm tubing.
- the vacuum pressure was regulated with a flow valve and measured as drops of collection media per minute entering the collection tube. The vacuum pressure was usually adjusted to 50 to 70 drops/minute.
- the complete puncture set was washed and rinsed ten times with tissue culture quality distilled water before gas sterilization, and one time with collection medium before use.
- the collection medium was TCM 199 supplemented with 0.05 mg/ml of heparin and 1% (v/v) fetal calf serum (FCS).
- FCS fetal calf serum
- the collection tube contained approximately 0.5 ml of this medium to receive the oocytes.
- the goats were fasted 24 hours prior to laparoscopy. Anaesthesia was induced by intravenous administration of diazepam (0.35 mg/kg body weight) and ketamine (5 mg/kg body weight), and maintained with isofluorane via endotrachial intubation. The animals were restrained in a cradle position for laparoscopic artificial insemination as described by Evans and Maxwell (Salomon's Artificial Insemination of Sheep and Goats, Sydney: Butterworths, 1987). The 3 trocars described above were inserted and the abdominal cavity was filled with filtered air. The ovary was exposed by pulling the fimbria in different directions and the follicles were punctured.
- the needle was inserted into the follicle and rotated gently to ensure that as much of the follicle contents as possible were aspirated. After 3 to 5 follicles were aspirated, the pipette and tubing were rinsed using collection media from a sterile tube.
- Results from LOPU procedures performed on two types of goats, standard dairy breeds (STD) and dwarf breeds (BELE), receiving hormonal treatments (as described in Example 1) in terms of number of follicles (FL) aspirated, and cumulus-oocyte complexes (COCs) recovered per donor (average ⁇ standard deviation) are presented in Table 1.
- OS* one shot; all the gonadotrophic stimulation was given in a single injection, either 36 or
- Oocyte Maturation In vitro oocyte maturation (IVM) was done as follows. Cumulus-oocyte complexes (COCs) obtained by LOPU were washed in Ml 99 supplemented with penicillin (100 IU/ml), streptomycin (0.1 mg/ml), kanamycin (0.05 mg/ml), and bovine serum albumin (BSA; 0.5%). COCs were placed into 1.5 ml vials containing 1 ml of the wash medium and transported to the laboratory in a thermos containing warm (34°C to 38°C) water.
- COCs Cumulus-oocyte complexes obtained by LOPU were washed in Ml 99 supplemented with penicillin (100 IU/ml), streptomycin (0.1 mg/ml), kanamycin (0.05 mg/ml), and bovine serum albumin (BSA; 0.5%). COCs were placed into 1.5 ml vials containing 1 ml of the wash medium and transported to the laboratory in a
- the COCs were then transferred into 50 ⁇ l drops of maturation medium, overlayed with mineral oil, and incubated at 38.5 °C to 39°C for 24 to 27 hours in air containing 5% C0 2 .
- An example of maturation medium consisted of M199 supplemented with bLH (0.02 U/ml; Sioux Biochemicals), bFSH (0.02 U/ml; Sioux Biochemicals), estradiol ⁇ -17 (1 ⁇ g/ml; Sigma), sodium pyruvate (0.2 mM; Sigma), kanamycin (50 ⁇ g/ml), and 10% heat- inactivated fetal calf serum (ImmunoCorp) or goat serum.
- In vitro fertilization was performed as follows. The expanded cumulus cells were partially or fully removed from the matured COCs by repeated pipetting with a fine bore pipette, dissection using 18 to 24G needles, or vortexing. Oocytes were washed in fertilization medium and transferred to drops of the same medium under mineral oil.
- SOFM synthetic oviduct fluid medium
- penicillamine 3 ⁇ g/ml
- hypotaurine 1 ⁇ g/ml
- 20% oestrus sheep serum was the original synthetic oviduct fluid medium described by Tervit et al. (J. Reprod.
- Sperm capacitation may be accomplished using either fresh or frozen semen.
- the semen may be either transgenic or non-transgenic.
- Several protocols exist for this procedure In one method, fresh semen was collected on the day of use. Motile sperm were separated on a 45%:90% Percoll gradient. Alternatively, frozen semen was thawed in a sterile glass tube and was overlaid on top of a 45%:90% Percoll gradient. The resulting pellet was washed in HEPES-SOFM or mDM (glucose free -DM; Brackett, Biol. Reprod. 12:260-274, 1975) and resuspended in fertilization medium or mDM containing the appropriate capacitation factors.
- Capacitation may be performed using a variety of protocols. Examples of such protocols follow.
- the sperm were resuspended in either SOFM containing penicillamine (3 ⁇ g/ml), hypotaurine (1 ⁇ g/ml), and 20% oestrus sheep serum, or in mDM containing 8-bromo-cAMP (0.5 mM), ionomycin (100 nM), and heparin (10 ⁇ g/ml), or in mDM supplemented with calcium lactate (7.75 mM), caffeine (0.4 mg/ml), and heparin (10 ⁇ g/ml).
- the sperm concentration was then adjusted to a final concentration of 10 6 sperm/ml, and the sperm was added to fertilization drops containing the oocytes.
- IVC In vitro culture
- Example 4 Transgenes Used for the Generation of Transgenic Goats and the Production of Heterologous or Homologous Protein in Milk, Urine, or Blood of the Transgenic Animal
- a genetic construct suitable for use in the present invention generally includes the following elements:
- an insulator element e.g., chicken ⁇ -globin or chicken lysozyme MARS elements
- a gene dosage effect i.e., more copies of the transgene yield increased protein expression
- a position-independent expression which is a result of the insulating effect from surrounding chromatin.
- Useful promoters include as 1 -casein (as described, for example, in U.S. Patent No. 5,304,489), ⁇ s2-casein, ⁇ -casein, ⁇ -casein, ⁇ -lactoglobulin (as described, for example, in U.S. Patent No. 5,322,773), ⁇ -lactalbumin, and whey acidic protein (WAP).
- the promoter may be linked to enhancer elements (such as CMV or SV40) or insulator elements (such as chicken ⁇ -globin).
- a DNA expression cassette using the WAP promoter for example, as described in WO 92/22644, and insulator elements operably linked to a heterologous gene (in this case, a gene from a spider encoding components of spider silk) can be used as illustrated in WO 99/47661 A2.
- This genetic construct also includes a transcription termination region.
- the termination region includes a poly-adenylation site at the 3' end of the gene from which the promoter region of the genetic construct was derived.
- the heterologous or homologous gene may be either a cDNA or genomic clone containing introns (all or a subset). If the gene is a cDNA clone, the genetic construct preferably also includes an intron which may increase the level of expression of the particular gene.
- Useful introns for example, are those found in genes encoding caseins or immunoglobulins.
- Urine-specific expression of a heterologous or homologous protein Useful promoters for the urine-specific expression of a heterologous or homologous protein are those disclosed in PCT/US96/08233, and U.S. Patent No.
- the uroplakin II promoter has been shown to direct the expression of hGH in the urine of transgenic mice in detectable levels.
- Other useful promoters include kidney-specific promoters such as rennin and uromodulin.
- Blood-specific expression of human immunoglobulin A genetic construct that directs the blood-specific expression of human immunoglobulin may also be prepared. Such a construct would include human Ig loci containing plural variable V h and V k regions either as a mini-locus region or as a large portion of the Ig locus, as described in PCT/US97/23091 and references therein. Such a construct may be created using, for example, yeast artificial chromosomes (YACS) or mammalian artificial chromosomes.
- YACS yeast artificial chromosomes
- mammalian artificial chromosomes mammalian artificial chromosomes.
- the genetic construct may be microinjected into the pronucleus of a zygote or used to make retroviral particles which can infect an oocyte or zygote or embryo.
- the resulting embryo is transferred to a recipient (surrogate) mother and an offspring is born.
- This animal will have functional, mature B-cells expressing high affinity antibodies in the animal.
- a specific antigen e.g., anthrax
- human antibodies will be produced, which may be purified from the plasma of these animals and subsequently used for the treatment of animals according to techniques described in WO 98/24893, hereby incorporated by reference.
- Example 5 Microinjection of DNA into Zygotes Produced from LOPU-derived Oocytes Zygotes produced as described in Example 3 are used in microinjection experiments in which one or both pronuclei are injected with a DNA construct of interest, as described in Example 4.
- oocytes were observed for pronuclear (PN) formation using a microscope equipped with DIC optics.
- oocytes may be microcentrifuged. Zygotes with visible PN were microinjected with the DNA construct (1-2 ng/ml in a Tris-EDTA-NaCl buffer; pH 7.5) of interest.
- transfer of embryos is preferably performed 2-4 hours following microinjection.
- the following protocol is an example of an in vitro culture method known to those skilled in the art.
- Embryo transfer Injected embryos at the pronuclear or 2- to 8-cell stage may be transferred to the oviduct of synchronized recipients. If the embryos are at the morula or blastocyst stage they may be transferred to the uterus of synchronized recipients. Pregnancies were determined by ultrasound monitoring at 30 and 60 days of gestation.
- Prepubertal goats Ten prepubertal Nubian goats were hormonally stimulated and oocytes were collected by LOPU (610 follicles were aspirated and 500 oocytes recovered). Twenty-four zygotes were transferred after IVF, to 4 recipients, of which, 2 became pregnant (50% initial pregnancy rate). Another set of 35 IVM/IVF-produced zygotes were microinjected and transferred to 5 recipients, of which, 3 became pregnant (60% initial pregnancy rate).
- Recipients are synchronized by any established regime known by those skilled in the art. They should be observed on standing heat during the day that the oocytes are in vitro fertilized.
- the following hormonal protocol is one example of a method which may be used. Intravaginal sponges containing 60 mg of medoxyprogesterone acetate were inserted into the vagina of recipient goats and left in place for 7 to 10 days with an injection of 125 ⁇ g closprostenol given 48 hours before sponge removal. Sponges were removed and an injection of 400 IU of eCG was administered on the same day as the LOPU took place.
- microinjected embryos were either incubated for a short period (2-5 hours), overnight, or for 5 days, and then transferred to synchronized recipients.
- the microinjected embryos may be cultured in vitro following any established protocol known by those skilled in the art.
- the following protocol is an example of one such method for the in vitro culture of microinjected embryos.
- Microinjected embryos were cultured under mineral oil, in 40 ⁇ l drops of culture medium consisting of SOFM supplemented with 8 mg/ml BSA-Fatty Acid Free. This in vitro culture was carried out at 39 °C in a humidified incubator with 5% C0 2 , 7% 0 2 , and 88% N 2 .
- the embryos were moved every 48 hours into fresh drops of medium prepared 3 to 4 hours in advance.
- the recipient goats were fasted 24 hours prior to surgery.
- Anesthesia was induced by intravenous administration of diazepam (0.35 mg/kg body weight) and ketamine (5 mg/kg body weight), and maintained with isofluorane via endotrachial intubation.
- a laparoscopic exploration was then performed to confirm if the recipient had one or more recent ovulations/corpora lutea (CL) present in the ovaries and a normal oviduct and uterus.
- the laparoscopic exploration was carried out to avoid performing a laparotomy on an animal which had not responded properly to the hormonal synchronization protocol and to which an embryo should not be transferred.
- the embryos may be transferred to the oviduct of recipient goats.
- a mid-ventral laparotomy of approximately 10 cm in length is established, the reproductive tract is exteriorized, and the embryos are implanted into the oviduct ipsilateral to ovulation/s by means of a TomCat catheter threaded into the oviduct from the fimbria. If embryos are cultured for 5 days, the resulting morula/blastocyst- staged embryos may be transferred to the uterus.
- a mid- ventral laparotomy of approximately 5 cm in length is established and the uterine horn ipsilateral to the CLs is exteriorized using a surgical clamp under laparoscopic observation. A small perforation is made with an 18G needle in the oviduct third of the horn, and the embryos are then implanted by means of a TomCat catheter threaded into the uterine lumen.
- Tables 2 and 3 summarize the results obtained when a series of LOPU procedures were performed on the same animals, as a demonstration of the reproducibility of the technique. The interval between collections was 30 to 60 days. These results demonstrate that the LOPU procedure can be repeated on the same animal for several months without a decrease in the number of oocytes recovered.
- Example 9 Infection of Oocytes Using Retroviruses
- This example describes the introduction of exogenous DNA sequences into the genome of unfertilized oocytes recovered as described in the present invention.
- the DNA is introduced by microinjection of retroviruses into the peri-vitelline space of the oocytes.
- the integration of retroviral genome into the genome of a pre- maturation or pre-fertilization oocyte followed by fertilization may be achieved as taught in WO 98/141,615, hereby incorporated by reference.
- the resulting embryo may be cultured or transferred to a recipient goat as described above.
- Morula and blastocyst-staged embryos derived from LOPU oocytes which underwent IVM/IVF/IVC were transferred into 2 recipients, one of which became pregnant giving birth to two normal kids (one male, one female). Seven hundred twenty-one COCs were recovered from donor goats. After in vitro fertilization, 246 developed to the pronuclear stage, and of those, 222 were microinjected with a WAP-BIOSTEEL DNA construct and transferred to 36 synchronized recipients (an average of 6.2 embryos were transferred per recipient). Twenty recipients kidded 30 kids and of those 2 were transgenic for the WAP-BIOSTEEL construct, as tested by PCR and Southern blotting analyses. One of the transgenic kids was derived from an oocyte obtained from a pre-pubertal goat, while the other was derived from an oocyte obtained from an adult goat. A summary of all of the recipient goat pregnancy rates now follows.
- oocytes derived from adult donor goats 564 DNA microinjected zygotes were generated. These zygotes were transferred into 98 recipients (an average of 5.7 embryos per recipient), and 51 recipients became pregnant (54% pregnancy rate).
- oocytes derived from prepubertal donor goats oocytes were recovered from 10 different donors. A total of 610 follicles were aspirated, and 500 oocytes were recovered. Twenty- four non-microinjected zygotes were transferred to 4 recipients, of which, 2 became pregnant (50% pregnancy rate). One hundred forty-seven DNA microinjected zygotes were transferred into 25 recipients, and 11 recipients became pregnant (44% pregnancy rate).
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
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CA002388758A CA2388758A1 (en) | 1999-10-29 | 2000-10-27 | Transgenic animal production using lopu-derived oocytes |
AU14413/01A AU1441301A (en) | 1999-10-29 | 2000-10-27 | Transgenic animal production using lopu-derived oocytes |
EP00976673A EP1224276A4 (en) | 1999-10-29 | 2000-10-27 | Transgenic animal production using lopu-derived oocytes |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US16253099P | 1999-10-29 | 1999-10-29 | |
US60/162,530 | 1999-10-29 |
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WO2001032855A1 true WO2001032855A1 (en) | 2001-05-10 |
WO2001032855A9 WO2001032855A9 (en) | 2001-09-27 |
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Application Number | Title | Priority Date | Filing Date |
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PCT/US2000/029751 WO2001032855A1 (en) | 1999-10-29 | 2000-10-27 | Transgenic animal production using lopu-derived oocytes |
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EP (1) | EP1224276A4 (en) |
AU (1) | AU1441301A (en) |
CA (1) | CA2388758A1 (en) |
WO (1) | WO2001032855A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8530168B2 (en) | 2007-06-21 | 2013-09-10 | Helmholtz Zentrum Munich Deutsches Forschungszentrum Fuer Gesundheit Und Umwelt (Gmbh) | Fusion protein comprising a Caspase domain and a nuclear hormone receptor binding domain and methods and uses thereof |
WO2016054032A1 (en) * | 2014-09-29 | 2016-04-07 | The Jackson Laboratory | High efficiency, high throughput generation of genetically modified mammals by electroporation |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
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US5366888A (en) * | 1990-07-09 | 1994-11-22 | Amrad Corporation Limited | Enhanced maintenance of pregnancy using leukaemia inhibitory factor in embryo culturing |
US5693534A (en) * | 1993-02-23 | 1997-12-02 | Genentech, Inc. | Enhancement of fertilization capability of oocytes |
US5895833A (en) * | 1993-01-28 | 1999-04-20 | Cohesion Technologies, Inc. | Production of human recombinant collagen in the milk of transgenic animals |
US5907080A (en) * | 1995-11-30 | 1999-05-25 | Nexia Biotechnologies, Inc. | Method for development of transgenic dwarf goats |
US6110741A (en) * | 1996-05-29 | 2000-08-29 | Wisconsin Alumni Research Foundation | Gonadotrophin releasor hormone-containing composition for embryo culture and method for in vitro fertilization |
-
2000
- 2000-10-27 EP EP00976673A patent/EP1224276A4/en not_active Withdrawn
- 2000-10-27 WO PCT/US2000/029751 patent/WO2001032855A1/en not_active Application Discontinuation
- 2000-10-27 CA CA002388758A patent/CA2388758A1/en not_active Abandoned
- 2000-10-27 AU AU14413/01A patent/AU1441301A/en not_active Abandoned
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5366888A (en) * | 1990-07-09 | 1994-11-22 | Amrad Corporation Limited | Enhanced maintenance of pregnancy using leukaemia inhibitory factor in embryo culturing |
US5895833A (en) * | 1993-01-28 | 1999-04-20 | Cohesion Technologies, Inc. | Production of human recombinant collagen in the milk of transgenic animals |
US5693534A (en) * | 1993-02-23 | 1997-12-02 | Genentech, Inc. | Enhancement of fertilization capability of oocytes |
US5907080A (en) * | 1995-11-30 | 1999-05-25 | Nexia Biotechnologies, Inc. | Method for development of transgenic dwarf goats |
US6110741A (en) * | 1996-05-29 | 2000-08-29 | Wisconsin Alumni Research Foundation | Gonadotrophin releasor hormone-containing composition for embryo culture and method for in vitro fertilization |
Non-Patent Citations (1)
Title |
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See also references of EP1224276A4 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8530168B2 (en) | 2007-06-21 | 2013-09-10 | Helmholtz Zentrum Munich Deutsches Forschungszentrum Fuer Gesundheit Und Umwelt (Gmbh) | Fusion protein comprising a Caspase domain and a nuclear hormone receptor binding domain and methods and uses thereof |
WO2016054032A1 (en) * | 2014-09-29 | 2016-04-07 | The Jackson Laboratory | High efficiency, high throughput generation of genetically modified mammals by electroporation |
Also Published As
Publication number | Publication date |
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EP1224276A4 (en) | 2004-04-07 |
EP1224276A1 (en) | 2002-07-24 |
AU1441301A (en) | 2001-05-14 |
CA2388758A1 (en) | 2001-05-10 |
WO2001032855A9 (en) | 2001-09-27 |
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