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Science 11 December 1998:
Vol. 282. no. 5396, pp. 2095 - 2098
DOI: 10.1126/science.282.5396.2095
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Reports

Eight Calves Cloned from Somatic Cells of a Single Adult

Yoko Kato, Tetsuya Tani, Yusuke Sotomaru, Kazuo Kurokawa, Jun-ya Kato, Hiroshi Doguchi, Hiroshi Yasue, Yukio Tsunoda *

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.

Y. Kato, Y. Sotomaru, Y. Tsunoda, Laboratory of Animal Reproduction, College of Agriculture and Research Institute for Animal Developmental Biotechnology, Kinki University, 3327-204, Nakamachi, Nara, 631-8505, Japan. T. Tani, Laboratory of Animal Reproduction, College of Agriculture, Kinki University, 3327-204, Nakamachi, Nara, 631-8505, Japan. K. Kurokawa and J. Kato, Division of Molecular Oncology, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara, 630-0101, Japan. H. Doguchi and H. Yasue, Department of Animal Breeding and Genetics, National Institute of Animal Industry, Ministry of Agriculture, Forestry, and Fisheries (MAFF), Tsukuba, Ibaraki 305-0901 Japan.
*   To whom correspondence should be addressed. E-mail: tsunoda{at}nara.kindai.ac.jp

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).

Fig. 1. Labeling of bovine oviductal (A to C) 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. [View Larger Version of this Image (172K GIF file)]

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.

Origin of donor cells No. of oocytes
 No. of oocytes developed to
Fused/total Cultured Two-cell Eight-cell Morula Blastocyst
Cumulus 47/99 37 31 25 21 18
Oviduct 94/150 88 77 58 39 20

Table 2. Calves cloned from somatic cells. OVI and CUM designate the origin of the donor cells: oviduct and cumulus cells, respectively.

Calf number Born at day Weight at birth (kg) Status
OVI-1 242 18.2 Living
OVI-2 242 17.3 Living
CUM-3 266 32.0 Dead (day 3)
CUM-4 267 17.3 Dead (day 0)
CUM-5 267 34.8 Dead (day 0)
CUM-6 276 23.0 Living
CUM-7 276 27.5 Living
OVI-8 287 30.1 Dead (day 0)

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.

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.

REFERENCES AND NOTES

  1. R. Briggs and T. J. King, Proc. Natl. Acad. Sci. U.S.A. 38, 455 (1952) [Free Full Text] .
  2. ___, Dev. Biol. 2, 252 (1960) [CrossRef] [Web of Science] [Medline] .
  3. M. A. Di Berardino, in Genomic Potential of Differentiated Cells (Columbia Univ. Press, New York, 1997).
  4. I. Wilmut, A. E. Schnieke, J. McWhir, A. J. Kind, K. H. S. Campbell, Nature 385, 810 (1997) [CrossRef] [Medline] .
  5. T. Wakayama, A. C. F. Perry, M. Zuccotti, K. R. Johnson, R. Yanagimachi, ibid. 394, 369 (1998) [CrossRef] [Medline].
  6. A. E. Schnieke, et al., Science 278, 2130 (1997) [Abstract/Free Full Text] .
  7. J. B. Cibelli, et al., ibid. 280, 1256 (1998) [Abstract/Free Full Text].
  8. M. S. Joshi, J. Reprod. Fertil. 83, 249 (1988) [Abstract/Free Full Text] .
  9. Epithelial cell clusters from the mucosal tissue were squeezed from the oviduct and cultured.
  10. They were passaged six times for cumulus and four times for oviductal epithelial cells. D-ME medium modified for mouse embryonic stem cell culture (ES-D-MEM) and supplemented with 10% fetal bovine serum (FBS) was used for the cell culture [E. J. Robertson, Ed., Teratocarcinomas and Embryonic Stem Cells (IRL Press, Oxford, 1987).
  11. Cells cultured in 0.5% or less FBS for longer than 3 days attained a quiescent state (14).
  12. For nuclear transfer, in vitro-matured oocytes were enucleated at 22 to 24 hours after maturation. A single donor cell was electrically fused with an oocyte immediately after enucleation with two pulses of 150 v/mm of dc for 25 µs in Zimmerman fusion medium. Pulses were repeated twice with an interval of 15 min until fusion occurred. Fused oocytes were again electrically stimulated (20-v/mm dc pulses for 20 µs) to ensure activation. Nuclear transplant oocytes were immediately treated with cyclohexamide (10 µg/ml) in CR1-aa medium [C. F. Rosenkraus and N. L. First, Theriogenology 35, 266 (abstr.) (1991)] with 3 mg of bovine serum albumin (fatty acid free) for 5 to 6 hours. After treatment, the oocytes were cultured in cyclohexamide-free medium. On day 3 (day 1 being the day of nuclear transfer), the nuclear transplant embryos were transferred to dishes containing CR-1aa medium supplemented with 10% FBS and mouse fetal fibroblast cells pretreated with mitomycin C (10 µg/ml) for 2.5 hours. On days 8 and 9 of in vitro culture, visually normal blastocysts were selected and transferred to recipient cows.
  13. Genomes of recipient cows, nuclear donor cells, and cloned calves were typed for microsatellites by means of 23 primer sets that were provided by Shirakawa Institute of Animal Genetics, Livestock Technology Association of Japan [ M. M. Inoue , et al., Anim. Sci. Technol. 68, 443 (1997) ].
  14. Y. Kato et al., unpublished data.
  15. Even after 8 to 15 passages when they stopped dividing, most (71 to 82%) maintained normal diploid chromosomes.
  16. W. Reik, et al., Development 119, 933 (1993) [Abstract/Free Full Text] .
  17. M. Dorland, S. Hol, R. J. van Kooij, E. R. te Velde, J. Reprod. Fertil. 20 (abstr.), 31 (1997).
  18. F. B. Garry, R. Adams, J. P. McCann, K. G. Odde, Theriogenology 45, 141 (1996) [CrossRef] [Web of Science] .
  19. The antiserum to keratin was obtained from Transformation Research, Farmingham, MA (catalog number 1007), and antibody to vimentin was from Diagnostic BioSystems, Freemont, CA (catalog number PDR 001).
  20. We thank M. DiBerardino for the critical review of the manuscript and M. Kita, G. Tachiura, and other staff members of the Ishikawa Prefecture Livestock Station and Animal Public Health Center for embryo transfer, assistance and management of recipient animals, and assistance in postmortem analyses. This work was supported by grants from the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) and the Special Coordination Funds for Promoting Science and Technology from the Ministry of Science and Technology.
11 September 1998; accepted 3 November 1998


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   Abstract »    Full Text »    PDF »
Utility of Rapidly Matured Oocytes as Recipients for Production of Cloned Embryos from Somatic Cells in the Pig.
K. Miyoshi, S. J. Rzucidlo, S. L. Pratt, and S. L. Stice (2002)
Biol Reprod 67, 540-545
   Abstract »    Full Text »    PDF »
Production of Nuclear Transfer Horse Embryos by Piezo-Driven Injection of Somatic Cell Nuclei and Activation with Stallion Sperm Cytosolic Extract.
Y.H. Choi, C.C. Love, Y.G. Chung, D.D. Varner, M.E. Westhusin, R.C. Burghardt, and K. Hinrichs (2002)
Biol Reprod 67, 561-567
   Abstract »    Full Text »    PDF »
Dependence of DNA Synthesis and In Vitro Development of Bovine Nuclear Transfer Embryos on the Stage of the Cell Cycle of Donor Cells and Recipient Cytoplasts.
S. Kurosaka, Y. Nagao, N. Minami, M. Yamada, and H. Imai (2002)
Biol Reprod 67, 643-647
   Abstract »    Full Text »    PDF »
Nuclei of Nonviable Ovine Somatic Cells Develop into Lambs after Nuclear Transplantation.
P. Loi, M. Clinton, B. Barboni, J. Fulka Jr., P. Cappai, R. Feil, R. M. Moor, and G. Ptak (2002)
Biol Reprod 67, 126-132
   Abstract »    Full Text »    PDF »
Production of Cloned Cattle from In Vitro Systems.
E. J. Forsberg, N. S. Strelchenko, M. L. Augenstein, J. M. Betthauser, L. A. Childs, K. J. Eilertsen, J. M. Enos, T. M. Forsythe, P. J. Golueke, R. W. Koppang, et al. (2002)
Biol Reprod 67, 327-333
   Abstract »    Full Text »    PDF »
Clinical, Hormonal, and Hematologic Characteristics of Bovine Calves Derived from Nuclei from Somatic Cells.
P. Chavatte-Palmer, Y. Heyman, C. Richard, P. Monget, D. LeBourhis, G. Kann, Y. Chilliard, X. Vignon, and J.P. Renard (2002)
Biol Reprod 66, 1596-1603
   Abstract »    Full Text »    PDF »
Remarkable Differences in Telomere Lengths among Cloned Cattle Derived from Different Cell Types.
N. Miyashita, K. Shiga, M. Yonai, K. Kaneyama, S. Kobayashi, T. Kojima, Y. Goto, M. Kishi, H. Aso, T. Suzuki, et al. (2002)
Biol Reprod 66, 1649-1655
   Abstract »    Full Text »    PDF »
In Vitro Development of Bovine Nuclear Transfer Embryos from Transgenic Clonal Lines of Adult and Fetal Fibroblast Cells of the Same Genotype.
S. Arat, J. Gibbons, S. J. Rzucidlo, D. S. Respess, M. Tumlin, and S. L. Stice (2002)
Biol Reprod 66, 1768-1774
   Abstract »    Full Text »    PDF »
In Vitro Development of Horse Oocytes Reconstructed with the Nuclei of Fetal and Adult Cells.
X. Li, L. H.-A. Morris, and W.R. Allen (2002)
Biol Reprod 66, 1288-1292
   Abstract »    Full Text »
Age-Related Changes of the Somatotropic Axis in Cloned Holstein Calves.
K. E. Govoni, X. C. Tian, G. W. Kazmer, M. Taneja, B. P. Enright, A. L. Rivard, X. Yang, and S. A. Zinn (2002)
Biol Reprod 66, 1293-1298
   Abstract »    Full Text »    PDF »
Hypertonic Medium Treatment for Localization of Nuclear Material in Bovine Metaphase II Oocytes.
J.-L. Liu, L.-Y. Sung, M. Barber, and X. Yang (2002)
Biol Reprod 66, 1342-1349
   Abstract »    Full Text »
Rhesus Monkey Embryos Produced by Nuclear Transfer from Embryonic Blastomeres or Somatic Cells.
S. M. Mitalipov, R. R. Yeoman, K. D. Nusser, and D. P. Wolf (2002)
Biol Reprod 66, 1367-1373
   Abstract »    Full Text »
From intestine to muscle: Nuclear reprogramming through defective cloned embryos.
J. A. Byrne, S. Simonsson, and J. B. Gurdon (2002)
PNAS 99, 6059-6063
   Abstract »    Full Text »    PDF »
Enhanced Survivability of Cloned Calves Derived from Roscovitine-Treated Adult Somatic Cells.
J. Gibbons, S. Arat, J. Rzucidlo, K. Miyoshi, R. Waltenburg, D. Respess, A. Venable, and S. Stice (2002)
Biol Reprod 66, 895-900
   Abstract »    Full Text »    PDF »
Porcine Sperm Factor Supports Activation and Development of Bovine Nuclear Transfer Embryos.
J. G. Knott, K. Poothapillai, H. Wu, C. L. He, R. A. Fissore, and J. M. Robl (2002)
Biol Reprod 66, 1095-1103
   Abstract »    Full Text »    PDF »
Reproductive Characteristics of Cloned Heifers Derived from Adult Somatic Cells.
B.P. Enright, M. Taneja, D. Schreiber, J. Riesen, X.C. Tian, J.E. Fortune, and X. Yang (2002)
Biol Reprod 66, 291-296
   Abstract »    Full Text »    PDF »
Bovine Somatic Cell Nuclear Transfer Using Recipient Oocytes Recovered by Ovum Pick-Up: Effect of Maternal Lineage of Oocyte Donors.
K. Bruggerhoff, V. Zakhartchenko, H. Wenigerkind, H.-D. Reichenbach, K. Prelle, W. Schernthaner, R. Alberio, H. Kuchenhoff, M. Stojkovic, G. Brem, et al. (2002)
Biol Reprod 66, 367-373
   Abstract »    Full Text »    PDF »
Frequency and Occurrence of Late-Gestation Losses from Cattle Cloned Embryos.
Y. Heyman, P. Chavatte-Palmer, D. LeBourhis, S. Camous, X. Vignon, and J.P. Renard (2002)
Biol Reprod 66, 6-13
   Abstract »    Full Text »
Production of Cloned Goats after Nuclear Transfer Using Adult Somatic Cells.
C.L. Keefer, R. Keyston, A. Lazaris, B. Bhatia, I. Begin, A.S. Bilodeau, F.J. Zhou, N. Kafidi, B. Wang, H. Baldassarre, et al. (2002)
Biol Reprod 66, 199-203
   Abstract »    Full Text »
Placentomegaly in Cloned Mouse Concepti Caused by Expansion of the Spongiotrophoblast Layer.
S. Tanaka, M. Oda, Y. Toyoshima, T. Wakayama, M. Tanaka, N. Yoshida, N. Hattori, J. Ohgane, R. Yanagimachi, and K. Shiota (2001)
Biol Reprod 65, 1813-1821
   Abstract »    Full Text »    PDF »
Cloned Transgenic Offspring Resulting from Somatic Cell Nuclear Transfer in the Goat: Oocytes Derived from Both Follicle-Stimulating Hormone-Stimulated and Nonstimulated Abattoir-Derived Ovaries.
B. C. Reggio, A. N. James, H. L. Green, W. G. Gavin, E. Behboodi, Y. Echelard, and R. A. Godke (2001)
Biol Reprod 65, 1528-1533
   Abstract »    Full Text »    PDF »
Feasibility of Producing Porcine Nuclear Transfer Embryos by Using G2/M-Stage Fetal Fibroblasts as Donors.
L. Lai, T. Tao, Z. Machaty, B. Kuhholzer, Q.-Y. Sun, K.-W. Park, B. N. Day, and R. S. Prather (2001)
Biol Reprod 65, 1558-1564
   Abstract »    Full Text »    PDF »
Assessment of the developmental totipotency of neural cells in the cerebral cortex of mouse embryo by nuclear transfer.
Y. Yamazaki, H. Makino, K. Hamaguchi-Hamada, S. Hamada, H. Sugino, E. Kawase, T. Miyata, M. Ogawa, R. Yanagimachi, and T. Yagi (2001)
PNAS
   Abstract »    Full Text »    PDF »
Epigenetic Instability in ES Cells and Cloned Mice.
D. Humpherys, K. Eggan, H. Akutsu, K. Hochedlinger, W. M. Rideout III, D. Biniszkiewicz, R. Yanagimachi, and R. Jaenisch (2001)
Science 293, 95-97
   Abstract »    Full Text »    PDF »
Evaluation of Gestational Deficiencies in Cloned Sheep Fetuses and Placentae.
P. A. De Sousa, T. King, L. Harkness, L. E. Young, S. K. Walker, and I. Wilmut (2001)
Biol Reprod 65, 23-30
   Abstract »    Full Text »    PDF »
Parthenogenetic Activation of Rhesus Monkey Oocytes and Reconstructed Embryos.
S. M. Mitalipov, K. D. Nusser, and D. P. Wolf (2001)
Biol Reprod 65, 253-259
   Abstract »    Full Text »    PDF »
Clonal Lines of Transgenic Fibroblast Cells Derived from the Same Fetus Result in Different Development When Used for Nuclear Transfer in Pigs.
B. Kühholzer, R.J. Hawley, L. Lai, D. Kolber-Simonds, and R.S. Prather (2001)
Biol Reprod 64, 1695-1698
   Abstract »    Full Text »
Effect of Fibroblast Donor Cell Age and Cell Cycle on Development of Bovine Nuclear Transfer Embryos In Vitro.
P. Kasinathan, J. G. Knott, P. N. Moreira, A. S. Burnside, D. Joseph Jerry, and J. M. Robl (2001)
Biol Reprod 64, 1487-1493
   Abstract »    Full Text »
Generation of Dwarf Goat (Capra hircus) Clones Following Nuclear Transfer with Transfected and Nontransfected Fetal Fibroblasts and In Vitro-Matured Oocytes.
C.L. Keefer, H. Baldassarre, R. Keyston, B. Wang, B. Bhatia, A.S. Bilodeau, J.F. Zhou, M. Leduc, B.R. Downey, A. Lazaris, et al. (2001)
Biol Reprod 64, 849-856
   Abstract »    Full Text »
Cloned Mice from Fetal Fibroblast Cells Arrested at Metaphase by a Serial Nuclear Transfer.
Y. Ono, N. Shimozawa, M. Ito, and T. Kono (2001)
Biol Reprod 64, 44-50
   Abstract »    Full Text »
Development of Cloned Embryos from Adult Rabbit Fibroblasts: Effect of Activation Treatment and Donor Cell Preparation.
A. Dinnyés, Y. Dai, M. Barber, L. Liu, J. Xu, P. Zhou, and X. Yang (2001)
Biol Reprod 64, 257-263
   Abstract »    Full Text »
Direct Exposure of Chromosomes to Nonactivated Ovum Cytoplasm Is Effective for Bovine Somatic Cell Nucleus Reprogramming.
T. Tani, Y. Kato, and Y. Tsunoda (2001)
Biol Reprod 64, 324-330
   Abstract »    Full Text »
Evidence for Placental Abnormality as the Major Cause of Mortality in First-Trimester Somatic Cell Cloned Bovine Fetuses.
J. R. Hill, R. C. Burghardt, K. Jones, C. R. Long, C. R. Looney, T. Shin, T. E. Spencer, J. A. Thompson, Q. A. Winger, and M. E. Westhusin (2000)
Biol Reprod 63, 1787-1794
   Abstract »    Full Text »
In Vitro Development of Reconstructed Porcine Oocytes after Somatic Cell Nuclear Transfer.
D.-B. Koo, Y.-K. Kang, Y.-H. Choi, J. S. Park, S.-K. Han, I. Y. Park, S.-U. Kim, K.-K. Lee, D.-S. Son, W.-K. Chang, et al. (2000)
Biol Reprod 63, 986-992
   Abstract »    Full Text »
Advances in Livestock Nuclear Transfer.
B. Kühholzer and R. S. Prather (2000)
Experimental Biology and Medicine 224, 240-245
   Abstract »    Full Text »
Pig Cloning by Microinjection of Fetal Fibroblast Nuclei.
A. Onishi, M. Iwamoto, T. Akita, S. Mikawa, K. Takeda, T. Awata, H. Hanada, and A. C. F. Perry (2000)
Science 289, 1188-1190
   Abstract »    Full Text »
Ubiquitinated Sperm Mitochondria, Selective Proteolysis, and the Regulation of Mitochondrial Inheritance in Mammalian Embryos.
P. Sutovsky, R. D. Moreno, J. Ramalho-Santos, T. Dominko, C. Simerly, and G. Schatten (2000)
Biol Reprod 63, 582-590
   Abstract »    Full Text »
Postnatal Growth and Behavioral Development of Mice Cloned from Adult Cumulus Cells.
K. L.K. Tamashiro, T. Wakayama, R. J. Blanchard, D. C. Blanchard, and R. Yanagimachi (2000)
Biol Reprod 63, 328-334
   Abstract »    Full Text »
Establishment of a Porcine Cell Line from In Vitro-Produced Blastocysts and Transfer of the Cells into Enucleated Oocytes.
K. Miyoshi, Y. Taguchi, Y. Sendai, H. Hoshi, and E. Sato (2000)
Biol Reprod 62, 1640-1646
   Abstract »    Full Text »
Development Rates of Male Bovine Nuclear Transfer Embryos Derived from Adult and Fetal Cells.
J. R. Hill, Q. A. Winger, C. R. Long, C. R. Looney, J. A. Thompson, and M. E. Westhusin (2000)
Biol Reprod 62, 1135-1140
   Abstract »    Full Text »
Extension of Cell Life-Span and Telomere Length in Animals Cloned from Senescent Somatic Cells.
R. P. Lanza, J. B. Cibelli, C. Blackwell, V. J. Cristofalo, M. K. Francis, G. M. Baerlocher, J. Mak, M. Schertzer, E. A. Chavez, N. Sawyer, et al. (2000)
Science 288, 665-669
   Abstract »    Full Text »
Analysis of Gene Transcription in Bovine Nuclear Transfer Embryos Reconstructed with Granulosa Cell Nuclei.
R. Daniels, V. Hall, and A.O. Trounson (2000)
Biol Reprod 63, 1034-1040
   Abstract »    Full Text »
Epigenetic Modifications Necessary for Normal Development Are Established During Oocyte Growth in Mice.
S. Bao, Y. Obata, J. Carroll, I. Domeki, and T. Kono (2000)
Biol Reprod 62, 616-621
   Abstract »    Full Text »
From the Cover: Six cloned calves produced from adult fibroblast cells after long-term culture.
C. Kubota, H. Yamakuchi, J. Todoroki, K. Mizoshita, N. Tabara, M. Barber, and X. Yang (2000)
PNAS 97, 990-995
   Abstract »    Full Text »    PDF »
Cell Cycle Synchronization of Porcine Fetal Fibroblasts: Effects of Serum Deprivation and Reversible Cell Cycle Inhibitors.
W.A. Kues, M. Anger, J.W. Carnwath, D. Paul, J. Motlik, and H. Niemann (2000)
Biol Reprod 62, 412-419
   Abstract »    Full Text »
Production of Live Calves Derived from Embryonic Stem-Like Cells Aggregated with Tetraploid Embryos.
S. Iwasaki, K. H.S. Campbell, C. Galli, K. Akiyama, and S. Iwasaki (2000)
Biol Reprod 62, 470-475
   Abstract »    Full Text »
Clonal Propagation of Primate Offspring by Embryo Splitting.
A. W. Chan, T. Dominko, C. M. Luetjens, E. Neuber, C. Martinovich, L. Hewitson, C. R. Simerly, and G. P. Schatten (2000)
Science 287, 317-319
   Abstract »    Full Text »
Reprogramming nuclei: insights from cloning, nuclear transfer and heterokaryons.
N Kikyo and A. Wolffe (2000)
J. Cell Sci. 113, 11-20
   Abstract »    PDF »
Genetic Analysis of Inherited Hypertension in the Rat.
J. P. Rapp (2000)
Physiol Rev 80, 135-172
   Abstract »    Full Text »    PDF »
Dynamic Imaging of the Metaphase II Spindle and Maternal Chromosomesin Bovine Oocytes: Implications for Enucleation Efficiency Verification, Avoidanceof Parthenogenesis, and Successful Embryogenesis.
T. Dominko, A. Chan, C. Simerly, C.M. Luetjens, L. Hewitson, C. Martinovich, and G. Schatten (2000)
Biol Reprod 62, 150-154
   Abstract »    Full Text »
Mice cloned from embryonic stem cells.
T. Wakayama, I. Rodriguez, A. C. F. Perry, R. Yanagimachi, and P. Mombaerts (1999)
PNAS 96, 14984-14989
   Abstract »    Full Text »    PDF »
Developmental Potential of Mouse Follicular Epithelial Cells and Cumulus Cells After Nuclear Transfer.
Y. Kato, A. Yabuuchi, N. Motosugi, J.-y. Kato, and Y. Tsunoda (1999)
Biol Reprod 61, 1110-1114
   Abstract »    Full Text »
From intestine to muscle: Nuclear reprogramming through defective cloned embryos.
J. A. Byrne, S. Simonsson, and J. B. Gurdon (2002)
PNAS 99, 6059-6063
   Abstract »    Full Text »    PDF »
Assessment of the developmental totipotency of neural cells in the cerebral cortex of mouse embryo by nuclear transfer.
Y. Yamazaki, H. Makino, K. Hamaguchi-Hamada, S. Hamada, H. Sugino, E. Kawase, T. Miyata, M. Ogawa, R. Yanagimachi, and T. Yagi (2001)
PNAS 98, 14022-14026
   Abstract »    Full Text »    PDF »


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Science. ISSN 0036-8075 (print), 1095-9203 (online)