Eight Calves Cloned from Somatic Cells of a Single Adult

Abstract

lation with anti-CD28 enhanced NFATc nuclear accumulation (Fig. 4B), in keeping with the finding that T H 2 cytokine induction in wildtype T H cells requires costimulation (Fig. 2C). In contrast, anti-CD3 treatment alone led to an increase in nuclear NFATc in Jnk1-/-T H cells and a decrease in cytoplasmic NFATc (Fig. 4, A and B), consistent with the high T H 2 cytokine production by CD3-activated Jnk1-/cells (Fig. 2C). The enhanced accumulation of nuclear NFATc in Jnk1-/-T H cells was observed in cells 8, 24, and 48 hours after stimulation, but was not observed in nonactivated cells (10). NFATc accumulation was specific because the amount of nuclear NFATp, a proposed negative regulator of T H 2 cytokine genes (21), was the same in wild-type and Jnk1-/cells (Fig. 4A). Enhanced nuclear accumulation of NFATc in Jnk1-/-T cells was not blocked by anti-IL-4 (Fig. 4A); hence, increased IL-4 production and NFATc nuclear localization is intrinsic to T cell receptor signaling and is not secondary to IL-4 production. Because NFATc can bind to the IL-4 promoter and is required for IL-4 production and T H 2 differentiation (20, 22), the greatly enhanced amount of nuclear NFATc could account for the increased IL-4 production in CD3activated Jnk1-deficient mice. The mechanism by which JNK1 negatively regulates NFATc nuclear accumulation remains to be resolved. The isoform NFAT4 is phosphorylated and negatively regulated by JNK, leading to nuclear exclusion (23). This regulation appears to be specific to the NFAT4 isoform; evidence for JNK regulation of NFATc was not reported (23). An indirect mechanism may therefore account for the altered regulation of NFATc in Jnk1-/-T H cells. NFATc and NFATp can bind to the IL-4 promoter NFAT sites (22). Both Jnk1 and NFATp knockout mice have enhanced T cell proliferation and T H 2 cytokine production (21, 24), precisely the opposite of the NFATc knockout. It is therefore possible that these two NFAT factors antagonize each other in the regulation of the IL-4 gene. The apparent similarity between NFATp-/and Jnk1-/phenotypes supports a functional linkage between JNK1 and NFAT. Our results further reveal a novel mechanism by which TCR signaling negatively regulates T H 2 cytokines through JNK1. Positive and negative regulation of JNK1 activity may affect the decision of T H cells to differentiate into T H 1 or T H 2 effectors, and therefore may affect the type of immune response that is initiated. The function of JNK1 demonstrated in this study is distinct from that of JNK2, which is required for IFN-␥ production in T H 1 cells (14). Moreover, the related p38 mitogen-activated protein kinase pathway is T H 1 specific and drives IFN-␥ transcription (25). Together, these pathways potentiate the T H 1 response and provide a potential target for pharmaceutical intervention.

Pig transgenesis by Sleeping Beauty DNA transposition

“…With the successful cloning of animals by somatic cell nuclear transfer (SCNT), it is now possible to produce transgenic pigs from genetically engineered somatic donor cells. In SCNT, reported for a variety of animal species including the mouse (Wakayama et al 2000; Yanagimachi 1999), the sheep (Wilmut et al 1997), the cow (Cibelli et al 1998;Kato et al 1998;Kubota et al 2000), and the pig (Betthauser et al 2000;Onishi et al 2000;Polejaeva et al 2000), the nucleus from a single differentiated somatic cell is transferred into an enucleated oocyte (unfertilized egg cell), and the reconstructed embryo is subsequently transferred to a surrogate mother. This procedure allows modification of the somatic donor cell in culture by transgene insertion or introduction of loss-offunction gene knockout mutations by homologous recombination.…”

Reprogramming of genome function through epigenetic inheritance

“…The most pronounced manifestation of this erasure occurs when a differentiated somatic nucleus is transplanted back into an oocyte, which results in the restoration of totipotency [4][5][6][7] . The reconstituted egg can then progress forward to generate a new organism that is a genetic copy or a clone of the individual nuclear donor.…”

Use of strontium in the activation of bovine oocytes reconstructed by somatic cell nuclear transfer

“…An increased number of births of cloned animals using somatic cells has been reported in several species (Wilmut et al, 1997;Kato et al, 1998;Wakayama et al, 1998;Baguisi et al, 1999;Polejaeva et al, 2000), but the process of cloning is still inefficient. Birth rates were, in general, lower than 7% (Heyman et al, 2002), and many cloned offspring die just after birth or within few days or weeks of life (Hill et al, 1999;Heyman et al, 2002;Wilmut et al, 2002).…”