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Eight Calves Cloned from Somatic Cells of a Single Adult

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Science  11 Dec 1998:
Vol. 282, Issue 5396, pp. 2095-2098
DOI: 10.1126/science.282.5396.2095

Abstract

Eight calves were derived from differentiated cells of a single adult cow, five from cumulus cells and three from oviductal cells out of 10 embryos transferred to surrogate cows (80 percent success). All calves were visibly normal, but four died at or soon after birth from environmental causes, and postmortem analysis revealed no abnormality. These results show that bovine cumulus and oviductal epithelial cells of the adult have the genetic content to direct the development of newborn calves.

Nuclear transfer is an efficient technique for assessing the developmental potential of a nucleus and for analyzing the interactions between the donor nucleus and the recipient cytoplasm. In amphibians, successful nuclear transfer was first reported by Briggs and King who used blastula cells for nuclear transfer to oocytes, which proceeded to develop into tadpoles (1) and later juvenile frogs (2). Other cell types, including germ cells and somatic cells from tadpoles, have also been shown to have developmental totipotency (3): their nuclei directed the formation of fertile amphibians. However, despite extensive studies in amphibians, progeny could not be generated from adult cell nuclei (3). This obstacle was recently overcome in sheep (4) and mice (5), and nuclei from fetal fibroblast cells have directed the formation of lambs (4, 6) and calves (7). Wakayama et al. (5) used nuclear transfer to produce fertile mice from cumulus cells collected from metaphase II oocytes. Here, we report cloning of calves at a high rate using cumulus cells and oviductal epithelial cells that were passaged several times in vitro.

Oviducts and ovaries used as the donor nuclear source were obtained from a local slaughterhouse from a single cow of Japanese beef cattle in an unknown stage of the estrous cycle. Cumulus cells from ovarian oocytes at the germinal vesicle stage and oviductal epithelial cells (8, 9) were collected and cultured for several passages (10), and cells quiescent in the G0-G1 phase by serum starvation for 3 to 4 days (4, 11) were used for nuclear transfer (12). The characteristics of donor cells were determined by labeling with vimentin and cytokeratine (Fig. 1).

Figure 1

Labeling of bovine oviductal (A toC) and cumulus (D to F) cells with vimentin B and E and cytokeratin (C and F). Panels (A) and (D) are negative controls. All oviductal epithelial cells were visually positive for a marker of epithelial cells, cytokeratin (C) (detected with rabbit antiserum to keratin), and for vimentin (B) (detected with rabbit antibody to vimentin) (19). All cumulus cells were also visually positive for vimentin (E) and cytokeratin (F), though the latter was very weak. Original magnification, × 100.

Forty-seven percent of the enucleated oocytes fused with cumulus cells and 63% did so with oviductal epithelial cells (Table 1). Among these constructs, 37 cumulus and 88 oviductal nuclear transplants were selected for culture in vitro for 8 to 9 days, by which time 49% of the cumulus-derived and 23% of the oviductal-derived nuclear transplants had developed into blastocysts. A total of 10 blastocysts originating from both cell types were nonsurgically transferred into surrogate cows at day 7 or 8 after the onset of estrous. Six blastocysts derived from cumulus cells were transferred into three females, and four from oviductal cells were placed into two females. All five females became pregnant. Two of the three surrogates containing cumulus nuclear transplants and one of the two with oviductal transplants had multiple pregnancies. Of the 10 blastocysts transferred to cows, 8 cloned female fetuses completed gestation and were born (Table 2). Calves OVI-1, -2, CUM-3, -4, -5, -6, -7, and OVI-8 were delivered 242, 242, 266, 267, 267, 276, 276, and 287 days of gestation, respectively (OVI and CUM indicate origin from oviductal or cumulus cells). All calves were born vaginally except calf OVI-8, which was delivered by cesarean section because of dystocia. The average length of pregnancy of Japanese beef cattle with a female fetus is 286.6 ± 0.9 days and the average body weight at birth is 27.0 ± 0.8 kg. The pregnancy period is often shorter when there are two fetuses. The calves of OVI-1 and OVI-2 were born prematurely.

Table 1

Developmental potential of somatic nuclear transplants in vitro.

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Table 2

Calves cloned from somatic cells. OVI and CUM designate the origin of the donor cells: oviduct and cumulus cells, respectively.

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Four of the eight calves died. Postmortem analysis did not reveal any abnormality; however, environmental factors appeared to account for their deaths. Calf CUM-3 died 3 days after birth from pneumonia apostematosa stemming from heatstroke, CUM-4 and -5 died just after birth from drawing in superfluous amniotic fluid, and OVI-8 died at birth from dystocia and delayed delivery. The other four calves were healthy. In addition, most surrogate mothers showed no or few symptoms of parturition such as labor pains and mammary development. On 1 November 1998, OVI-1 and -2 calves were 120 days old and CUM-6 and -7 calves were 85 days old. The results of microsatellite-typing (13) indicated that the genomes of the cloned calves were identical to those of the donor cells, and different from those of the surrogate mothers (Table 3).

Table 3

DNA microsatellite analysis. The values indicate the fragment size in base pairs. DIK024, AG223, DIK069, DIK089, AG035, AG233, AG053, DIK106, DIK096, DIK020, DIK097, AG310, DIK102, AG119, DIK039, AG133, AG140, AG273, AG147, DIK010, AG160 and DIK068 are on the chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, 20, 21, 22, 23, 24, 26, and 28, respectively. ND, not determined.

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Nuclear transfer of adult somatic cells from farm animals is the most efficient technique for obtaining large numbers of genetically identical animals. Although preimplantation embryonic cells and fetal fibroblasts are also useful for cloning, the economic potential of the donor is not predictable. In contrast, adult somatic cells can be selected from animals already proven to be ideal milk or meat producers. In particular, cumulus cells are especially appropriate for cloning females, because they can be easily obtained without injury to the animals.

In our study, the percentage of nuclear transplants developing into blastocysts was quite high (23% from oviductal cells and 49% from cumulus cells) compared with that of bovine fetal fibroblasts (12%) reported by Cibelli et al. (7). Our higher efficiency may relate to our culture system in which 30% of the control oocytes matured and fertilized in vitro developed into blastocysts (14). Thus, our nuclear transplants were about equal to the controls in developmental ability to the blastocyst stage. Furthermore, the quality of the nuclear transplant blastocysts was evidenced by the fact that they had normal cell numbers (69 to 114 cells) (14).

The high percentage of nuclear transplant embryos developing to term may be due to a number of factors. First, both donor cell populations maintained an apparent normal karyotype during the in vitro culture before use for nuclear transfer (15). Second, nucleo cytoplasmic interactions might be more compatible in this bovine experiment than in previous mouse experiments where the genetic type of the donor nucleus was critically important for later development (16). Third, although it was hypothesized that the donor cytoplasm of some somatic cell types might interfere with the development of nuclear transplants (5), the cumulus cytoplasm used in this study may have been compatible with the oocyte cytoplasm. The precursor cells of cumulus cells were connected by cytoplasmic bridges of microvilli and processes, through which cytoplasmic factors were exchanged. This exchange of factors might account for the higher percentage of nuclear transplant blastocysts from cumulus cell (49%) compared with oviductal cells (23%). Although, the telomerase activity of bovine cumulus cells is unclear, human cumulus cells, known to exhibit telomerase activity (17), might suffer fewer aging affects than other cell types and serve as an ideal adult donor cell for cloning. Fourth, twinning all embryos may have improved the survival rates of the embryos.

A problem for investigation concerns the cytoplasmic contribution of the oocyte to the properties of the clone. Bovine ovaries are often obtained from a slaughterhouse and the genetic background of the oocytes is unknown. In mice, cytoplasmic factors do affect the phenotype of nuclear transplants (16), but whether the effect stems from mitochondrial or maternal gene products is unknown. Two technical factors regarding the donor cells also require consideration, namely, freezing and the cell cycle stage. Large-scale cloning requires freezing of the donor cells. In our study, both donor cell types were freshly prepared and used before freezing. Although freezing of donor cells does not affect the in vitro development of nuclear transplants (14), the later developmental potential of such transplants is unknown. As cells are often damaged during freezing and thawing, this process should be carefully examined.

The application of somatic cell nuclear transfer to animal breeding poses many unanswered questions. Future studies are needed to reduce the death rate from environmental causes and also to reveal whether surviving calves grow normally into fertile adults. The low survival rate of calves might also be in part due to an epigenic component resulting from cloning and related procedures such as culture conditions, because the previous study on cloning bovine by nuclear transfer of embryonic nuclei reported similar postnatal problems (18). Whether these problems were caused by the nuclear transfer procedure or other factors is not known. Also, yet to be determined is whether other adult cell types can be reprogrammed to direct the development of fertile animals.

  • * To whom correspondence should be addressed. E-mail: tsunoda{at}nara.kindai.ac.jp

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