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Biomedical subjects

Ian Wilmut

Publications and source records attributed to Ian Wilmut.

At least 19 recordsLinked to original sources
PubMed2004

The effect of interspecific oocytes on demethylation of sperm DNA.

In contrast to mice, in sheep no genome-wide demethylation of the paternal genome occurs within the first postfertilization cell cycle. This difference could be due either to an absence of a sheep demethylase activity that is present in mouse ooplasm or to an increased protection of methylated cytosine residues in sheep sperm. Here, we use interspecies intracytoplasmic sperm injection to demonstrate that sheep sperm DNA can be demethylated in mouse oocytes. Surprisingly, mouse sperm can also be demethylated to a limited extent in sheep oocytes. Our results suggest that the murine demethylation process is facilitated either by a sperm-derived factor or by male pronuclear chromatin composition.

Animals↗
PubMed2004

Effect of limited DNA methylation reprogramming in the normal sheep embryo on somatic cell nuclear transfer.

Active demethylation of cytosine residues in the sperm genome before forming a functional zygotic nucleus is thought to be an important function of the oocyte cytoplasm for subsequent embryonic development in the mouse. Conversely, this event does not occur in the sheep or rabbit zygote and occurs only partially in the cow. The aim of this study was to investigate the effect of limited methylation reprogramming in the normal sheep embryo on reprogramming somatic nuclei. Sheep fibroblast somatic nuclei were partially demethylated after electrofusion with recipient sheep oocytes and undergo a stepwise passive loss of DNA methylation during early development, as determined by 5-methylcytosine immunostaining on interphase embryonic nuclei. A similar decrease takes place with in vivo-derived sheep embryos up to the eight-cell stage, although nuclear transfer embryos exhibit a consistently higher level of methylation at each stage. Between the eight-cell and blastocyst stages, DNA methylation levels in nuclear transfer embryos are comparable with those derived in vivo, but the distribution of methylated DNA is abnormal in a high proportion. By correlating DNA methylation with developmental potential at individual stages, our results suggest that somatic nuclei that do not undergo rapid reorganization of their DNA before the first mitosis fail to develop within two to three cell cycles and that the observed methylation defects in early cleavage stages more likely occur as a direct consequence of failed nuclear reorganization than in failed demethylation capacity. However, because only embryos with reorganized chromatin appear to survive the 16-cell and morula stages, failure to demethylate the trophectoderm cells of the blastocyst is likely to directly impact on developmental potential by altering programmed patterns of gene expression in extra-embryonic tissues. Thus, both remodeling of DNA and epigenetic reprogramming appear critical for development of both fertilized and nuclear transfer embryos.

Animals↗
PubMed2004

Nuclear transfer in rodents.

Cloning is the asexual reproduction of an individual, such that the offspring have an essentially identical nuclear genome. Nuclear transfer and cloning have been achieved in a number of species, namely sheep, cows, goats, rabbits, cats and mice, but have been largely unsuccessful, so far, in dogs, primates and rats. Clearly, contributory factors which affect the outcome of successful cloning experiments are not universally applicable to all species. One theme common to all cloning experiments, however, is the overall inefficiency of the process, typically 0-4%. A number of factors contribute to nuclear transfer inefficiency, and we will review mouse cloning experiments, which address these problems, highlighting the importance of donor nucleus choice (somatic or ES cell, fetal or adult, quiescent or actively dividing). Finally, we will summarize the emerging principles which appear to govern nuclear reprogramming and production of clones, and will consider the application of nuclear transfer to the rat.

Animals↗
PubMed2003

Application of reproductive biotechnology in animals: implications and potentials. Applications of reproductive cloning.

The development of new methods of nuclear transfer in mammals is creating many new opportunities in research, medicine and agriculture. The method of cloning is repeatable and has been established in many laboratories worldwide. However, the present procedure is inefficient with fewer than 4% of embryos becoming viable offspring. A considerable improvement in efficiency is required before wide scale use for livestock improvement. The opportunity to introduce precise genetic changes to livestock is available for the first time through the use of gene targeting procedures in cultured cells that are used as nuclear donors. This has potential application in the production of organs for transplantation to humans, studies of human genetic disease and basic research in to the control of gene expression and function.

Agriculture↗
PubMed2003

Telomerase-immortalized sheep fibroblasts can be reprogrammed by nuclear transfer to undergo early development.

Telomere shortening and lack of telomerase activity have been implicated in cellular senescence in human fibroblasts. Expression of the human telomerase catalytic reverse transcriptase subunit (hTERT) in these cells reconstitutes telomerase activity and immortalizes the cells without tumor transformation. In this report, we show that sheep fibroblasts are similar to human cells. They do not have detectable telomerase activity and undergo only a finite numbers of cell divisions before replicative senescence. Telomere lengths in sheep fibroblasts are similar to those reported for human cells and shorten at a rate of 50-200 base pairs (bp) each cell division. Expression of the human telomerase catalytic subunit restored the telomerase activity in the sheep cells and extended their proliferative life span. None of the telomerase positive sheep fibroblasts exhibited a transformed phenotype after 200 days of continuous culture, and the higher hTERT expressing cells maintained their telomere lengths and normal cell characteristics for more than 500 days in culture. In cloning experiments using one of these cell lines as a nuclear donor, the reconstructed karyoplasts were reprogrammed and developed to the blastocyst stage at a similar frequency to that observed with the parental, telomerase negative cell line. After embryo transfer the blastocysts exhibited a relatively high frequency of implantation, early fetal development, and organogenesis. No fetuses survived beyond 40 days of development, however, showing that although these cells could be substantially reprogrammed, they were not fully competent for nuclear transfer.

Animals↗
PubMed2003

Effects of donor oocytes and culture conditions on development of cloned mice embryos.

Mice have been successfully cloned from somatic and embryonic stem (ES) cells using the "Honolulu method." In the present study, different donor oocytes and different culture conditions were compared to evaluate the developmental potential of nuclear transfer embryos reconstructed with an inbred ES cell line HM-1. Oocytes were recovered from two different F1 donors B6D2F1 (C57BL/6 x DBA/2) and B6CBAF1 (C57BL/6 x CBA). There was no effect of oocyte origin on development of cloned embryos to the morulae/blastocyst stage (B6D2F1 44.1% vs. B6CBAF1 45.0%), and the transferred embryos could develop to term. Two culture conditions were compared to show their ability to support development to the morulae/blastocyst stage of reconstructed embryos with B6D2F1 oocytes. The total cell number in the cloned blastocysts cultured in M16 with 20% oxygen was much higher than that observed in CZB with 20% oxygen. Low oxygen concentration during culture of nuclear transfer embryos in CZB medium showed no beneficial effect on pre-implantation development, no embryos developed to term after transfer to surrogate mothers. Our results demonstrated that not only B6D2F1, but B6CBAF1 oocytes, can be used for nuclear transfer. M16 medium is superior for culture of nuclear transfer embryos and low oxygen concentration with CZB medium during culture shows no benefit on development of cloned embryos.

Animals↗
PubMed2003

Conservation of IGF2-H19 and IGF2R imprinting in sheep: effects of somatic cell nuclear transfer.

In different mammalian species, in vitro culture and manipulation can lead to aberrant fetal and peri-natal development. It has been postulated that these diverse abnormalities are caused by epigenetic alterations and that these could affect genes that are regulated by genomic imprinting. To explore this hypothesis relative to somatic cell nuclear transfer in sheep, we investigated whether the ovine H19-IGF2 and IGF2R loci are imprinted and analysed their DNA methylation status in cloned lambs. A comparison between parthenogenetic and control concepti established that imprinting at these two growth-related loci is evolutionarily conserved in sheep. As in humans and mice, IGF2R and H19 comprise differentially methylated regions (DMRs) that are methylated on one of the two parental alleles predominantly. In tongue tissue from 12 out of 13 cloned lambs analysed, the DMR in the second intron of IGF2R had strongly reduced levels of DNA methylation. The DMR located upstream of the ovine H19 gene was found to be similarly organised as in humans and mice, with multiple CTCF binding sites. At this DMR, however, aberrant methylation was observed in only one of the cloned lambs. Although the underlying mechanisms remain to be determined, our data indicate that somatic cell nuclear transfer procedures can lead to epigenetic deregulation at imprinted loci.

Alleles↗
PubMed2003

Proliferative lifespan is conserved after nuclear transfer.

Cultured primary cells exhibit a finite proliferative lifespan, termed the Hayflick limit. Cloning by nuclear transfer can reverse this cellular ageing process and can be accomplished with cultured cells nearing senescence. Here we describe nuclear transfer experiments in which donor cell lines at different ages and with different proliferative capacities were used to clone foetuses and animals from which new primary cell lines were generated. The rederived lines had the same proliferative capacity and rate of telomere shortening as the donor cell lines, suggesting that these are innate, genetically determined, properties that are conserved by nuclear transfer.

Animals↗
PubMed2003

Human cloning: can it be made safe?

There are continued claims of attempts to clone humans using nuclear transfer, despite the serious problems that have been encountered in cloning other mammals. It is known that epigenetic and genetic mechanisms are involved in clone failure, but we still do not know exactly how. Human reproductive cloning is unethical, but the production of cells from cloned embryos could offer many potential benefits. So, can human cloning be made safe?

Animals↗