Multicellular living organisms and unmodified parts thereof and – Method of making a transgenic nonhuman animal
Reexamination Certificate
2000-03-29
2004-01-06
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of making a transgenic nonhuman animal
C800S003000, C800S008000, C800S004000, C800S014000
Reexamination Certificate
active
06673987
ABSTRACT:
RELATED APPLICATIONS
This application claims priority under 35 USC §119(a) to an application with the same title filed in the UK Patent Office on Mar. 24, 2000.
TECHNICAL FIELD
This invention relates generally to the field of embryo transfer. More particularly, it relates to factors that allow pregnancy to reach term following artificial engraftment of a fertile embryo in animal species such as ungulates.
BACKGROUND
There are now a variety of powerful techniques for developing new artificial strains of animals. These strains hold considerable promise for producing biological material for use in human and veterinary medicine, research, and agriculture.
Many of these techniques involve embryo transfer, in which a fertile embryo is engrafted into the uterus of a surrogate host. Pregnancy is a complex physiological transformation involving a number of signaling events, some of which emanate from the embryos in utero. In some species, a plurality of embryos must be present in order for pregnancy to be maintained. If less than the critical number of embryos is present, the corpora lutea regress and the embryos are not carried to term. When artificial embryo transfer is employed to initiate pregnancy in these species, it is often too difficult or too costly to transfer enough embryos for the pregnancy to be viable.
The estrous cycle of the sow is reviewed in the following publications: I. Gordon, pp. 60-76 in “Controlled Reproduction in Pigs” Vol. 3, CAB International, 1997; P. Dziuk et al., pp. 471-489 in “Reproduction in Domestic Animals”, 4
th
ed. P. T. Cupps, Academic Press Inc., 1991; R. D. Geisert et al., J. Reprod. Fert., Suppl. 40:293-305, 1990; and W. W. Thatcher et al., J. Animal Sci. 62 (suppl. 2):25-46, 1986.
Pigs normally deliver a litter of about 11 piglets. Following fertilization, a dialogue between endometrial epithelium and trophectoderm of the developing conceptus is important for pregnancy to be maintained. The normal reproductive cycle lasts 21 days, and unless specific signals are received the uterus secretes prostaglandin into the circulation 15 days after ovulation. This causes the corpora lutea (formed from follicles after they have ovulated) to regress, allowing the development of another group of follicles that grow to a pre-ovulatory size by day 21. The follicles release estrogen, which causes a surge in luteonizing hormone, triggering ovulation and starting the whole cycle over again.
If the eggs released by the follicles are successfully fertilized, they migrate around the uterus of the pig, and by 10-11 days are evenly spaced throughout the tract. The growing embryos provide signals that act locally or systemically. One such signal (thought to be mediated by estrogen) redirects secretion of prostaglandin from uterine cells to within the lumen of the uterus. This prevents regression of the corpora lutea, which in turn maintains raised levels of progesterone, preventing resumption of the estrus cycle and allowing the pregnancy to continue.
If there are not embryos occupying about 50% of the uterus before day 10 of the cycle, then the corpora lutea regresses and the estrus cycle is resumed. The critical number of embryos for maintaining pregnancy in the pig is about 3-4. However, if all the embryos are removed from one of the two horns after day 15, pregnancy is still successfully maintained to term (Dziuk et al., supra).
Current methods for artificially maintaining pregnancy typically rely on administering hormones that mimic the signals released by embryos or artificially maintain progesterone levels, preventing resumption of the estrous cycle.
Spies et al. (J. Anim. Sci. 19:114, 1960) conducted studies to determine the hormonal requirements for maintaining pregnancy in swine. Subcutaneous injections of progesterone and estrone were started 72 hours after breeding with a fertile bore. The percentages of embryo survival and the numbers of live embryos per pregnant gilt were significantly less for the treated animals than for the untreated controls.
Ellicott et al. (Biol. Reprod. 9:300, 1973a) conducted studies to measure the minimum quantity of estrogen and progesterone necessary to maintain pregnancy. Gilts were bred artificially or naturally on the first day of estrus, and ovariectomized under halothane anesthesia. Hormone was administered by inserting silicone capsules in the abdominal cavity. Pregnancy was maintained at a progesterone dose as low as 28.6 mg/day, and there was no added benefit of including 5 &mgr;g estradiol. No ovarian estrogen was necessary after day 10. A concentration of 4 ng/mL of progesterone in peripheral plasma appeared to be the minimum to maintain pregnancy.
Ellicott et al. (J. Animal Sci. 37:971, 1973b) studied the effects of various hormone combinations on the induction of an accessory set of corpora lutea for maintaining pregnancy. A significantly higher proportion of inseminated gilts were pregnant at day 30 when treated with estrogen-progestogen combinations. Oral administration of 200 &mgr;g melengestrol acetate and 1 mg estradiol on day 12 resulted in pregnancy in 4 out of 10 inseminated gilts. Five gilts pregnant at day 60 were removed from treatment, and two of these farrowed 5-6 piglets to term.
Christenson et al. (J. Animal Sci. 32:282, 1971) studied the maintenance of unilateral pregnancy in the pig with induced corpora lutea. The gilts were injected with pregnant mare serum and human chorionic gonadotrophin prior to the 11
th
day of pregnancy to induce a second set of corpora lutea. This increased the percentage of gilts with a non-pregnant uterine horn that maintained pregnancy to 25-37 days of gestation.
U.S. Pat. No. 5,366,888 reports enhanced maintenance of pregnancy using leukemia inhibitory factor (LIF) in embryo culturing. Following introduction into foster mothers of embryos cultured in vitro in the presence of LIF, the maintenance of pregnancy is enhanced relative to that seen following introduction of embryos that had not been cultured with LIF. Embryos cultured in a medium optimally containing 1000-5000 units/mL of LIF is proposed to help maintain pregnancy in sheep.
In practice, administering hormones to a surrogate mother has a sub-optimal success rate in maintaining pregnancy in animals that have received an embryo transfer. In the laboratory where the present invention was made, the best treatment regime involving hormones alone was found to maintain pregnancy in only 26% of treated pigs. Embryos were also transferred into pigs that have already been mated. In these experiments, 37% of the pigs carried the transferred embryos. However, ultrasound at day 35 is unable to distinguish the transferred embryos from the embryos resulting from insemination—so it was not possible to determine which of the females were carrying the high value transferred embryos.
In view of the limitations of currently available technology, there is a pressing need to develop new artificial methods to maintain pregnancy.
SUMMARY OF THE INVENTION
This invention provides a technique for maintaining pregnancy in a female to which a fertile embryo has been transferred. The technique involves placing additional infertile embryos into the uterus, which have the effect of generating signals that allow the pregnancy to continue. The additional embryos typically maintain pregnancy through the early critical period, and then are reabsorbed or otherwise eliminated before the end of term. This allows viability of the engrafted fertile embryos to be monitored later in pregnancy, and helps avoid overcrowding of the uterus.
Embodiments of this invention include methods for maintaining pregnancy in a female pregnant with one or more fertile embryos, by engrafting into the uterus of the female one or more infertile embryos, thereby allowing the pregnancy to reach term, or at least until the fertile embryo is at a stage that it is viable ex utero. Also embodied in the invention are pregnant animals and birthed animals that result from the application of such methods.
The female is treated according to the invention generally because she is
Crouch Deborah
Earp David J.
Geron Corporation
Schiff J. Michael
Woitach Joseph
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