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...

Cloning by somatic cell nuclear transfer has been successfully achieved by both fusing of a donor cell with and injecting an isolated donor cell nucleus into an enucleated oocyte. However, each of the above methods involves extended manipulation of either the oocytes (fusion) or the donor cells (nucleus isolation). Additionally, cloning efficiency can be reduced by low fusion rate of the cell fusion method, and specialized micromanipulation equipment and exacting nucleus isolation techniques are required for the nucleus injection method. Here we report a whole-cell injection technique for nuclear transfer in pigs and the production of cloned piglets with comparable, if not higher, efficiency than the other two nuclear transfer procedures. First, we tested the feasibility of this technique with three types of frequently used donor cells (cumulus, mural granulosa, and fibroblasts) and obtained the optimal nuclear reprogramming conditions for these cells. We further improved our protocol by avoiding ultraviolet exposure during enucleation and achieved a 37% blastocyst rate. We then conducted whole-cell injection using skin fibroblasts from the ear of a sow transgenic for two genes, the porcine lactoferrin and the human factor IX, and produced four live-born cloned transgenic piglets from three recipients. The present study demonstrated the applicability of producing normal, cloned piglets by the simple and less labor-intensive whole-cell intracytoplasmic injection.

Soluble epoxide hydrolase (sEH) is a phase-I xenobiotic metabolizing enzyme having both an N-terminal phosphatase activity and a C-terminal epoxide hydrolase activity. Endogenous hydrolase substrates include arachidonic acid epoxides, which have been involved in regulating blood pressure and inflammation. The subcellular localization of sEH has been controversial. Earlier studies using mouse and rat liver suggested that sEH may be cytosolic and/or peroxisomal. In this study we applied immunofluorescence and confocal microscopy using markers for different subcellular compartments to evaluate sEH colocalization in an array of human tissues. Results showed that sEH is both cytosolic and peroxisomal in human hepatocytes and renal proximal tubules and exclusively cytosolic in other sEH-containing tissues such as pancreatic islet cells, intestinal epithelium, anterior pituitary cells, adrenal gland, endometrium, lymphoid follicles, prostate ductal epithelium, alveolar wall, and blood vessels. sEH was not exclusively peroxisomal in any of the tissues evaluated. Our data suggest that human sEH subcellular localization is tissue dependent, and that sEH may have tissue- or cell-type-specific functionality. To our knowledge, this is the first report showing the subcellular localization of sEH in a wide array of human tissues.

This research was to study the in vitro and in vivo development of cloned embryos derived from adult rabbit fibroblasts following various activation protocols. Effects of serum starvation and passage number of donor cells on the efficiency of cloning were also examined. In experiment I, oocytes were activated either by electric pulses or by electric pulses followed by culture with 6-dimethylaminopurin (DMAP). For experiment II, the best activation protocol from experiment I was employed for cloning using adult rabbit fibroblasts that were cultured for 0-15 passages. In experiment III, the effect of serum starvation of the donor cells on cloning was examined. Finally, in experiment IV, embryo transfers were conducted. These experiments showed that combined electrical pulse and DMAP treatment resulted in superior parthenogenetic blastocyst development (up to 29%), and that activation of the cytoplast before versus after fusion was not different in supporting the in vitro development of nuclear transferred embryos (16%-18% blastocysts). Adult fibroblasts derived from nonpassaged cells were less capable of developing into blastocysts than passaged cells (6% vs. 17%). Serum starvation of donor cells improved cleavage (up to 71%) but did not improve blastocyst development (13%), and no progeny was obtained, irrespective of the treatment. Cell-cycle analysis of adult rabbit fibroblast cells showed that passage 6 and 12 cells were more likely to be in G(0)/G(1) than passage 0 cells, which agrees with the improved embryo development in the passaged-cell groups.

Due to its association with low-quality milk and a decrease in milk production in bovines, mastitis is a major cause of economic loss. Additionally, mastitis can be harmful to suckling newborns and can cause damage to the mammary gland. In mastitic mammary secretions there is a substantial increase in somatic cells, specifically neutrophils. In this study we examined the ability of mastitic and nonmastitic mammary secretions to cause in vitro neutrophil chemotaxis using a microchemotaxis assay. Also, the role of the inflammatory chemokine interleukin-8 (IL-8) in neutrophil recruitment during mastitis was addressed in these in vitro experiments. We found that both nonmastitic and mastitic mammary secretions were chemotactic, not chemokinetic, for neutrophils. The neutrophil chemotactic activity in mastitic, but not nonmastitic, mammary secretions was blocked by anti-IL-8 antibodies. Molecular mass separation of the active components showed that the chemotactic activity of the mastitic secretions was present in the 10-kDa-or-less fraction and was blocked by anti-IL-8 antibodies. These results indicate that IL-8 plays a major role in neutrophil recruitment during mastitis. An understanding of its role will be of help in designing strategies for immunomodulatory therapies for mastitis.