EP1242591A1

EP1242591A1 - Production of transgenic animals using nuclear transfer and oocytes recovered by lopu

Info

Publication number: EP1242591A1
Application number: EP00975410A
Authority: EP
Inventors: Hernan Baldassarre; Carol L. Keefer; Bin Wang; Costas N. Karatzas
Original assignee: Nexia Biotechnologies Inc
Current assignee: Nexia Biotechnologies Inc
Priority date: 1999-10-29
Filing date: 2000-10-27
Publication date: 2002-09-25
Also published as: WO2001032854A8; CA2388722A1; AU1346601A; WO2001032854A1

Abstract

Methods for generating transgenic non-human animals using enucleated oocytes obtained by laparoscopic ovum pick.

Description

PRODUCTION OF TRANSGENIC ANIMALS USING NUCLEAR TRANSFERAND OOCYTES RECOVERED BY LOPU

Background of the Invention The field of the invention is the development and propagation of transgenic animals.

Techniques to generate transgenic animals by the introduction of a recombinant DNA into zygotes, fetal cells, or oocytes are well known

(reviewed by Wall, Theriogenology 45:57-68, 1996). Methods to develop transgenic animals carrying a gene fused to a tissue-specific promoter, such as milk, are also known (WO 93/25567). The use of transgenic animals carrying such transgenes makes it possible to produce desired peptides in the animals. These peptides 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 by nuclear transfer, they or their offspring may be used in efficient, large quantity production of desired polypeptides. In this context, tissue-specific expression and production of proteins has been demonstrated.

Summary of the Invention The invention features methods for the generation of a transgenic animal using oocytes recovered through laparoscopic aspiration of follicles and nuclear transfer (NT) techniques. This oocyte recovery procedure is also known as laparoscopic ovum pick up (LOPU), laparoscopic follicle aspiration, or laparoscopic oocyte aspiration. A transgene may be introduced into the oocytes by enucleating the oocytes and fusing them with a donor cell containing the gene.

Accordingly, in a first aspect, the invention features a method for generating a non-human transgenic animal containing a desired gene, involving the steps of: (a) recovering an oocyte from a donor animal by laparoscopic ovum pick up;

(b) enucleating the oocyte;

(c) providing a cell containing the desired gene;

(d) fusing the cell with the oocyte to form a fused couplet; (e) activating the couplet to form a zygote;

(f) transferring the zygote, or a cleaved embryo, morulae, or blastocyst formed from culturing said zygote, into a recipient animal; and

(g) allowing the zygote, cleaved embryo, morulae, or blastocyst to develop to term. In a second aspect, the invention features a method for generating a non-human transgenic animal containing a desired gene, said method involving:

(a) recovering an oocyte from a donor animal by laparoscopic ovum pick up; (b) activating the oocyte;

(c) enucleating the activated oocyte;

(d) providing a cell containing a desired gene;

(e) fusing the cell with the enucleated activated oocyte to form a fused couplet; (f) allowing the couplet to form a zygote;

(g) transferring the zygote, or a cleaved embryo, morulae, or blastocyst formed from culturing the zygote, into a recipient animal; and (h) allowing the zygote, cleaved embryo, morulae, or blastocyst to develop to term.

In one embodiment of the above aspects of the invention, the cell and the oocyte are derived from the same animal or different animals. The animals can be from the same or different breeds. In another embodiment of the above aspects of the invention, the animal is a ruminant, such as a goat or a sheep.

By "transgenic animal" is meant a non-human animal containing a transgene.

By "transgene" is meant a DNA sequence introduced into the germline of a non-human animal by way of human intervention using any of the methods described herein.

By "tissue-specific expression" is meant the expression within a specifically desired tissue or a product which is released from a specific tissue. As used herein, "tissue-specific expression" refers to the production of a protein in the milk, urine, or blood specifically.

As referred to herein, by "prepubertal" is meant less than 5 months of age.

By "laparoscopic ovum pick up" or "LOPU" is meant a procedure used to recover oocytes. The invention provides a number of advantages. The LOPU procedure may be repeated on the same animal for several months without a reduction in the number of oocytes recovered. Also, no apparent adhesions develop as a result of LOPU procedures. In addition, the ability to generate a transgenic animal using oocytes generated from a prepubertal donor is advantageous because it reduces the time required for propagating generations of transgenic animals in the case that the same animal is the donor of both the oocyte (recovered by LOPU) and the nucleated cell to be used for the nuclear transfer process.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

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 technique.

Detailed Description 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 μg cloprostenol given 48 hours before sponge removal. Typically, for recovery of immature oocytes, the sponge was left in place until the oocyte collection, while for the recovery of oocytes more advanced in maturation, the sponge was removed up to 48 hours before the 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) starting 48 hours before sponge removal. Alternatively, 70 mg of NIH- FSH-P1 may be 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 which 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. For example, the FSH/LH contents of the hypophysis may be depleted using gonadotrophin releasing hormone (GnRH) agonists such as buserelin or deslorelin. Alternatively, 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.

Example 2 Laparoscopic Ovum Pick-Up

Oocytes from donor goats were recovered by aspiration of follicle contents (puncture or folliculocentesis) under laparoscopic observation. The laparoscopy equipment used (commercially available from 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, and was usually adjusted to 50-70 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). 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 surface was visualized and the follicles were punctured by pulling the fimbria in different directions with the grasping forceps. The needle was inserted into the follicle and rotated gently to ensure that as much of the follicle contents as possible were aspirated. After aspiration of 3 to 5 follicles, the pipette and tubing were rinsed using sterile collection media. Results from LOPU 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. Table 1. COCs Recovered from Goats Given Different Hormonal Treatments Prior to the LOPU Procedure

= ar y r y STD= standard breed

OS* = one shot; all the gonadotrophic stimulation was given in a single injection, either 36 or 48 hours before LOPU

Example 3 Culture and Enucleation of Oocytes Recovered from Goats by LOPU Oocyte preparation: Cumulus-oocyte complexes (COCs) were recovered from primed follicles by LOPU. The COCs were washed once in 2 ml of Ml 99 containing 0.5% BSA, placed into 50 μl drops of maturation medium, covered with an overlay of mineral oil (Sigma), and incubated at 38.5 °C-39°C in 5% C0₂. The maturation medium consisted of Ml 99 supplemented with bLH

(0.02 U; Sioux Biochemicals), bFSH (0.02 U; Sioux Biochemicals), estradiol β- 17 (1 μg/ml; Sigma), sodium pyruvate (0.2 mM; Sigma), kanamycin (50 μg/ml), and 10% heat-inactivated fetal calf serum (ImmunoCorp), goat serum, or estrous goat serum. After 23-24 hours of maturation, the cumulus cells were removed from the matured oocytes by placing the COCs in a 1.5 ml microcentrifuge tube containing 250 μl of EmCare supplemented with hyaluronidase (1 mg/ml), and vortexing for 1-2 minutes. The cumulus cells may be used in subsequent manipulations, for example, gene transfer, as donor cells for oocytes derived from the same animal or a different animal.

The denuded oocytes were washed in EmCare containing 1 % FCS and returned to maturation medium. Fifteen to twenty denuded oocytes were placed into a microdrop (50 μl) containing 5 μg of the fluorescent DNA dye Hoeschst 33342 (stock solution 1 mg/ml saline) in 1 ml of EmCare containing 1 % FCS. The oocytes were incubated in the Hoeschst-EmCare solution for 20- 30 minutes at 30 °C-36°C.

Manipulation of Oocytes: One manipulation drop (150 μl) of EmCare supplemented with 1% FCS was placed into a 100 mm Optics dish (Falcon), centered, and covered completely with mineral oil. Oocytes stained with the Hoeschst dye were placed into the center of the manipulation drop. Each oocyte was picked up using the holding pipette and rotated until the polar body (PB) was visualized between 3- and 6 o'clock. The edge of the oocyte- containing polar body was moved into a fluorescent UV light path and the location of the chromosomes were noted. The oocyte was pulled slightly out of the UV light path, and the cytoplasm in the area containing the chromosomes and polar body was removed using the manipulation pipette. The removed cytoplasm was checked for the presence of chromosomes and the polar body by moving the pipette into the UV light path. The process was repeated until all oocytes were enucleated. The enucleated oocytes were then placed into a droplet of EmCare containing 1% FCS, and overlaid with 2 ml of mineral oil in a Falcon 1008 dish. These dishes were kept on a warm surface (30°C-36°C). Alternatively, the enucleated oocytes were returned to the maturation drop if the nuclear transfer procedure was not immediate. Results: In 11 sessions, 734 LOPU-derived and in trø-matured oocytes were prepared for enucleation. The denuded oocytes were stained with Hoeschst 33342 and their stage of nuclear maturation was observed. Sixty-nine percent (235/338) of the oocytes matured in maturation medium supplemented with FCS were in metaphase II (Mil), while 86% (341/396) of the oocytes matured in maturation medium supplemented with goat serum were in MIL A total of 462 karyoplast-cytoplast couplets were produced using the LOPU-derived enucleated oocytes (462 cytoplasts/576 enucleated Mil oocytes; 80% production).

Isolation of Activated Oocytes: Alternatively, if desired, an activated oocyte may be used to carry out the present invention. To activate an oocyte, one would carry out the oocyte preparation and manipulation procedures as described above. Upon observation of the denuded oocytes stained with Hoeschst 33342, oocytes which are in the telophase stage of nuclear maturation are considered to be activated. These oocytes may be selected and fused with a cell to form a fused couplet which does not require further activation.

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:

(a) a promoter or transcription initiation regulatory unit;

(b) a transcription termination codon; (c) DNA encoding a useful protein, or a nucleotide consisting of a ribozyme or antisense oligonucleotide;

(d) optionally, a naturally-occurring or synthetic sequence encoding a signal polypeptide directing the secretion of the recombinant protein from the cell if secretion is desired; and (e) optionally, an insulator element (e.g., chicken β-globin or chicken lysozyme MARS elements) which may result in a gene dosage effect (i.e., more copies of the transgene yield increased protein expression) or may allow for position-independent expression which is a result of the insulating effect from surrounding chromatin. Conventional molecular biology methods are used to generate and assemble the above elements.

Milk-specific expression of a heterologous or homologous protein: Useful promoters include as 1 -casein (as described, for example, in U.S. Patent No. 5,304,489), αs2-casein, β-casein, κ-casein, β-lactoglobulin (as described, for example, in U.S. Patent No. 5,322,773), α-lactalbumin, and whey acidic protein (WAP). If desired, the promoter may be linked to enhancer elements (such as CMV or SV40) or insulator elements (such as chicken β-globin).

An example of 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. Preferably, 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.

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.

5,824,543, such as uroplakins I, II, and III, hereby incorporated by reference.

The uroplakin II promoter, for example, 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 includes 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 cited therein. Such a construct can be created using, for example, yeast artificial chromosomes (YACS) or mammalian artificial chromosomes.

Preferably a construct containing the Ig locus or loci is introduced into a cell in which the endogenous Ig loci have been interrupted and rendered non-functional. This cell may then be used for embryo reconstruction using nuclear transfer techniques. This will allow the development of functional, mature B-cells expressing high affinity antibodies in the animal. Upon challenge with 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 98/24,893, hereby incorporated by reference.

Example 5 Introduction of a Transgene into a Cell Line from which a Donor Nucleus is Used in Nuclear Transfer Experiments

In all of the above examples, the genetic construct may be introduced into a cell type of interest, for example, a fetal fibroblast (using, for example, the methods of Cibelli et al., Science 280: 1256-1528, 1998) or cumulus cells (using, for example, the methods of Kato et al., Science 282:2095-2098, 1998) by a variety of techniques, including electroporation, lipofection, calcium phosphate transfection, viral infection, and microinjection. Preferably the transgene is transfected with a selectable marker so selection of cells containing the transgene may be achieved. Such selection markers include, but are not limited to G418, hygromycin, and puromycin. It may also be desirable for the transgene to specifically target an area of the genome of the cell by using, for example, the Cre-lox system (Melton, Bioessays 16:633-638, 1994; Guo et al., Nature 389:40-46, 1997). In all of the examples described above the selected cell line is used in the subsequent step of fusion with an enucleated LOPU- derived oocyte.

Example 6

Nuclear Transfer (Fusion and Activation) and Culture of the Nuclear

Transfer-derived Embryo Culture

Preparation of donor cells by serum starvation to generate GO cells: Fetal fibroblasts were isolated from day 27 to day 30 fetuses from the dwarf breed of goat BELE^® (Breed Early Lactate Early). The cells were transfected with a construct containing the enhanced green fluorescent protein (GFP) gene. Four GFP positive clones (one male and three female, including the donor cell line BELE-FF3-GFP) and one non-transfected male line (BELE-FF4) were used as donor cells in nuclear transfer. Eight days prior to the nuclear transfer, 2.5 x 10⁴ donor cells were plated in one well of a 24-well plate in 1.5 ml of complete media (DMEM supplemented with 10% FBS, 0.1 miM β-mercaptoethanol, and 0.1% gentamycin) and incubated in a humidified atmosphere at 37 °C and 5% C0₂. The next day, fresh complete media was added to the well. Two days later the media was again replaced with fresh media. Four to eight days prior to nuclear transfer, the cells were washed twice, placed into low serum media (DMEM supplemented with 0.5% FBS, 0.1 mM β-mercaptoethanol, and 0.1 % gentamycin), and returned to the incubator (37 °C and 5% CO,) until the day of nuclear transfer. Low serum media was replaced with fresh low serum media every 24-48 hours.

On the day of nuclear transfer the donor cells were prepared as follows. Thirty minutes before they were needed, the cells were rinsed quickly with pre-warmed 0.05% trypsin/EDTA, and incubated with 200 μl of the same solution for 3 minutes in the incubator. The cells were recovered from the well and placed into a cryovial with EmCare supplemented with 1% FCS. The cells were pelleted by centrifugation (875xg for 3 min) and resuspended twice in EmCare supplemented with 1 % FCS. The final donor cell suspension (500 μl per ml of EmCare containing 1 % FCS) was placed in a 35 mm suspension dish and the cells were used immediately for nuclear transfer.

Oocyte preparation: Cumulus-ooctyes complexes (COCs) were recovered from primed follicles by LOPU as described in Example 2. Manipulation of Oocytes: The enucleation of LOPU-derived oocytes was achieved as described in Example 3.

Fusion: A donor cell was picked up with the manipulation tool and slipped into the perivitelline space. Cell-cytoplast couplets were fused using electrofusion as soon after enucleation of the oocytes as possible. The couplets were moved through dishes containing (i) EmCare supplemented with 1 mg of BSA/ml; (ii) a 1: 1 dilution of sorbitol fusion medium (0.25 M sorbitol, 0.1 mM calcium acetate, 0.5 mM magnesium acetate, 0.1% bovine serum albumin) and EmCare; and (iii) sorbitol fusion medium. Groups of four to six couplets were aligned between the electrodes of a BTX fusion chamber (catalog No. 450) in a 100 mm plate containing sorbitol fusion medium. A brief fusion pulse was administered by a BTX and optimizer. A typical pulse of 17 μsec at 2.39 kV/cm (90 V peak) was applied.

The couplets were moved through the sorbitol fusion medium/EmCare solution and the EmCare/BSA solution, and then placed in microdrops of EmCare supplemented with 1% FCS. After all couplets had been exposed to the fusion pulse they were placed into culture drops of the appropriate medium (SOFM according to Tervit et al. (J. Reprod. Fertility 30:493-497, 1972); Gl according to Gardner and Lane (Human Reprod., Update 3:367-382, 1997); or TCM containing 10% fetal calf serum, and incubated at 38.5 °C-39°C in 5% C0₂, 7% 0₂, and 88% N₂.

After 2-3 hours, the fused couplets were activated using the calcium ionophore and DMAP method of Susko-Parrish et al. (Biol. Reprod. 51 : 1099- 1 108, 1994) or by application of additional electrical pulses (1.26 kV/cm, 80 μsec), followed by incubation in nocodozole or cytochalasin B (Campbell et al., Nature 380:64-66, 1996). After being cultured for 2.5 to 4 hours in DMAP, nocodazole, or cytochalasin B, activated nuclear transfer-derived zygotes were returned to culture drops containing SOFM or Gl. Cleavage development (2- to 4-cell stages) was observed at 22 hours (the night before embryo transfer) and 36 hours (the morning of embryo transfer). Nuclear transfer-derived embryos were transferred into synchronized recipients between days 1 and 12 post fusion (day 0 = day of fusion).

In Vitro Culture: Reconstructed embryos were placed into microdrops of 25 μl of Gl or low phosphate (0.35 mM) SOFM embryo culture medium (Gardner et al., Biol. Reprod. 50:390-400, 1994) under an oil overlay. After 48-72 hours, cleaved embryos were moved to fresh microdrops of embryo culture medium. On day 4 or 5 (day 0 = day of fusion) embryos were moved to microdrops of G2 medium or high phosphate (1.2 mM) SOFM.

Embryo transfer: Nuclear transfer-derived zygotes, or cleaved embryos at the 2- to 8-cell stage were transferred into the oviduct of a synchronized recipient. Morulae and blastocysts were transferred into the uterus of a synchronized recipient. Pregnancies were determined at 30 and 60 days of gestation.

Results: A total of 462 karyoplast-cytoplast couplets received one or more fusion pulses, with 54% appearing fused and 43% undergoing initial cleavage (2- to 8-cell stages). A total of 153 nuclear transfer-derived embryos were transferred into 21 recipients; 21 morula-staged embryos into 4 recipients and 132 cleavage-staged embryos into 17 recipients. Five of the recipients receiving cleavage-staged embryos were confirmed to be pregnant by ultrasound. Table 2 summarizes the effects of donor cell source on pregnancy rates. These results indicate that this system is in fact efficient.

* cleavage-stage transfers

Example 7 Synchronization of Animals to be Used as Recipients of Nuclear Transfer- reconstructed Embryos Derived Using Oocytes From LOPU Procedures

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 enucleated. 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 μg 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 takes place.

Example 8 Transfer of Embryos Reconstructed by Nuclear Transfer Using LOPU- derived Oocytes to Recipient Goats

Reconstructed nuclear transfer embryos were either incubated for a short period (42-48 hours) or 5 days and then transferred to synchronized recipient goats. 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. If the short culture period is preferred

(overnight following nuclear transfer/fusion), the embryos may be transferred to the oviduct of recipient goats. For this purpose, 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. For this purpose, 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 oviductal third of the horn, and the embryos are then implanted by means of a TomCat catheter threaded into the uterine lumen.

Example 9 Pregnancies and Births

A total of 21 recipients were transferred, of which 5 (24% of embryos transferred) became pregnant and gave birth to 6 kids. Four of these pregnancies (5.7% of embryos transferred) were derived from the non- transfected male line (BELE-FF4), while one (7.6% of embryos transferred) was derived from a GFP transfected female line (BELE-FF3-GFP). The BELE-FF4 donor cells used to generate the embryos which were transferred to recipients, which then became pregnant, were from passage 2 cells maintained in low serum for 4 days. The BELE-FF3-GFP donor cells which were used to produce cleavage-stage nuclear transfer-reconstructed embryos which were then transferred to a donor recipient, which then became pregnant, were derived from passage 5 donor cells maintained in low serum for 6 days. Four recipients receiving morula-staged embryos did not become pregnant. The source of the oocyte (Dwarf/Pygmy or Saanen/ Alpine) did not affect the initial pregnancy rate (P>0.05).

In an additional set of experiments, 155 nuclear transfer- reconstructed embryos using LOPU-derived oocytes and cumulus, granulosa, or fetal fibroblast lines as the donor cells were transferred to 15 recipients. Of these recipients and other subsequent sets of recipients, 6 kids have been born. Of these 6 kids, 2 express biosteel (a spider silk protein) in their milk.

What is claimed is:

Claims

1. A method for generating a non-human transgenic animal containing a desired gene, said method comprising:

(a) recovering an oocyte from a donor animal by laparoscopic ovum pick up; (b) enucleating said oocyte;

(c) providing a cell containing said desired gene;

(d) fusing said cell with said oocyte to form a fused couplet;

(e) activating said couplet to form a zygote;

(f) transferring said zygote, or a cleaved embryo, morulae, or blastocyst formed from culturing said zygote, into a recipient animal; and

(g) allowing said zygote, cleaved embryo, morulae, or blastocyst to develop to term.

2. A method for generating a non-human transgenic animal containing a desired gene, said method comprising: (a) recovering an oocyte from a donor animal by laparoscopic ovum pick up;

(b) activating said oocyte;

(c) enucleating activated said oocyte;

(d) providing a cell containing said desired gene; (e) fusing said cell with said enucleated activated oocyte to form a fused couplet;

(f) allowing said couplet to form a zygote;

(g) transferring said zygote, or a cleaved embryo, morulae, or blastocyst formed from culturing said zygote, into a recipient animal; and (h) allowing said zygote, cleaved embryo, morulae, or blastocyst to develop to term.

3. The method of claim 1 or 2, wherein said cell and said oocyte are derived from the same animal or different animals.

4. The method of claim 3, wherein said animals are of the same or different breeds.

i 5. The method of claim 1 or 2, wherein said animal is a ruminant.

6. The method of claim 5, wherein said animal is a goat.

7. The method of claim 5, wherein said animal is a sheep.