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.

Precisely regulated radial migration out of the ventricular zone is essential for corticogenesis. Here, we identify a mechanism that can tether ventricular zone cells in situ. FILIP interacts with Filamin A, an indispensable actin-binding protein that is required for cell motility, and induces its degradation in COS-7 cells. Degradation of Filamin A is identified in the cortical ventricular zone, where filip mRNA is localized. Furthermore, most ventricular zone cells that overexpress FILIP fail to migrate in explants. These results demonstrate that FILIP functions through a Filamin A F-actin axis to control the start of neocortical cell migration from the ventricular zone.

Cattle are the species used most frequently for the development of assisted reproductive technologies, such as nuclear transfer. Cattle cloning can be performed by a large number of laboratories around the world, and the efficiency of nuclear transfer in cattle is the highest among 25 all species in which successful cloning has been achieved. However, an understanding of the expression of imprinted genes in this important species is lacking. In the present study, real time reverse-transcription polymerase chain reaction (RT-PCR) was utilized to quantify the expression of the bovine Igf2, Igf2r and H19 genes in eight major organs (brain, bladder, heart, kidney, liver, lung, spleen and thymus) of somatic cell cloned calves that died shortly after birth, 30 in three tissues (skin, muscle and liver) of healthy clones that survived to adulthood, and in corresponding tissues of control animals from natural reproduction. We found that, deceased bovine cloned calves exhibited abnormal expression of all three genes studied in various organs.Large variations in the expression levels of imprinted genes were also seen among these clones, which were produced from the same genetic donor. In surviving adult clones, however, the 35 expression of these imprinted genes was largely normal, except for the expression of the Igf2 gene in muscle, which was highly variable. Our data showed disruptions of expression of imprinted genes in bovine clones, which is possibly due to incomplete reprogramming of donor cell nuclei during nuclear transfer, and these abnormalities may be associated with the high neonatal mortality in cloned animals; clones that survived to adulthood, however, are not only 40 physically healthy but also relatively normal at the molecular level of those three imprinted genes.3