WO2002029000A2 - Methods of minimizing immunological rejection of a nuclear transfer fetus - Google Patents

Abstract

The present invention relates to a method of minimizing immunological rejection of a nuclear transfer ('NT') fetus which includes transferring a NT embryo into an embryo recipient under conditions effective for development of a NT fetus with minimal risk of immunological rejection of the fetus due to a maternal anti-fetal MHC-I immune response. After determining an MHC-I antigen type for a NT embryo and an MHC-I antigen type for embryo recipients, the NT embryo is either (i) transferred into a first embryo recipient having a compatible MHC-I antigen type under conditions effective for development of a NT fetus from the NT embryo, or (ii) transferred into a second embryo recipient having an incompatible MHC-I antigen type, followed by regulating MHC-I expression of the NT embryo or suppressing an immune response of the embryo recipient under conditions effective for development of a nuclear transfer fetus.

Description

METHODS OF MINIMIZING IMMUNOLOGICAL REJECTION OF A NUCLEAR TRANSFER FETUS

This application claims the benefit of U.S. Provisional Application Serial No. 60/237,673, filed October 3, 2000, which is hereby incoφorated by reference in its entirety.

This invention was made, at least in part, with funding received from the U.S. Department of Agriculture, NRICGP, Grant No. 96-35203-3356. The U.S. government may have certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates generally to animal cloning and, more specifically, to methods of minimizing immunological rejection of a nuclear fransfer ("NT") fetus.

BACKGROUND OF THE INVENTION

Success has now been achieved with somatic cell cloning in several species using a variety of cell types (Campbell et al., 1996; Schnieke et al., 1997;

Wells et al., 1997; Wilmut et al., 1997; Cibelli et al., 1998; Kato et al., 1998;

Wakayama et al., 1998; Baguisi et al., 1999; Renard et al., 1999; Wells et al., 1999;

Wakayama, Yanagimachi, 1999a). This technology has great potential for use in agriculture, animal and human medicine, and for the propagation of rare animals. These potential uses of cloning technology clearly have great commercial and conservational benefit.

The efficiency of this process, however, is quite poor, resulting in less than one animal born per 100 reconstructed NT embryos (Schnieke et al., 1997).

Much of this inefficiency is due to low initial pregnancy rates and early pregnancy losses. First trimester losses of greater than 50% are common for nuclear fransfer pregnancies (Wells et al., 1997; Wilmut et al., 1997; Cibelli et al., 1998; Baguisi et al., 1999; Wells et al., 1999), whereas only 2-4% of naturally conceived Day 30 bovine pregnancies and 11% of in vitro produced embryos are expected to be lost by Day 60 (Alexander et al., 1995; Hasler et al, 1995; Forar et al., 1996). This lack of normal in vivo development has occurred in each species so far studied and is delaying the transfer of this new technology into commercial practice.

In general, early fetal losses may be due to abnormalities of the embryo or its placenta, alterations in maternal uterine environment or feto-maternal interactions (Wilmut et al., 1986). In normal pregnancies, fetal abnormalities are known to be a major cause of pregnancy loss (Wilmut et al., 1986). Fetal abnormalities, predominantly fetal oversize, have been observed as a result of in vitro embryo culture and this syndrome is believed to result from serum containing media (Thompson et al., 1995; Walker et al., 1996; Young et al., 1998). In shaφ confrast, NT fetuses that die during the first trimester are undersized, which probably represents the effects of "starvation" due to inadequate maternal-fetal contact and poor transfer of nutrients (Hill et al. 2000b). The fetuses that die appear not to lose viability because of inherent fetal problems, but due to starvation from an inadequate placental nutrient transfer.

In cloned animals, normal placental development appears to be rare, as placental abnormalities occur at a high incidence in early and late term cloned fetuses (Stice et al., 1996; Hill et al., 1998; Wells et al., 1998). Stice et al. (1996) observed a lack of placentome development in Day 35-50 cloned bovine fetuses and suggested that this caused a high rate of first trimester death. Even in NT fetuses that survive beyond Day 50, the number of placentomes may be reduced from normal by as much as 80% (Hill et al., 1998). This suggests that the completeness of placental development in cloned animals varies widely.

King et al. (1979) documented the normal development of Day 30-60 bovine placentas. The placental attachment phase in ruminants is progressive and extends almost throughout the first trimester in confrast to the more rapid and invasive attachment phase in humans and rodents. At Day 30, placentomes are visible using light microscopy with tenuous attachment of maternal and fetal epithelia and formation of micro villi. Contact with the maternal caruncle areas of the endometrium induces growth of villous processes that undergo hypertrophy and hypeφlasia to form cotyledons (Noden, de Lahunta, 1990) and by Day 42 larger, more complex placentomes develop (King et al., 1979). Placentomes are formed from extensive and complex branching of fetal villi and maternal crypts, serve as specialized areas for supplying nutrition to the developing conceptus. Villous projections assist in maintaining apposition and facilitate subsequent union. Binucleate cells form transient feto-maternal syncytia in the cow, which has been proposed to be central to villous expansion (Wooding, Flint, 1994). Chorioallantoic villous formation at the cotyledons is thought to be the primary site of transport of easily diffusible small molecules such as oxygen, carbon dioxide and also amino acids and glucose, whereas macromolecules are transported in the inteφlacentomal areas adjacent to uterine gland openings.

Cloned pregnancies fail at a higher than normal rate during each trimester of pregnancy (Wilmut et al. 1997). Although in vitro development rates to the blastocyst stage approach that of in vitro fertilized ("IVF") embryos, subsequent in vivo development drops dramatically. Pregnancy rates at Day 30 in recipient cows can approach 50%, but only with fransfer of two or more cloned embryos. Single embryo transfer of cloned embryos results in almost negligible pregnancy rates whereas IVF embryos transferred singly achieve 50-70% pregnancies. The cause of these losses have not been determined and may be due to failure of maternal recognition, placental development, or inherently low cloned embryo viability. Following on from these losses is a well documented period of embryonic loss from Day 30-60 that results in a minority of first trimester pregnancies maintaining their viability into the second trimester (Wells et al., 1997; Wilmut et al., 1997; Cibelli et al., 1998; Baguisi et al., 1999; Wells et al., 1999)Hill et al. 2000b). During the second and third trimesters there are sporadic losses of cloned fetuses, often accompanied by the development of major placental abnormalities such as hydrops allantois. Postnatal viability is also markedly lower for many cloned calves (Kato et al. 1998; Hill et al. 1999a; Renard et al. 1999; Kubota et al. 2000; Kato et al. 2000). It is uncertain if the cause(s) of fetal loss in the first trimester is related to later losses.

For somatic cell NT to become a viable technique, its efficiency must be improved. Although the numbers of cloned calves born worldwide since 1998 has rapidly increased into the hundreds and press reports often detail the latest successful birth, these successes gloss over the huge amount of resources that must be devoted to producing each cloned calf. If the cloning technique can be improved so that pregnancy rates increase and fetal losses decrease to approximate those of in vitro produced embryos and fetuses, noted above, utilization of the technique would immediately increase. This would enable the use of cloning in commercial agriculture, facilitate production of transgenic animals, and dramatically reduce the costs to research institutions in maintaining recipient cows for cloned embryos. The present invention is directed to overcoming the above-noted deficiencies in art and otherwise minimizing the failure of NT pregnancies.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a method of minimizing immunological rejection of a nuclear transfer fetus which includes transferring a nuclear transfer embryo into an embryo recipient under conditions effective for development of a nuclear fransfer fetus with minimal risk of immunological rejection of the fetus due to a maternal anti-fetal MHC class I ("MHC-I") immune response.

A second aspect of the present invention relates to a method of performing embryo transfer which includes: determining an MHC-I antigen type for a nuclear fransfer embryo and an MHC-I antigen type for embryo recipients and either (i) transferring the nuclear transfer embryo into a first embryo recipient having a compatible MHC-I antigen type under conditions effective for development of a nuclear transfer fetus from the nuclear fransfer embryo, or (ii) transferring the nuclear transfer embryo into a second embryo recipient having an incompatible MHC-I antigen type and (a) regulating MHC-I expression of the nuclear transfer embryo or (b) suppressing an immune response of the embryo recipient, under conditions effective for development of a nuclear transfer fetus from the nuclear fransfer embryo. Ultimately, development of a healthy neonate from the nuclear transfer fetus/embryo is desired.

A third aspect of the present invention relates to an MHC-I microarray typing system which includes: a substrate and a plurality of oligonucleotide probes bound to the substrate, each of the plurality of oligonucleotides binding to at least one MHC-I allele, wherein each MHC-I allele binds to different oligonucleotide probes.

Trophoblast cells in NT embryos display abnormal expression of MHC-I antigen. The abnormal MHC class I expression results in immunological rejection of these fetuses in a large proportion of NT pregnancies, particularly during the first trimester. This is in shaφ contrast to normal pregnancies, where the rate of early embryonic loss is low and MHC incompatible pregnancies do not have a significantly increased amount of early embryonic loss. Presumably, the reason for this distinction is that in normal bovine pregnancy, there is no frophoblast MHC-I antigen expression in early pregnancy (Davies et al., 2000). Consequently, MHC-I antigen expression is not a target for immunologically mediated fetal rejection in normal pregnancies. To overcome this problem acutely associated with NT fransfer, the present invention identifies two approaches for avoiding immunological rejection of MHC-I incompatible NT pregnancies. The first approach involves matching NT donor cells and NT recipients for their MHC-I haplotype expression prior to transfer. According to a second approach, which can be employed independently of the first approach (i.e., with or without prior matching), MHC-I antigen expression by NT frophoblasts is down-regulated, returning the NT frophoblasts to their normal MHC-I negative state. Both of these approaches minimize rejection of the NT fetus, particularly during the first trimester.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-J illustrate the nucleotide sequence alignment of different

MHC-I alleles. This sequence alignment was prepared by George Russell of the Roslin Institute and made available at the Internet site for the Bovine Leucocyte Antigens (BoLA) nomenclature committee (standing committee of the International Society for Animal Genetics). dl8-2 (SEQ ID No: 1, Genbank Accession No. Y09206); alO (SEQ ID No: 2, Genbank Accession No. M69026); jspl (SEQ ID No: 3, Genbank Accession No. X92870); pbolal (SEQ ID No: 4, Genbank Accession No. M24090); bl3-6 (SEQ ID No: 5, Genbank Accession No. M21044); bsa (SEQ ID No: 6, Genbank Accession No. L02832); bsc (SEQ ID No: 7, Genbank Accession No. L02833); dl8-l (SEQ ID No: 8, Genbank Accession No. Y09205); bsn (SEQ ID No: 9, Genbank Accession No. L02835); manl (SEQ ID No: 10, Genbank Accession No. AJ010863); bsf (SEQ ID No: 11, Genbank Accession No. L02834); man8 (SEQ ID No: 12, Genbank Accession No. AJ010866); dl8-3 (SEQ ID No: 13, Genbank Accession No. Y09207); pbolal 9 (SEQ ID No: 14, Genbank Accession Nos. X82671-X82675); bl3-7 (SEQ ID No: 15, Genbank Accession No. M21043); hd7 (SEQ ID No: 16, Genbank Accession No. X80935); hd6 (SEQ ID No: 17, Genbank Accession No. X80934); 3349 (SEQ ID No: 18, Genbank Accession No. AJ010862); man2 (SEQ ID No: 19, Genbank Accession No. AJO 10861); man3 (SEQ ID No: 20, Genbank Accession No. AJ010864); 4221 (SEQ ID No: 21, Genbank Accession No. AJ010865); hdl (SEQ ID No: 22, Genbank Accession No. X80933); bsx (SEQ ID No: 23, Genbank Accession No. U01187); dl8-4 (SEQ ID No: 24, Genbank Accession No. Y09208); pbola4 (SEQ ID No: 25, Genbank Accession Nos. X87645 and X97646-X97649); knl04 (SEQ ID No: 26, Genbank Accession No. M69204); and hdl 5 (SEQ ID No: 27, Genbank Accession No. X80936). All Genbank Accessions are hereby incoφorated by reference in their entirety.

Figures 2A-D are photomicrographs comparing normal and cloned embryo development. Photomicrographs were originally photographed at 200X. In Figure 2 A, the endometrium and attached chorioallantois from a normal bovine pregnancy are shown at 39 days gestation (H+E stain). Note trophoblast cells forming a pseudocolumnar layer of cells and the subjacent endometrium lined by an irregular layer of endometrial epithelial cells. Two endometrial glands and moderately cellular endometrial interstitium are evident in the endometrium. In Figure 2B, the endometrium of a cow pregnant 35 days with a cloned embryo (fetal membranes are not shown; H+E stain) is shown containing a marked lymphoplasmacytic cellular infiltrate extending from just beneath the endometrial epithelium to deep within the endometrium. This is in marked confrast to the normal cellularity demonstrated in Figure 2 A. Figures 2C-D illustrate sections of normal day 39 chorioallantois and endometrium (2C) and day 35 cloned embryonic placenta and opposing maternal endometrium (2D), respectively. Immunohistochemistry staining was performed with ILA19 antibody for bovine MHC-I antigen. Note the mild staining of the endometrial epithelial cells and complete absence of staining of trophoblast cells in Figure 2C. Confrast this to the intense class I staining of the trophoblast and endometrial cells in fetal and maternal tissues from a cow carrying a cloned fetus shown in Figure 2D. The trophoblast and endometrial cells show marked upregulation of MHC-I expression. Figure 3 is a graph illustrating the interaggregate cd3 positive cells located in the endometrium of 3 cloned pregnancies (hatched bars) and 7 controls (clear bars). The counts are the number of cd3 positive cells per 0.584 mm² field at 1 Ox magnification.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to new approaches for performing nuclear transfer ("NT") embryo fransfer into embryo recipients and, as a result, minimizing the immunological rejection of a developing NT fetus. By minimizing the immunological rejection, it is intended that the incidence of NT fetus rejection when practicing an embodiment of the present invention is less than the historical incidence of NT fetus rejection, which is greater than about 80 percent for bovine during the first trimester (Hill et al., 2000a).

The NT embryo is prepared using donor and recipient cells from a non- human mammal, preferably a ruminant such as a cow, sheep, goat, buffalo, water buffalo, llama, alpaca, camel, giraffe, etc., or other mammals such as pig, horse, rabbit, mouse, or rat. Procedures for preparing the NT embryo are known in the art (Campbell et al., 1996; Schnieke et al, 1997; Wells et al., 1997; Wilmut et al., 1997; Cibelli et al., 1998; Kato et al., 1998; Wakayama et al., 1998; Baguisi et al., 1999; Renard et al., 1999; Wells et al., 1999; Wakayama, Yanagimachi, 1999a) and further been have been described in U.S. Patent No. 6,147,276 to Campbell et al. and U.S. Patent No. 6,235,970 to Stice et al.

Suitable donor cells, i.e., cells useful in the subject invention, may be obtained from any cell or organ of the body. This includes all somatic or germ cells. All cells of normal karyotype, including embryonic, fetal and adult somatic cells, may prove totipotent. Donor cells may be, but do not have to be, in culture. Cultured bovine primary fibroblasts, an embryo-derived ovine cell line (TNT4), an ovine mammary epithelial cell derived cell line (OME) from a 6 year old adult sheep, a fibroblast cell line derived from fetal ovine tissue (BLWF1), and an epithelial-like cell line derived from a 9-day old sheep embryo (SECL) have been employed for nuclear fransfer and described elsewhere. A class of embryo-derived cell lines useful in the invention, which includes the TNT4 cell line, are also the subject of PCT Publication No. WO 96/07732 to Campbell et al. All can be utilized in the present invention. Donor cells may be, but do not have to be quiescent. Cultured cells can be induced to enter the quiescent state by various methods including chemical treatments, nutrient deprivation, growth inhibition or manipulation of gene expression. Presently, the reduction of serum levels in the culture medium has been used successfully to induce quiescence in both ovine and bovine cell lines. In this situation, the cells exit the growth cycle during the Gl phase and arrest in the so- called GO stage. Such cells can remain in this state for several days (possibly longer depending upon the cell) until re-stimulated when they re-enter the growth cycle. Quiescent cells arrested in the GO state are diploid. The GO state is the point in the cell cycle from which cells are able to differentiate.

The recipient cell to which the nucleus from the donor cell is transferred may be an oocyte or another suitable cell. Recipient cells at a variety of different stages of development can be used, from oocytes at metaphase I through metaphase II to zygotes and two-cell embryos. Methods for isolation of oocytes are well known in the art. Essentially, this includes isolating oocytes from the ovaries or reproductive tract of a mammal. A readily available source of bovine oocytes is slaughterhouse materials.

Typically, oocytes should be matured in vitro before these cells may be used as recipient cells for nuclear transfer. This process generally requires collecting immature (prophase I) oocytes from mammalian ovaries and maturing the oocytes in a maturation medium prior to enucleation until the oocyte attains the metaphase II stage, which in the case of bovine oocytes generally occurs about 18-24 hours post- aspiration (the "maturation period").

Additionally, 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 cows or heifers 35 to 48 hours past the onset of estrus or past the injection of human chorionic gonadotropin (hCG) or similar hormone.

The stage of maturation of the oocyte at enucleation and nuclear transfer has been reported to be significant to the success of NT methods (Prather et al. 1991). In general, successful mammalian embryo cloning practices use the metaphase II stage oocyte as the recipient oocyte, because at this stage it is believed that the oocyte can be or is sufficiently "activated" to treat the introduced nucleus as it does a fertilizing sperm. In domestic animals, and especially cattle, the oocyte activation period generally ranges from about 10 to about 52 hours, preferably about 16 to about 42 hours post-aspiration.

Enucleation can be effected by known methods, such as described in U.S. Patent No. 4,994,384 to Prather et al. For example, enucleation may be accomplished microsurgically using a micropipette to remove the polar body and the adjacent cytoplasm. The oocytes may then be screened to identify those of which have been successfully enucleated. This screening may be effected by staining the oocytes with 1 μg/ml 33342 Hoechst dye in HECM, and then viewing the oocytes under ultraviolet irradiation for less than 10 seconds. The oocytes that have been successfully enucleated can then be placed in a suitable culture medium, e.g., CRlaa plus 10% serum.

Once suitable donor and recipient cells have been identified, it is necessary for the nucleus of the former to be transferred to the latter. Suitable procedures for nuclear transfer include donor/recipient cell fusion (i.e., via PEG treatment, inactivated Sendai virus, or elecfrofusion) and microinjection.

In donor/recipient cell fusion protocols, the donor cell is first transferred into the perivitelline space of the enucleated oocyte. Thereafter, the cells can be fused by providing a pulse of electricity that is sufficient to cause a transient breakdown and subsequent reformation of the plasma membrane. If upon reformation the lipid bilayers intermingle, small channels will open between the two cells and, due to the thermodynamic instability of such a small opening, it enlarges until the two cells become one (U.S. Patent No. 4,997,384 to Prather et al.). A variety of elecfrofusion media can be used including e.g., sucrose, mannitol, sorbitol and phosphate buffered solution. Alternatively, fusion can also be accomplished using Sendai virus as a fusogenic agent (Graham 1969).

In microinjection protocols, the donor nuclei is simply removed from the donor cell and injected into the recipient cell (Collas & Barnes 1994).

Either before or, preferably, after nuclear fransfer (or in some instances concomitantly therewith), parthenogenetic activation is typically required, at least if the cell is an oocyte, to stimulate the recipient cell into development. Parthenogenic activation is typically achieved using electrical stimulation of the diploidized oocyte, which is believed to allow for increases in intracellular calcium concentration. There is evidence that the pattern of calcium transients varies with species and it can be anticipated that the optimal pattern of electrical pulses will vary in a similar manner. The interval between pulses for rabbit oocytes is approximately 4 minutes (Ozil 1990), and in the mouse 10 to 20 minutes (Cuthbertson & Cobbold 1985), while observations in the cow suggest that the interval is approximately 20 to 30 minutes (Robl et al. 1992). In most published experiments activation was induced with a single electrical pulse, but new observations suggest that the proportion of reconstituted embryos that develop is increased by exposure to several pulses (Collas and Robl 1990). In any individual case, routine adjustments may be made to optimize the number of pulses, the field strength and duration of the pulses, and the calcium concentration of the medium.

Alternative approaches for parthenogenic activation include culturing the recipient oocyte of NT embryo at sub-physiological temperature (e.g., room temperature) and chemical shock. Suitable oocyte activation methods are further described in U.S. Patent No. 5,496,720 to Susko-Parrish et al.

By way of example, activation can be effected by briefly exposing the fused NT embryo to a TL-HEPES medium containing 5 μM ionomycin and 1 mg/ml BSA, followed by washing in TL-HEPES containing 30 mg/ml BSA within about 24 hours after fusion, and preferably about 4 to 9 hours after fusion. The reconstituted NT embryo may then give rise to one or more mammals, whether transgenic or non-transgenic. Preferably, the NT embryo will be cultured to a size of at least 2 to 400 cells, preferably 4 to 128 cells, and most preferably to a size of at least about 50 cells. Development to blastocyst stage can be carried out in vitro or in vivo (i.e., using a temporary pre-blastocyst recipient). After preparing the NT embryo (and optionally developing the NT embryo to the blastocyst stage), it is transferred into the uterus of an embryo recipient using known transfer procedures. The embryo recipient is preferably from the same species as the donor and recipient cells used to prepare the NT embryo, although dams from related species can, at least in some instances, be utilized to support gestation of the NT fetus. Synchronous transfers are desirable for success of the transfer, i.e., the stage of the NT embryo is in synchrony with the estrus cycle of the recipient female (Siedel 1981). Transfer procedures are described in detail in PCT Publication No. WO 94/26884 to Wheeler et al, PCT Publication No. WO 94/24274 to Smith et al., PCT Publication No. WO 90/03432 to Evans et al., U.S. Patent No. 4,944,384 to Prather et al., and U.S. Patent No. 5,057,420 to Massey.

According to one embodiment, the method for minimizing immunological rejection of a NT fetus involves transferring, into an embryo recipient, an NT embryo having an MHC-I antigen type which is compatible with an MHC-I antigen type of the embryo recipient. Prior to transferring the NT embryo, the MHC-I antigen type of the NT embryo and the embryo recipient are determined. Matching of the NT embryo and the embryo recipient (into which transfer will subsequently occur) is based on the determined MHC-I antigen haplotypes thereof. The determination of MHC-I antigen haplotype can be performed separately on individual NT embryos or it can be performed on a number of NT embryos in a single screening event. The same is true for making the determination of MHC-I antigen haplotype for the embryo recipients. A number of approaches can be utilized to perform the haplotyping, either alone or in combination. These include, without limitation, serological typing (Lewin 1996; Davies & Antczak 1991; Davies et al. 1994a); one dimensional-isoelectric focusing (Joosten et al. 1988; Davies et al. 1994a; Lewin 1996); DNA sequencing (Garber et al. 1993; Pichowski et al. 1996; Ellis et al. 1999); polymerase chain reaction amplification using allele specific primers (Ellis et al. 1998); and polymoφhism analysis using oligonucleotide probes (Davies et al. 2001). With respect to the use of oligonucleotide probes (Davies et al. 2001), hybridization arrays can be created with probes that are specific to a number of different MHC-I genes and the resulting hybridization array patterns can be analyzed using computer software, e.g. the Cytofile genotyping software (Davies 1988). Figures 1 A-J illustrate a nucleotide sequence alignment for a number of known MHC- I alleles. Probes can be selected based on the polymoφhism which exists among the various MHC-I alleles. Haplotype assignments for the NT embryo and the embryo recipient can be based on one or more of these methods.

For the polymoφhism analysis, an MHC-I microarray typing system can be used. This typing system includes a substrate and a plurality of oligonucleotide probes bound to the substrate, each of the plurality of oligonucleotides binding to at least one MHC-I allele, wherein each MHC-I allele binds to different oligonucleotide probes (i.e., a different subset of oligonucleotide probes). According to a second embodiment, the method for minimizing immunological rejection of an NT fetus involves transferring, into an embryo recipient, an NT embryo having an MHC-I antigen type which is incompatible with an MHC-I antigen type of the embryo recipient. A first approach to minimize immunological rejection in this situation, a involves down-regulation of MHC-I expression in the placenta of the NT embryo or fetus. Down-regulation of MHC-I expression by placental trophoblast cells is preferred, although down-regulation of MHC-I expression by other placental cells is also beneficial. Down-regulation of MHC-I expression (in placental cells of NT embryos) can be achieved by (i) modulating expression of an MHC-I transcription factor in the NT embryo or fetus; (ii) treating the NT embryo or fetus with a cytokine, a growth factor, or combinations thereof which is suitable to inhibit MHC-I expression; or (iii) both (i) and (ii). Without being bound by theory, it is believed that the regulation of

MHC-I genes in bovine trophoblast cells may involve many of the same positive regulatory elements as human MHC-I genes (Harms & Splitter 1994; Harms et al. 1995; Barker et al. 1997). In humans, down regulation of expression of "classical" MHC-I antigens on trophoblast cells involves both the absence of key transcription factors (CIITA and NF-κB/Rel family members p50 and p65) and the presence of specific negative regulatory factors (Gobin & van den Elsen 1999, 2000; Chiang & Main 1994; Coady et al. 1999; Peyman 1999). Introduction of a transgene expressing the RNA suppressor element (TSU) described by Peyman (1999) would be one option for the down regulation of MHC-I expression in the trophoblast cells of NT embryos. TSU is a particularly good candidate as an homologous goat expressed sequence tag (EST) was described in the original paper.

When treating the NT embryo or fetus with a cytokine or growth factor, the treatment can be carried out prior to transfer (i.e., in vitro), after fransfer (i.e., in utero), or both. For in vitro treatment, a suitable cytokine or growth factor is introduced into the growth medium in which the NT embryo resides following nuclear transfer, such as the above-described medium utilized for activation. For in vivo treatment, a suitable cytokine or growth factor can be administered via intrauterine delivery or intravenous injection. Suitable cytokines that can be employed to down-regulate MHC-I expression levels include, without limitation, several interleukins such as IL-4, IL-10 and IL-13, leukemia inhibitory factor ("LIF") and transforming growth factor-β ("TGF-β"), which has both cytokine and growth factor activities, or combinations thereof (Mitchell et al. 1993 ; Robertson et al. 1994; Moreau et al. 1999). While IL- 10 can directly down-regulate MHC-I expression (see Moreau et al. 1999), it is believed that the other cytokines act indirectly by inhibiting the production of inflammatory cytokines (particularly INF-gamma) that induce MHC-I expression.

Suitable growth factors that can also be employed to down-regulate MHC-I expression levels include, without limitation, insulin, epidermal growth factor ("EGF"), granulocyte/macrophage colony-stimulating factor ("GM-CSF"), TGF-β, insulin-like growth factor(s) ("IGFs"), interleukin-3 ("IL-3"), or combinations thereof (Mitchell el al. 1993; Robertson et al. 1994).

A second approach to minimize immunological rejection in this situation involves suppressing an immune response of the embryo recipient.

Suppression of the embryo recipient's immune response to the MHC-I incompatible embryo or fetus is effected by administering an amount of an immunosuppresive drug to the embryo recipient under conditions effective to suppress the anti-MHC-I immune response. Suitable immunosuppressive drugs include, without limitation, cyclosporin A, tacrolimus, and sirolimus. These exemplary immunosuppressive drugs are believed to cause immunosuppression by blocking signaling pathways in lymphocytes, thereby blocking immunological rejection. These immunosuppressive drugs can be administered systemically (i.e., intravenous) to the embryo recipient. These two approaches for minimizing immunological rejection in MHC-I incompatible NT pregnancies can be utilized alone or in combination.

EXAMPLES

The following examples are intended to illustrate, but by no means are intended to limit, the scope of the present invention as set forth in the appended claims. Materials & Methods

Leukocyte immunohistochemistry Immunoperoxidase staining for leukocyte differentiation antigens was performed on 8 μm sections of frozen uterine and placental tissues. For each pregnancy, sections from a minimum of two placentomal and two inteφlacentomal blocks were assessed. Staining was performed using the three-stage avidin-biotin system described under the SBU3 staining. The following antibodies can be used: anti-CD2 (mAb CC42; BioSource), CD3 (mAb MM1A; VMRD), CD4 (mAb CC30; BioSource), CD8β (mAb CC58; Serotec), TCR-γ/δ (mAb GB21 A; VMRD), CD21

(mAb CC21; Serotec), CD25 (IL-2 receptor, mAb CACTI 16A, VMRD), CD68 (mAb EMB 11 ; DAKO), and MHC class II (mAb H42 A; VMRD). Antigen positive cells in the placentomal and inteφlacentomal endometrium are enumerated by digital image processing with NIH Image software (Grϋnig et al. 1995).

Cytokine immunohistochemistry Immunohistochemistry can be used to compare cytokine production between groups and to identify cytokine producing cells at the uterine/placental interface. For each pregnancy, sections from at least two placentomal and two inteφlacentomal blocks would be assessed. Staining can be done using the three- stage avidin-biotin system described above. Antibodies against the following cytokines can be used: IL-2 (mAb 14.1, VMRD), IL-4 (mAb CC303, Serotec; Weynants et al. 1998), IL-10 (goat anti-human IL-10, R & D Systems; Brown et al. 1994), IL-12 (mAb CC301, Serotec), IFN-γ (mAb CC302, Serotec), TNF-α (mAb 2C4-1D3 and polyclonal rabbit anti-bovine TNF-α, generously provided by Dr. Ted Elsasser; Palmer et al. 1998; Kenison et al. 1990; Sileghem et al. 1992), TGFβl and TGFβ2 (rabbit anti-human TGFβl and TGFβ2 from R & D Systems; Munson et al. 1996), and GM-CSF (mAb CC305, Serotec). Normal mouse ascites, rabbit serum or goat serum can be used as a negative control. Identification of cytokine positive cells can be based on cell location and moφhologic features. The leukocyte differentiation antigen immunohistochemistry described above would be invaluable in the inteφretation of the cytokine immunohistochemistry. The number of positive cells and the intensity of staining would be assessed using digital image analysis with NIH Image software (Grϋnig et al. 1995).

Example 1 - Microarray MHC-I Typing

A bovine MHC-I microarray typing system was prepared by providing

17-22 bp oligonucleotides spotted on epoxy-silane treated, 12-well, Teflon masked, glass slides (Erie Scientific) using an Affymetrix 417 arrayer (Call et al. 2001). The

MHC-I typing array is based on 118 known cDNA or genomic sequences from the BoLA Nomenclature Web Site and GenBank. As shown in Tables 1-4 below, two series of exon 2 probes and two series of exon 3 probes are provided. The exon 2 probes include 25 series A probes for codons 61-68 and 30 series B probes for codons 71-78 (see also Figures 1A-J). The exon 3 probes include 27 series A probes for codons 111-118 and 31 series B probes for codons 151-158 (see also Figures 1 A-J). Together, these probes (and the corresponding polymoφhisms) define an undetermined number of MHC-I haplotypes.

Table 1 : BoLA Class I, Exon 2, Series A Probes

Oligo Name Sequence # bp TM GC% BoLA-ClEx2A01 CGGGAGACGCAAAGGGCC 18 57 72 BoLA-ClEx2A02 CAGGAGACGCGAAAGGCC 18 55 67 BoLA-ClEx2A03 CGGAACACGCGAAACGCC 18 55 67 BoLA-ClEx2A04L ATCGAAACACGAGAATCTACAA 22 49 36 BoLA-ClEx2A05 GAGCAGACGCGAATAGTC 18 50 56 BoLA-ClEx2A06 CGCGAGACGCGAAACTCC 18 55 67 BoLA-ClEx2A07 CGCGAGACTCAAATCTCC 18 50 56 BoLA-ClEx2A08 CGCGAGACGCGAATCTCC 18 55 67 BoLA-ClEx2A09 CAGAACACGCGAAACTCC 18 50 56 BoLA-ClEx2A10 GAGGAGACGTGGAGAGCC 18 55 67 BoLA-ClEx2Al l CAGGAGACGCAGAGAACT 18 50 56 BoLA-ClEx2A12 CAAGAGACGCGGATACAA 18 48 50 BoLA-ClEx2A13 CAGGCGACGCAGAGAACT 18 53 61 BoLA-ClEx2A14 CAGGAGACGCGAAACGCC 18 55 67 BoLA-ClEx2A15 GAGATGACACGAGATGCC 18 50 56 BoLA-ClEx2A17 GACGAGACGCGAATCTCC 18 53 61 BoLA-ClEx2A18R CGGTGTCCTTGAAGTTTCGC 20 54 55 BoLA-ClEx2A19R CGGCGCCCTTTAAGTTTCG 19 53 58 BoLA-ClEx2A20 GCGATGACAAGAGATGCC 18 50 56 BoLA-ClEx2A21 CAGAACACGCGAAACGCC 18 53 61 BoLA-ClEx2A22 CAGGAGACGCAGAGGACT 18 53 61 BoLA-ClEx2A23L CATCAGGAGACGCAGATAACT 21 52 48 BoLA-ClEx2A25 GAGGAAACGCAAAGGGCC 18 53 61 BoLA-ClEx2A26 GAGGAGACGCAAAGGGCC 18 55 67 BoLA-ClEx2A27 TCGAAACACGAGGATCTACA 20 50 45

Table 2: BoLA Class I, Exon 2, Series B Probes

ID No Oligo Name Sequence # bp TM GC%

58 BoLA-ClEx2B01L CAGATTTTCCGAGTGAGCC 19 51 53

59 BoLA-ClEx2B02L CAATTTTTCCGAGTGAGCCT 20 50 45

60 BoLA-ClEx2B03 CAGACTTTCCGGGCGAAC 18 53 61

61 BoLA-ClEx2B04L CAGAGTTTCCGAGTGAACCT 20 52 50

62 BoLA-ClEx2B05L CAGACTTTCCGAGTGGACC 19 53 58

63 BoLA-ClEx2B07 CAGACTTTCCGAGCGAAC 18 50 56

64 BoLA-ClEx2B08L CAGACTTTCCGAGTGTACC 19 51 53

65 BoLA-ClEx2B09 CAGATTTTCCGGGCGAAC 18 50 56

66 BoLA-ClEx2B10L CAGATTTTCCGAGTGGACC 19 51 53

67 BoLA-ClEx2Bl l CAGTCTTTCCGAGTGGGC 18 53 61

68 BoLA-ClEx2B12 CTGTGGTACCGAGAGGCC 18 55 67

69 BoLA-ClEx2B13 CTGGTGTATCGAGGGAGC 18 53 61

70 BoLA-ClEx2B14L ACTGGTGTATCGAGAGAGC 19 51 53

71 BoLA-ClEx2B15L CTGGTATATCGAGAGAGCC 19 51 53

72 BoLA-ClEx2B16L CAATTTTTCCGACGGGGCC 19 53 58

73 BoLA-ClEx2B17L ACAATTTTTCCGAGTGTACCT 21 49 38

74 BoLA-ClEx2B18L CAGAATTTCCGAGTGGGCC 19 53 58

75 BoLA-ClEx2B19 CAGACTTTCCGAGCAAAC 18 48 50

76 BoLA-ClEx2B22L CTGCTGTATCGAGAGAACC 19 51 53

77 BoLA-ClEx2B23 CTGAAGTACCGAGAGGCC 18 53 61

78 BoLA-ClEx2B24L CAGAAATCCCGATTATGCTTG 21 50 43

79 BoLA-ClEx2B25L CAGGAATCCCGATTATGCTT 20 50 45

80 BoLA-ClEx2B26L2 CTGCTGTATCGAAAGAACCT 20 50 45

81 BoLA-ClEx2B27 CAGAGATTGCGAACGGGC 18 53 61

82 BoLA-ClEx2B28L CAGACTTTCCGAGTGAACC 19 51 53

83 BoLA-ClEx2B29L CAGAGATCCCAATTATGCTTG 21 50 43

84 BoLA-ClEx2B30L CAGTCTTTCCGAGTGAACC 19 51 53

85 BoLA-ClEx2B31L CAGTTTCCGAGTGAACCTGA 20 52 50

86 BoLA-ClEx2B32L CAGGTTTTCCAAGTGAACCT 20 50 45

87 BoLA-ClEx2B33L CAGGTTTTCCGAGTGAACC 19 51 53

Table 3: BoLA Class I, Exon 3, Series A Probes

ID No Oligo Name Sequence # bp TM GC%

88 BoLA-ClEx3A01R GGCGTCCTGCCTGTATCC 18 55 67

89 BoLA-ClEx3A02R GCCGAACTGCTCATAGCC 18 53 61

90 BoLA-ClEx3A03R GGCGTTCTGCCAGATCCC 18 55 67

91 BoLA-ClEx3A04R GGCGTCCTGCCTGTACC 17 54 71

92 BoLA-ClEx3A05R AGCGTCCTGCCTGTACCC 18 55 67

93 BoLA-ClEx3A06R GGCGTACTGCCTGTACCC 18 55 67

94 BoLA-ClEx3A07R GGCGTCCTGACTGTACCC 18 55 67

95 BoLA-ClEx3A08R GGCGAACTGATCGTACCC 18 53 61

96 BoLA-ClEx3A09R GGCGAGCTGATTATACCCG 19 53 58

97 BoLA-ClEx3A10R GGCGTCCTGATTATACCCG 19 53 58

98 BoLA-ClEx3Al lR GCCGAACTGCGTATACCC 18 53 61

99 BoLA-ClEx3A12R GGCGTCCTGCTCATACCC 18 55 67

100 BoLA-ClEx3A13R GCCGTACTGCTCATACCC 18 53 61

101 BoLA-ClEx3A14R GCCGTACTGATCATACCCG 19 53 58

102 BoLA-ClEx3A15R GGCTAACTGATCATACCCG 19 51 53

103 BoLA-ClEx3A16R GGCGAACTGATCATACCCG 19 53 58

104 BoLA-ClEx3A17R GCCGTACTGCTAATACCCG 19 53 58

105 BoLA-ClEx3A18R GGCGAACTGCTTGAACCC 18 53 61

106 BoLA-ClEx3A19R GCCGAACTGCGTGAACCC 18 55 67

107 BoLA-ClEx3A20R GGCGTCCTGCATGAACCC 18 55 67

108 BoLA-ClEx3A21R GCCGTACTGCATGAACCC 18 53 61

109 BoLA-ClEx3A22R GGCGAACTGCATGAACCC 18 53 61

110 BoLA-ClEx3A23R GCCGAACTGCATGAACCC 18 53 61

111 BoLA-ClEx3A24R GCCGAACTGCCAGAACCC 18 55 67

112 BoLA-ClEx3A25R GCCGAACTGCCAAAACCC 18 53 61

113 BoLA-ClEx3A26R GGCGAACTGATCATACCGC 19 53 58

114 BoLA-ClEx3A27R GGCCTTCTGCCAGAATCCA 19 53 58

Table 4: BoLA Class I, Exon 3, Series B Probes

ID No Oligo Name Sequence # bp TM GC%

115 BoLA-ClEx3B01 CGCTGAGGAGAGACACAC 18 53 61

116 BoLA-ClEx3B06L GGCAGGCAAAGATCCAACG 19 53 58

117 BoLA-ClEx3B07 GGAGGCAGAGTTCCAACG 18 53 61

118 BoLA-ClEx3B08 TAATGCGGAGAGCGAGAG 18 50 56

119 BoLA-ClEx3B09 TAATGCGGAGAGCGGGAG 18 53 61

120 BoLA-ClEx3B10 TCGTGCGGAGAGATTCAG 18 50 56

121 BoLA-ClEx3Bl lL GTGAAGCTGAGGTACAGAG 19 51 53

122 BoLA-ClEx3B12N TGAGGCGGAGAGACACAG 18 53 61

123 BoLA-ClEx3B13 TGAGGCGGAGAGACGCAG 18 55 67

124 BoLA-ClEx3B15 TGAGGCGGAGAGATTCAG 18 50 56

125 BoLA-ClEx3B16 TGATGCCGCGCGTGTGAG 18 55 67

126 BoLA-ClEx3B17L GTGATGCGGAGACTTGGAG 19 53 58

127 BoLA-ClEx3B18L GTGATGCGGAGAGACAGAG 19 53 58

128 BoLA-ClEx3B19L GGTGATGCGGAGAGATTAAG 20 52 50

129 BoLA-ClEx3B20L GTGATGCGGAGAGATTCAG 19 51 53

130 BoLA-ClEx3B21 TGATGCGGAGGGACACAG 18 53 61

131 BoLA-ClEx3B22 TGATGCGGCGCGTGTGAG 18 55 67

132 BoLA-ClEx3B23 TGCTGCGAAGGGCGAGAG 18 55 67

133 BoLA-ClEx3B24 TGCTGCGGAGACTTGGAG 18 53 61

134 BoLA-ClEx3B25 TGCTGCGGAGAGACAGAG 18 53 61

135 BoLA-ClEx3B26L GTGCTGCGGAGAGATTAAG 19 51 53

136 BoLA-ClEx3B27 TGCTGCGGAGAGATTCAG 18 50 56

137 BoLA-ClEx3B28 TGCTGCGGAGCGTGTGAG 18 55 67

138 BoLA-ClEx3B29S TGCTGCGGAGGGCGAGA 17 54 71

139 BoLA-ClEx3B30L GTGTTGCGGAGAGATTCAG 19 51 53

140 BoLA-ClEx3B31L GTTACGCTGAGGTACAGAG 19 51 53

141 BoXA-ClEx3B32L GTTATGCTGAGGTACAGAG 19 49 47

142 BoLA-ClEx3B33L CAGATTATGCTGAGTCTTTGA 21 49 38

143 BoLA-ClEx3B34L GGTTCTACGGACTTTTACAG 20 50 45

144 BoLA-ClEx3B35 TTCTGCGGAGAGCGGGAG 18 55 67

145 BoLA-ClEx3B38L AAGGTTATGCTGAGTCTTTGA 21 49 38

The nucleotide sequences of MHC-I alleles appearing in the following Genbank Accession Nos. were used to prepare the probes listed in Tables 1-4 above: M69204, AB008573-AB008654 inclusive, AB009347-AB009349 inclusive, AB009359, AB009360, AB009655, AB013099, AJ010861-AJ010867 inclusive, AJ271292, AJ271294, L02832-L02835 inclusive, M21043, M21044, M24090, M69206, U01187, X80933-X80936 inclusive, X82672, X92870, X97645, and Y09205-Y09208 inclusive. (In addition to the above-reported nucleotide sequences, probes for these same regions can be utilized for any new alleles identified hereafter.)

A hemi-nested PCR protocol was used to amply exons 2 and 3 together from genomic DNA (primers BoClFP-E2A/E2B and BoClRP-E3C) followed by amplification of each exon independently. For second stage amplifications biotinylated forward and reverse primers are be used to amplify each exon. The primer sequences are as follows:

Class I exon 2 mixture of BoClFP-E2A/E2B (SEQ ID Nos: 28, 29) acgtggacga cacg (c/g) agttc 20

and BoClRP-E2A (SEQ ID No: 30) ctcgctctgg ttgtagtagc c 21

Class I exon 3

BoClFP-E3D (SEQ ID No: 31) tggtcggggc gggtcagggt ctcac 25

and BoClRP-E3C (SEQ ID No: 32) ccttcccgtt ctccaggtat ctgcggagc 29

Following 10 cycles of first round PCR amplification and 35 cycles of second round amplification, 20 μl of the 25 μl PCR reaction is diluted to 100 μl with blocking buffer (150 mM Na-Citrate, 5x Denharts), denatured for 5 minutes at 95°C, and 35 μl of diluted PCR product hybridized to a well of a corresponding microarray slide overnight at 50°C. Slides are washed in room temperature, O.lx SSPE, incubated for 1 hour at room temperature with 35 μl Sfreptavidin-Alexa Fluor^® 546 conjugate (Molecular Probes) diluted 1:500 in blocking buffer, rinsed in O.lx SSPE, dried and scanned on an Applied Precision Array WoRx scanner. Spots are scored on a 5 -point scale from negative to strongly positive and data is inteφreted using Cytofile genotyping software (Davies 1988; Davies et al. 1994b).

Example 2 - Nuclear Transfer

Cryopreserved aliquots of cell suspensions from a Nellore fetus removed by hysterotomy at Day 45 of gestation were used to provide donor cells. The donor cells were derived from cells frozen at passage 2 (Day 10 of culture), then thawed and cultured in 4 well Nunc plates containing Dulbecco's Modified Eagles medium (DMEM-F12) + 10% v:v fetal bovine serum (FBS) + 1% v:v penicillin/streptomycin at 37°C in air containing 5% CO₂. At 50% confluence they were serum starved (0.5% FBS) for 5 days prior to NT.

Recipient oocytes were slaughterhouse derived and matured for 17 hours in Medium 199 (M 199; Gibco Laboratories Inc.; Grand Island, NY) supplemented with 10% v:v fetal calf serum (FCS; Gibco), FSH 0.1 units/ml (Sioux Biochem; Sioux City, IA), LH 0.1 units/ml (Sioux Biochem), estradiol 1 μg/ml (Sigma; St Louis, MO), 0.1 mM Cysteamine (Sigma M 9768), and 1% penicillin- streptomycin. The cumulus-oocyte complexes were vortexed 17 hours post maturation for 3 min in 0.1% hyaluronidase, washed, and then held in M 199 + 4 mg/ml BSA.

Oocytes were enucleated beginning at 19 h post maturation. Prior to enucleation, oocytes were placed for 15 min in Hepes-buffered Ml 99 containing Hanks salts (H-M199; Gibco) with 4 mg/ml fatty acid free BSA (Sigma) plus 7.5 μg/ml cytochalasin B (Sigma) and 5 μg/ml Hoechst 33342 (Sigma). Oocytes were selected for the presence of a polar body and homogeneous cytoplasm. Suitable oocytes were enucleated in H-M199 with 7.5 μg/ml cytochalasin B using a beveled 25 μm outside diameter glass pipette. Only oocytes in which the removal of both the polar body and metaphase nucleus was confirmed by observation under UV light were included in the experiment. Fibroblasts were combined with enucleated oocytes in H-M199 using a 25 μm outside diameter glass pipette, then returned to Ml 99 + 4 mg/ml BSA. The oocyte-fibroblast couplets were manually aligned with a mouth pipette in groups of 4-6 and fused in a 0.5 mm fusion chamber (BTX) that contained mannitol 270 mM and magnesium chloride 0.05 mM (Wells & Powell 2000). Fusion parameters were 1x40 μsec 2.25 kV/c DC fusion pulses delivered by a BTX Elecfrocell Manipulator 830 (BTX; San Diego, CA). Oocyte-fibroblast fusion was assessed 20 - 30 minutes later by light microscopy and unfused couplets were refused. Oocyte activation were performed 3-5 h after fusion at 27 h post maturation, by a 4 min incubation in Hepes buffered Ml 99 + 5 μM ionomycin (Calbiochem; San Diego, CA), then 4 minutes in 30 mg/ml H199 + BSA followed by washing in 4 mg/ml BSA in H-M199. The fused oocytes were transferred into 2 mM DMAP in Ml 99 + 3 mg/ml BSA for 4 h followed by transfer to the embryo culture medium for 7 days. Embryos were cultured in 50 :1 drops of a derivative of synthetic oviductal fluid serum-free medium (BARC-1; Wells and Powell, 2000) under mineral oil (Sage Biopharma, Bedminster, NJ) in a 5% CO₂,

5% O₂, 90% N₂ atmosphere.

Cloned embryos classified as Grade 1 or 2 blastocysts on Day 6 following NT were transferred. Two blastocysts were non-surgically transferred into each recipient at Day 6.5 after natural or induced heat. Recipient cows were evaluated for pregnancy at 21 days following NT (15 days after embryo transfer) by serum progesterone levels and the first direct pregnancy examination was by transrectal ulfrasonography at Day 32 following NT. Fetuses with a detectable heartbeat were recovered following slaughter at Day 35.

Example 3 - Comparison of MHC-I Expression in Non-MHC-I matched NT Fetuses and Control Fetuses

A fibroblast cell line was derived from an in vivo produced Day 45 Nellore fetus. To produce the fetus, three embryos recovered non-surgically from a donor cow were transferred the same day into three recipient cows, all of which were pregnant at Day 45. The Nellore cell line was selected with a goal of amplifying any differences that may arise between tissue types of the donor tissue (Bos indicus) and recipient cows (Bos taurus - Angus). Fetal fibroblasts were derived from passage 2 cells (10-15 days in culture) and serum starved for 5 days prior to NT. NT was performed as previously described (Hill et al. 2000a) except that embryos were cultured for 7 days in a defined serum- free medium (BARC-1; Wells & Powell 2000).

The development rate for cloned embryos to blastocyst prior to selection of embryos for fransfer into recipient cows is shown in Table 5 below.

Table 5: Development rate of cloned embryos to blastocyst

Oocytes Percent No. Blasts Percent Blasts Cell Line Enucleated Fusion (Day 8) (Day 8)

N 737F 312 78% 86 35.4%

Day 7 embryos were shipped in a temperature-controlled 39°C incubator to a commercial embryo fransfer center (Trans Ova, Iowa) for transfer into synchronous recipient cows. The per embryo survival rate to Day 35 was 23% when transferred in pairs and the recipient cow pregnancy rate was 50%. Six cloned fetuses were recovered from 5 recipient cows between Day 35-50 of gestation.

Tissue samples were collected within 30 minutes of slaughter. If feasible, separate placentomal and inteφlacentomal samples were collected. However, in the Day 35 placentas, distinction between cotyledonary and intercotyledonary areas by visual inspection is difficult. Tissues were be fixed in 4% paraformaldehyde for histology and for immunohistochemistry by freezing in OCT freezing compound. Fetal heart, liver, lung, kidney, gut, and flank muscle were also processed for histology. For immunohistochemistry, 2 x 2.5 cm rectangular sections of apposed placenta and uterus would be excised, anchored in plastic boats with OCT, and immediately frozen in isopentane chilled in liquid nitrogen. Frozen tissues were held on dry ice and then transferred to a -80°C freezer for storage. For sectioning, blocks were warmed to -30°C and cryostat sectioned at 8 μm. Sections were transferred to slides, dried at room temperature for 30 minutes, fixed in cold acetone for 15 minutes, air dried for 30 minutes, and returned to the freezer for storage. If "normal" placentomes, with villus crypt interdigitation, and "failing" placentomes, where attachment is not occurring, were present, at least two tissue blocks containing each type of placentome were collected.

Confrol tissues (Holstein origin) were collected from commercial dairy cows sent to slaughter at a commercial slaughterhouse (Taylor Packing,

Pennsylvania). Tissues were collected and processed on site as described above. Pregnant tracts were initially selected for gestational age by palpation of amniotic vesicle. After opening the uterus, the crown rump length was measured and the fetal age determined using a formula developed for purebred Holsteins by Rexroad et al. (1974). MHC-I immunocytochemistry was performed on frozen sections from

6 NT and 8 confrol placentas within the range of 35-55 days of gestation (see Tables 6 and 7 below) as previously described (Davies et al. 2000). Basically, cryostat sections were blocked with normal goat serum and incubated with a 1 :6000 dilution of IL-A19 anti-bovine MHC-I mAb (Bensaid et al. 1989; generously provided by Jan Naessens, ILRI, Nairobi, Kenya) or control antibody for two hours at 37°C. Detection of antigen/antibody complexes were achieved using a three stage avidin-biotin system and the AEC chromogen. For each pregnancy a minimum of two inteφlacentomal and two placentomal sections were examined. If both "normal" and "failing" placentomes were present, at least two placentomes of each type were assessed. The percent of MHC-I positive trophoblast and maternal epithelium was determined by visual assessment of a minimum often lOOx fields. A reticle was used to define a constant field length. To eliminate inter-operator error, a single investigator read all slides.

Table 6: MHC-I expression in the 6 cloned fetuses recovered from 5 recipient cows

MHC-I MHC-I

% of total % of total

NT Fetal Endometrial MHC-I trophoblast MHC-I endometrium

Fetus Viability Age CR lymphocytes Cotyledon positive Endometrium positive

1 dead 35 0.7 ++-H-+ +++++ 97 ++++ 74 foci

2 live 35 1.5 93 44_. foci

3 live 35 1.3 1 1 1 1 + +++ 58 +++ 68 foci

4 live 40 1.7 + - • 0 10

5a live 50 4 + - 0 15

5b dead 50 3.5 + 15

5a and 5b were twins.

Age of fetus calculated from known NT dates. Table 7: MHC-I expression in 8 control fetuses recovered from 7 cows

MHC-I MHC-1

% of total % of total

Control Fetal Endometrial MHC-I trophoblast MHC-I endometrium

Fetus Viability Age CR lymphocytes Cotyledon positive Endometrium positive la Dead 39 2 + 20 lb Live 39 2 + 20

2 Live 41 2.5 -H- ++ 37

3 Live 45 3.5 + ++ 35

4 Live 45 3.5 + 5

5 Live 45 3.5 ++ 0

6 Live 54 6 + + 10

7 Live 54 6 + + 5

Age of fetus calculated from the crown rump measurement using the formula of Rexroad et al. 1974.

Each of the 3 positive placentas was at 35 days of gestation while the 3 negative placentas were at 40 or 50 days. Based on these results, fetuses that do not express MHC-I are able to develop more normal placentation and have a higher probability of reaching the 2^nd trimester of pregnancy. Non- viable fetuses were present in the cloned group. Two of 6 cloned fetuses were non- viable (as determined by lack of heartbeat on ultrasonographic scan on the previous day and confirmed by presence of amniotic hemorrhage at slaughter). One of these non- viable fetuses was MHC-I positive (Day 35 single) whereas the other was negative (a Day 50 twin).

A striking feature of the endometrium of the recipient cows carrying the 3 cloned fetuses with MHC-I positive trophoblast was widespread endometrial inflammation. There were multiple foci of stromal lymphocyte and plasma cell accumulations in the caruncular and intercaruncular areas (compare Figures 2A-B). These areas were not subtle accumulations, but were instead strikingly obvious even at low power on hematoxylin and eosin ("H&E") sections. The degree of lymphocytic infiltrate was similar for each of the 3 MHC-I positive pregnancies. Additionally, no neutrophils were found that would indicate an infectious endometritis. This degree of lymphocytic infiltration was not present in the caruncles of the MHC-I negative cloned placentas or of the 8 control placentas. Some areas of minor lymphocyte accumulations were found in the stratum compactum. These accumulations were deeper and more segmental than the widespread, commonly focal sub-epithelial accumulations in the MHC-I positive group. CD3 immunostaining of endometrial sections from cloned and control pregnancies confirmed the H&E diagnosis that these cells were indeed lymphocytes. In the three initial Day 35 cloned pregnancies recovered (Table 6: NT fetuses 1, 2 and 3), far greater numbers of lymphocytes were apparent on histological examination of multiple fields from multiple sections. The most striking observation was that of lymphocyte (cd3 positive) aggregates in the stratum compactum of the intercotyledonary areas of endometrium. Interspersed between these aggregates were increased numbers of lymphocytes and plasma cells mainly distributed immediately beneath the epithelium and adjacent to the endometrial glands. Aggregates were defined as areas of cd3 positive cells where more than 20 cells were in contact with each other. Objective counts of numbers of aggregates and interaggregate cd3 positive lymphocytes were determined by visual estimation using a 0.292mm² reticle to delineate linear boundaries per field. Mean counts per field were totaled per section, and means per case were calculated. A minimum of 5 fields per section, 4 sections from inteφlacentomal tissues, per case, was scored.

The cd3 positive aggregates were rare in the seven controls (4/158; 0.03% of fields), but found in over half the fields examined in the three clones (39/62; 62.9% of fields, p<0.001, Chi-square test). The mean number of aggregates per field was thus significantly higher in clones than controls (0.639 V 0.09 vs 0.025 V 0.012; p<0.001, Mann- Whitney rank sum test). These aggregates contained hundreds of cd3 positive lymphocytes in cross section. As illustrated in Figure 3, cd3 positive lymphocytes located away from these aggregates (interaggregate cd3 positive cells) were also found to be significantly higher in the cloned pregnancies (p<0.001). Thus, the combined numbers of cd3 positive cells (aggregate + interaggregate) in the endometrium of cloned pregnancies were far higher than in confrols (pθ.001 ; One Way Anova with Tukey pairwise comparisons).

The possibility that the observed endometrial inflammatory reaction in the cloned pregnancies (i.e., elevated cd3 positive cells) is caused by fetal death is unlikely when the cd3 numbers are compared from the one dead clone and one dead control fetus in this data set. The dead clone had the highest number of cd3 positive aggregates (0.8 aggregates per field) and interaggregate cd3 cells (133 V 38 cells per field; bar 1 in Figure 3) whereas the dead confrol fetus had no aggregates and a nonnal number of interaggregate cells (28 V 7 cells per field; bar 10 in Figure 3). The crown rump length for the dead clone was less than half that expected for a Day 35 fetus (0.7 cm vs expected of 1.9 cm). This indicated either failure of fetal development or a hostile uterine environment. While lymphocytic infiltration in the uterus of the non- viable fetus may logically be explained by release of fetal antigens to the endometrium, no signs of inflammation were present in endometrium of the other non- viable fetus - the Day 50 MHC-I negative clone. Thus, trophoblast MHC-I expression correlated with endometrial lymphocytic accumulations. This small group of clones provides compelling evidence that a substantial proportion of the high early embryonic mortality observed in cloned pregnancies is due to inappropriate trophoblast MHC-I expression and immunologically mediated placental rejection.

Moreover, there is a remarkable similarity between the placental characteristics from NT and interspecies ET fetuses such as horse/donkey ((Allen, 1982) and sheep/goat (Hancock et al., 1968; Hancock, McGovern, 1970). We previously detailed an 82% loss rate in first trimester cloned fetuses, reduced placental vascularity, and rudimentary implantation sites (Hill et al. 2000b). Similar observations have been recorded in interspecies ET from donkeys into horses, where 80% (20/22) of first trimester fetuses failed by Day 90 and implantation sites were abnormal, as demonstrated above. The gross vascularity of the interspecies placentas was reduced, villous and crypt formation was rudimentary and there was widespread accumulation of lymphocytes in the endometrium. It was also determined that the only donkey foal that progressed to term possessed the same tissue type (MHC-I) as the recipient mare. In goat/sheep embryo fransfers, placental attachment either failed to be established or to be maintained (Hancock et al., 1968; Hancock, McGovern, 1970). Underdeveloped cotyledons and lack of villous formation were characteristic findings and the histological findings were suggestive of maternal immune rejection of the placental tissue (Dent et al., 1971). These observations detail intriguing similarities in placental pathology between interspecies and NT fetuses. In most mammals, trophoblast cells do not express major histocompatibility antigens. Davies et al. (2000) demonstrated trophoblast expression of MHC-I antigens, which first appeared during the sixth month of pregnancy and was limited to the inteφlacentomal and placentomal arcade regions, with no expression in the placentomal villus/crypt region. This region is the area of intimate fetal-maternal contact and it suggests that down regulation of MHC-I is necessary to avoid immunological rejection.

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Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims

What is Claimed:

1. A method of minimizing immunological rejection of a nuclear fransfer fetus comprising: transferring a nuclear transfer embryo into an embryo recipient under conditions effective for development of a nuclear fransfer fetus with minimal risk of immunological rejection of the fetus due to a maternal anti-fetal MHC-I immune response.

2. The method according to claim 1 , wherein the nuclear transfer embryo comprises an MHC-I antigen type which is compatible with an MHC-I antigen type of the embryo recipient.

3. The method according to claim 2, further comprising: determining the MHC-I antigen type for both the nuclear transfer embryo and the embryo recipient and matching the nuclear fransfer embryo suitable for transfer into the embryo recipient based on the MHC-I antigen types thereof.

4. The method according to claim 1 , wherein the nuclear fransfer embryo comprises an MHC-I antigen type which is incompatible with an MHC-I antigen type of the embryo recipient.

5. The method according to claim 4, further comprising: regulating MHC-I expression of the nuclear transfer embryo or nuclear fransfer fetus.

6. The method according to claim 5, wherein said regulating comprises: modulating expression of an MHC-I transcription factor in the nuclear fransfer embryo or nuclear transfer fetus.

7. The method according to claim 5, wherein said regulating comprises: treating the nuclear fransfer embryo or nuclear fransfer fetus with a cytokine, a growth factor, or combinations thereof, under conditions effective to inhibit MHC-I expression.

8. The method according to claim 7, wherein said treating is carried out in vitro on the nuclear transfer embryo.

9. The method according to claim 7, wherein said treating is carried out in utero on the nuclear fransfer embryo or the nuclear fransfer fetus.

10. The method according to claim 7, wherein said treating is carried out with a cytokine.

11. The method according to claim 10, wherein the cytokine is IL- 4, IL-10, IL-13, LIF, TGF-β, or combinations thereof.

12. The method according to claim 7, wherein said treating is carried out with a growth factor.

13. The method according to claim 12, wherein the growth factor is insulin, EGF, GM-CSF, TGF-β, IGF, IL-3, or combinations thereof.

14. The method according to claim 4, further comprising: suppressing an immune response of the embryo recipient.

15. The method according to claim 14, wherein said suppressing comprises: administering an amount of an immunosuppresive drug to the embryo recipient under conditions effective to suppress the anti-MHC-I immune response.

16. The method according to claim 15, wherein the immunosuppressive drug is cyclosporin A, tacrolimus, or sirolimus.

17. The method according to claim 1, wherein the embryo recipient is a mammal.

18. The method according to claim 17, wherein the mammal is a ruminant.

19. The method according to claim 1, wherein the nuclear fransfer embryo is developed from non-human mammalian cells.

20. The method according to claim 19, wherein the non-human mammalian cells are ruminant cells.

21. The method according to claim 1, wherein said transferring is carried out by infroducing the nuclear transfer embryo into the uterus of the embryo recipient.

22. A method of performing embryo fransfer comprising: determining an MHC-I antigen type for a nuclear transfer embryo and an MHC-I antigen type for embryo recipients and either (i) transferring the nuclear fransfer embryo into a first embryo recipient having a compatible MHC-I antigen type under conditions effective for development of a nuclear fransfer fetus from the nuclear transfer embryo, or (ii) transferring the nuclear transfer embryo into a second embryo recipient having an incompatible MHC-I antigen type and (a) regulating MHC-I expression of the nuclear fransfer embryo or (b) suppressing an immune response of the embryo recipient, under conditions effective for development of a nuclear transfer fetus from the nuclear fransfer embryo.

23. The method according to claim 22, wherein said transferring according to step (i) or step (ii) comprises implanting the nuclear transfer embryo in a uterus of the first or second embryo recipient.

24. The method according to claim 22, said method comprising transferring according to step (ii) and regulating MHC-I expression of the nuclear transfer embryo according to step (a).

25. The method according to claim 24, wherein said regulating comprises: modulating expression of an MHC-I transcription factor in the nuclear transfer embryo.

26. The method according to claim 24, wherein said regulating comprises: treating the nuclear transfer embryo with a cytokine, a growth factor, or combinations thereof, under conditions effective to inhibit MHC-I expression.

27. The method according to claim 26, wherein said treating is carried out in vitro on the nuclear transfer embryo.

28. The method according to claim 26, wherein said treating is carried out in utero on the nuclear transfer embryo.

29. The method according to claim 26, wherein said treating is carried out with a cytokine.

30. The method according to claim 29, wherein the cytokine is IL- 4, IL-10, IL-13, LIF, TGF-β, or combinations thereof.

31. The method according to claim 26, wherein said treating is carried out with a growth factor.

32. The method according to claim 31 , wherein the growth factor is insulin, EGF, GM-CSF, TGF-β, IGF, IL-3, or combinations thereof.

33. The method according to claim 24 further comprising: regulating MHC-I expression of the nuclear fransfer fetus under conditions effective for continued development of the nuclear transfer fetus.

34. The method according to claim 33, wherein said regulating comprises: modulating expression of an MHC-I transcription factor in the nuclear fransfer fetus.

35. The method according to claim 34, wherein said regulating comprises: treating the nuclear transfer fetus with a cytokine, a growth factor, or combinations thereof, under conditions effective to inhibit MHC-I expression.

36. The method according to claim 35, wherein said treating is carried out in utero on the nuclear fransfer fetus.

37. The method according to claim 35, wherein said treating is carried out with a cytokine.

38. The method according to claim 37, wherein the cytokine is IL-

4, IL-10, IL-13, LIF, TGF-β, or combinations thereof.

39. The method according to claim 35, wherein said treating is carried out with a growth factor.

40. The method according to claim 39, wherein the growth factor is insulin, EGF, GM-CSF, TGF-β, IGF, IL-3, or combinations thereof.

41. The method according to claim 22, said method comprising fransferring according to step (ii) and suppressing an immune response of the embryo recipient according to step (b).

42. The method according to claim 41, wherein said suppressing comprises: administering an amount of an immunosuppresive drug to the embryo recipient under conditions effective to suppress the anti-MHC-I immune response.

43. The method according to claim 42, wherein the immunosuppressive drug is cyclosporin A, tacrolimus, or sirolimus.

44. The method according to claim 22, wherein the first or second embryo recipients is a mammal.

45. The method according to claim 44, wherein the mammal is a ruminant.

46. The method according to claim 22, wherein the nuclear transfer embryo is developed from non-human mammalian cells.

47. The method according to claim 46, wherein the non-human mammalian cells are ruminant cells.

48. An MHC-I microarray typing system comprising: a substrate and a plurality of oligonucleotide probes bound to the substrate, each of the plurality of oligonucleotides binding to at least one MHC-I allele, wherein each MHC-I allele binds to different oligonucleotide probes.

49. The MHC-I microarray typing system according to claim 48, wherein the at least one MHC-I allele is a bovine allele.

50. The MHC-I microarray typing system according to claim 49, wherein the plurality of oligonucleotide probes comprise: a first set of oligonucleotide probes specific for MHC-I exon 2, codons 61-68; a second set of oligonucleotide probes specific for MHC-I exon

2, codons 71-78; a third set of oligonucleotide probes specific for MHC-I exon 3, codons 111-118; and a fourth set of oligonucleotide probes specific for MHC-I exon

3, codons 151-158.