Therapeutic cloning, whereby somatic cell nuclear transfer (SCNT) is used to generate patient-specific embryonic stem cells (ESCs) from blastocysts cloned by nuclear transfer (ntESCs), holds great promise for the treatment of many human diseases. ntESCs have been derived in mice and cattle, but thus far there are no credible reports of human ntESCs. Here we review the recent literature on nuclear reprogramming by SCNT, including studies of gene expression, DNA methylation, chromatin remodeling, genomic imprinting and X chromosome inactivation. Reprogramming of genes expressed in the inner cell mass, from which ntESCs are derived, seems to be highly efficient. Defects in the extraembryonic lineage are probably the major cause of the low success rate of reproductive cloning but are not expected to affect the derivation of ntESCs. We remain optimistic that human therapeutic cloning is achievable and that the derivation of patient-specific ntESC lines will have great potential for regenerative medicine.

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...

In mammals, epigenetic marks on the X chromosomes are involved in dosage compensation. Specifically, they are required for X chromosome inactivation (XCI), the random transcriptional silencing of one of the two X chromosomes in female cells during late blastocyst development. During natural reproduction, both X chromosomes are active in the female zygote. In somatic-cell cloning, however, the cloned embryos receive one active (Xa) and one inactive (Xi) X chromosome from the donor cells. Patterns of XCIhave been reported normal in cloned mice, but have yet to be investigated in other species. We examined allele-specific expression of the X-linked monoamine oxidase type A (MAOA) gene and the expression of nine additional X-linked genes in nine cloned XX calves. We found aberrant expression patterns in nine of ten X-linked genes and hypomethylation of Xist in organs of deceased clones. Analysis of MAOA expression in bovine placentae from natural reproduction revealed imprinted XCI with preferential inactivation of the paternal X chromosome. In contrast, we found random XCI in placentae of the deceased clones but completely skewed XCI in that of live clones. Thus, incomplete nuclear reprogramming may generate abnormal epigenetic marks on the X chromosomes of cloned cattle, affecting both random and imprinted XCI.

Development to blastocyst following nuclear transfer is dependent on the donor cell's ability to reprogram its genome to that of a zygote. This reprogramming step is inefficient and may be dependent on a number of factors, including chromatin organization. Trichostatin A (TSA; 0-5 microM), a histone deacetylase inhibitor, was used to increase histone acetylation and 5-aza-2'-deoxycytidine (5-aza-dC; 0-5 microM), a DNA methyl-transferase inhibitor, was used to decrease methylation of chromatin in donor cells in an attempt to improve their reprogrammability. Adult fibroblast cells treated with 1.25 or 5 microM TSA had elevated histone H3 acetylation compared to untreated controls. Cells treated with 0.3 microM 5-aza-dC had decreased methylation compared to untreated controls. Both drugs at 0.08 microM caused morphological changes of the donor cells. Development to blastocysts by embryos cloned from donor cells after 0.08 or 0.3 microM 5-aza-dC treatments was lower than in embryos cloned from untreated control cells (9.7% and 4.2%, respectively, vs. 25.1%), whereas 0.08 microM TSA treatment of donor cells increased blastocyst development compared to controls (35.1% vs. 25.1%). These results indicate that partial erasure of preexisting epigenetic marks of donor cells improves subsequent in vitro development of cloned embryos.

Successful cryopreservation of mammalian oocytes would provide a steady source of materials for nuclear transfer and in vitro embryo production. Our goal was to develop an effective vitrification protocol to cryopreserve bovine oocytes for research and practice of parthenogenetic activation, in vitro fertilization, and nuclear transfer. Bovine oocytes matured in vitro were placed in 4% ethylene glycol (EG) in TCM 199 plus 20% fetal bovine serum (FBS) at 39 degrees C for 12-15 min, and then transferred to a vitrification solution (35% EG, 5% polyvinyl-pyrrolidone, 0.4 M trehalose in TCM 199 and 20% FBS). Oocytes were vitrified in microdrops on a precooled (-150 degrees C) metal surface (solid-surface vitrification). The vitrified microdrops were stored in liquid nitrogen and were either immediately thawed or were thawed after storage for 2-3 wk. Surviving oocytes were subjected to 1) parthenogenetic activation, 2) in vitro fertilization, or 3) nuclear transfer with cultured adult fibroblast cells. Treated oocytes were cultured in KSOM containing BSA or FBS for 9 to 10 days. Embryo development rates were recorded daily and morphologically high-quality blastocysts were cryopreserved for nuclear transfer-derived embryos at Day 7 or Day 8 of culture. Immediate survival of vitrified/thawed oocytes varied between 77% and 86%. Cleavage and blastocyst development rates of vitrified oocytes following in vitro fertilization or activation were lower than those of the controls. For nuclear transfer, however, vitrified oocytes supported embryonic development as equally well as fresh oocytes.