Cloning whole animals with somatic cells as parents offers the possibility of targeted genetic manipulations in vitro such as ''gene knock-out'' by homologous recombination. However, such manipulation requires prolonged culture of nuclear donor cells. Previous successes in cloning have been limited to the use of cells collected either fresh or after short-term culture. Therefore, demonstration of genetic totipotency of cells after prolonged culture is pivotal to combining site-specific genetic manipulations and cloning. Here we report birth of six clones of an aged (17-year-old) Japanese Black Beef bull using ear skin fibroblast cells as nuclear donor cells after up to 3 months of in vitro culture (10 -15 passages). We observed higher developmental rates for embryos derived from later passages (10 and 15) as compared with those embryos from an early passage (passage 5). The four surviving clones are now 10 -12 months of age and appear normal, similar to their naturally reproduced peers. These data show that fibroblasts of aged animals remain competent for cloning, and prolonged culture does not affect the cloning competence of adult somatic donor cells. G enetic manipulation of mouse embryonic stem cells has revolutionized mouse genetic research. However, embryonic stem cells are not available in other species. Fortunately, animal cloning using cultured somatic cells offers the possibility of targeted genetic manipulations like those performed in the mouse, should those somatic cells remain competent for cloning after prolonged culture. Live clones have been obtained from adult somatic cells in sheep (1), mice (2), and cows (3, 4). Furthermore, transgenic animals have been produced by cloning gene-transfected fetal somatic donor cells (5, 6). However, to date, successful somatic cell cloning has been largely limited to the use of the donor cells either fresh (2) or after short-term (under 10 passages) in vitro culture (1, 3-6), which would not allow targeted gene manipulations.A recent report (7) indicates that Dolly, the cloned sheep, inherited the shortened telomeres of the adult nuclear donor animal. Moreover, the telomeres of Dolly were further shortened during the brief in vitro culture of the donor cells. These observations raise the questions of whether healthy clones may be obtained from aged donor animals, particularly after longterm cultures of the ''aged'' donor cells. This study was conducted to test the cloning competence of skin fibroblast cells after prolonged in vitro culture, using an aged (17-year-old) elite bull. In this paper, we report that normal live clones were produced from cultured adult somatic cells in a cattle model after up to 3 months of culture (passage 15). Our finding offers promise for producing site-specific genetically modified animals such as ''gene knockout'' animals by somatic cell cloning. Additionally, success in cloning live, aged animals opens the possibility to compare the telomere lengths, aging, and the ''biological age'' of the cloned animals. Materials and Methods...

We used a half-sib family of purebred Japanese Black (Wagyu) cattle to locate economically important quantitative trait loci. The family was composed of 348 fattened steers, 236 of which were genotyped for 342 microsatellite markers spanning 2,664 cM of 29 bovine autosomes. The genome scan revealed evidence of 15 significant QTL (<5% chromosome-wise level) affecting growth and carcass traits. Of the 15 QTL, six QTL were significant at the 5% experiment-wise level and were located in bovine chromosomes (BTA) 4, 5, and 14. We analyzed these three chromosomes in more detail in the 348 steers, with an average marker interval of 1.2 cM. The second scan revealed that the same haplotype of the BTA 4 region (52 to 67 cM) positively affected LM area and marbling. We confirmed the QTL for carcass yield estimate on BTA 5 in the region of 45 to 54 cM. Five growth-related QTL located on BTA 14, including slaughter and carcass weights, were positively affected by the same region of the haplotype of BTA 14 (29-51 cM). These data should provide a useful reference for further marker-assisted selection in the family and positional cloning research. The research indicates that progeny design with moderate genotyping efforts is a powerful method for detecting QTL in a purebred half-sib family.

Cryopreservation of bovine oocytes would be beneficial both for nuclear transfer and for preservation efforts. The overall objective of this study was to evaluate the viability as well as the cryodamage to the nucleus vs. cytoplasm of bovine oocytes following freezing-thawing of oocytes at immature (GV) and matured (MII) stages using in vitro fertilization (IVF), parthenogenetic activation, or nuclear transfer assays. Oocytes were collected from slaughterhouse ovaries. Oocytes at the GV, MII, or MII but enucleated (MIIe) stages were cryopreserved in 5% (v/v) ethylene glycol; 6% (v/v) 1,2-propanediol; and 0.1-M sucrose in PBS supplemented with 20% (v/v) fetal bovine serum. Frozen-thawed oocytes were subjected to IVF, parthenogenetic activation, or nuclear transfer assays. Significantly fewer GV oocytes survived (i.e., remained morphologically intact during freezing-thawing) than did MII oocytes (47% vs. 84%). Subsequent development of the surviving frozen-thawed GV and MII oocytes was not different (58% and 60% cleavage development; 7% and 12% blastocyst development at Day 9, respectively, P > 0.05). Parthenogenetic activation of frozen-thawed oocytes resulted in significantly lower rates of blastocyst development for the GV than the MII oocyte groups (1% vs. 14%). Nuclear transfer with cytoplasts derived from frozen-thawed GV, MII, MIIe, and fresh-MII control oocytes resulted in 5%, 16%, 14%, and 17% blastocyst development, respectively. However, results of preliminary embryo transfer trials showed that fewer pregnancies were produced from cloned embryos derived from frozen oocytes or cytoplasts (9%, n = 11 embryos) than from fresh ones (19%, n = 21 embryos). Transfer of embryos derived by IVF from cryopreserved GV and MII oocytes also resulted in term development of calves. Our results showed that both GV and MII oocytes could survive freezing and were capable of developing into offspring following IVF or nuclear transfer. However, blastocyst development of frozen-thawed oocytes remains poorer than that of fresh oocytes, and our nuclear transfer assay suggests that this poorer development was likely caused by cryodamage to the oocyte cytoplasm as well as to the nucleus.

The chromosomal location of the swine tenascin gene (HXB) was determined by fluorescence in situ hybridization and simultaneous R-banding. Swine tenascin cDNA was labeled with biotin, and used as a probe. Hybridization was detected by the FITC-labeled streptavidin/biotinylated anti-streptavidin antibody system. The signals revealed that the tenascin gene is localized on swine chromosome 1q21.1→q21.3.

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