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Biomedical subjects

K H Campbell

Publications and source records attributed to K H Campbell.

At least 19 recordsLinked to original sources
PubMed2001

Nuclear transfer in practice.

The technique of nuclear transfer (NT) allows the production of embryos, fetuses, and offspring from a range of embryonic, fetal, and adult derived cell types in a range of species. Successful development is dependent upon numerous factors, including type of recipient cell, source of recipient cell, method of reconstruction, activation, embryo culture, donor cell type, and donor and recipient cell cycle stages. The present review will discuss the uses of NT, the techniques presently available, and the factors affecting subsequent development.

Animals↗
PubMed2000

Cloned pigs produced by nuclear transfer from adult somatic cells.

Since the first report of live mammals produced by nuclear transfer from a cultured differentiated cell population in 1995 (ref. 1), successful development has been obtained in sheep, cattle, mice and goats using a variety of somatic cell types as nuclear donors. The methodology used for embryo reconstruction in each of these species is essentially similar: diploid donor nuclei have been transplanted into enucleated MII oocytes that are activated on, or after transfer. In sheep and goat pre-activated oocytes have also proved successful as cytoplast recipients. The reconstructed embryos are then cultured and selected embryos transferred to surrogate recipients for development to term. In pigs, nuclear transfer has been significantly less successful; a single piglet was reported after transfer of a blastomere nucleus from a four-cell embryo to an enucleated oocyte; however, no live offspring were obtained in studies using somatic cells such as diploid or mitotic fetal fibroblasts as nuclear donors. The development of embryos reconstructed by nuclear transfer is dependent upon a range of factors. Here we investigate some of these factors and report the successful production of cloned piglets from a cultured adult somatic cell population using a new nuclear transfer procedure.

Animals↗
PubMed2000

Production of gene-targeted sheep by nuclear transfer from cultured somatic cells.

It is over a decade since the first demonstration that mouse embryonic stem cells could be used to transfer a predetermined genetic modification to a whole animal. The extension of this technique to other mammalian species, particularly livestock, might bring numerous biomedical benefits, for example, ablation of xenoreactive transplantation antigens, inactivation of genes responsible for neuropathogenic disease and precise placement of transgenes designed to produce proteins for human therapy. Gene targeting has not yet been achieved in mammals other than mice, however, because functional embryonic stem cells have not been derived. Nuclear transfer from cultured somatic cells provides an alternative means of cell-mediated transgenesis. Here we describe efficient and reproducible gene targeting in fetal fibroblasts to place a therapeutic transgene at the ovine alpha1(I) procollagen (COL1A1) locus and the production of live sheep by nuclear transfer.

Animals↗
PubMed2000

New advances in somatic cell nuclear transfer: application in transgenesis.

The ability to produce live offspring by nuclear transfer from cultured somatic cells provides a route for the precise genetic manipulation of large animal species. Such modifications include the addition, or "knock-in", and the removal or inactivation, "knock-out", of genes or their control sequences. This paper will review some of the factors which affect the development of embryos produced by nuclear transfer, the advantages of using cultured cells as donors of genetic material, and methods that have been developed to enrich gene targeting frequency. Commercial applications of this technology in biomedicine and agriculture will also be addressed.

Animals↗
PubMed2000

Production of live calves derived from embryonic stem-like cells aggregated with tetraploid embryos.

To date, cloned farm animals have been produced by nuclear transfer from embryonic, fetal, and adult cell types. However, mice completely derived from embryonic stem (ES) cells have been produced by aggregation with tetraploid embryos. The objective of the present study was to generate offspring completely derived from bovine ES-like cells. ES-like cells isolated from the inner cell mass of in vitro-produced embryos were aggregated with tetraploid bovine embryos generated by electrofusion at the 2-cell stage. A total of 77 embryo aggregates produced by coculture of two 8-cell-stage tetraploid embryos and a clump of ES-like cells were cultured in vitro. Twenty-eight of the aggregates developed to the blastocyst stage, and 12 of these were transferred to recipient cows. Six calves representing 2 singletons and 2 sets of twins were produced from the transfer of the chimeric embryos. Microsatellite analysis for the 6 calves demonstrated that one calf was chimeric in the hair roots and the another was chimeric in the liver. However, unfortunately, both of these calves died shortly after birth. Two of the placentae from the remaining pregnancies were also chimeric. These results indicate that the bovine ES-like cells used in these studies were able to contribute to development.

Animals↗
PubMed1999

Nuclear transfer in farm animal species.

CLONE 'a group of two or more individuals with identical genetic makeup derived, by asexual reproduction, from a single common parent or ancestor' (The Chambers Dictionary 1993, Chambers Harrap). The term clone was originally applied to plants but has subsequently been used in a much broader context to include a person or thing closely similar to another, a copy or replica. In animals, true clones, as defined above, may be produced by embryo splitting or blastomere separation either artificially, or as occurs naturally in the production of identical twins. In these individuals all of the components making up the individual, including nuclear genetic material (the genome) and other maternally derived factors are derived from a single unique embryo which is the result of sexual reproduction. The term clone has been applied to animals produced by the technique of nuclear transfer. In this asexual process, nuclear genetic material is transferred from a donor cell (karyoplast) into a recipient cell (cytoplast) from which the genetic material has been removed. In farm animals the cytoplast of choice is the matured oocyte (or unfertilised egg) thus the animals developing from this technique are not true clones as each cytoplast is often derived from a different animal. The resultant animals may therefore be more aptly described as 'genomic copies'. In mammals, successful development of embryos reconstructed by nuclear transfer was originally restricted to using early embryos as nuclear donors, however, recent progress has demonstrated successful development using nuclei from embryonic, foetal and adult derived cell populations. Numerous factors affect the development of embryos reconstructed by nuclear transfer including; the cell cycle stage of the recipient cell, the cell cycle stage of the donor nucleus, the differentiated state of the donor nucleus, activation of the recipient cell, the culture method. In addition, there are variations in success between species, these may be related to differences in organisation of the cytoskeleton and/or the meiotic spindle in the recipient cell,differences in cell cycle control during early development, the onset of zygotic transcription or differences in the metabolic requirements of early embryos in vitro. The aim of this article is to describe and discuss some of these factors in relation to the successful development of nuclear transfer reconstructed embryos and in particular to the 'reprogramming' or 'remodeling' of the donor genetic material to attain successful development.

Animals↗
PubMed1999

Analysis of telomere length in Dolly, a sheep derived by nuclear transfer.

We have used a (TTAGGG) oligonucleotide probe to demonstrate that ovine telomeres are composed of (TTAGGG) repeat arrays and to compare the terminal restriction fragment lengths of sheep derived by natural mating and nuclear transfer. Here we show that ovine somatic telomeres decrease in length with age, and that Dolly, derived by the transfer of 6-year-old adult somatic nucleus, exhibits diminished terminal restriction fragment lengths. The decrease is consistent with the age of the donor tissue and telomere erosion during in vitro culture. Nuclear transfer does not restore telomere lengths. Dolly otherwise appears physiologically and phenotypically normal for her breed and age. We further report on apparent telomere lengthening in sheep, occurring during the first year in naturally derived lambs.

Journal Article↗
PubMed1999

Nuclear equivalence, nuclear transfer, and the cell cycle.

The last 20 years have seen the development of techniques for the production of mammals by nuclear transfer. Originally limited to the swapping of pronuclei and the use of early cleavage-stage embryos as nuclear donors, nuclear transfer came of age in 1995 with the birth of 2 Welsh Mountain lambs, Megan and Morag, that were produced using cultured differentiated cells as donors of genetic material. In 1996, Dolly was the first animal to be produced using the genetic material from an adult-derived somatic cell. The techniques used in the production of these animals have now been reproduced in both sheep and cattle, and as predicted, successful development has been obtained using donor cells taken directly ex vivo. This article reviews the current status of mammalian nuclear transfer and the biological background to these successes.

Animals↗
PubMed1999

Nuclear transfer from somatic cells: applications in farm animal species.

The reconstruction of mammalian embryos by transfer of a blastomere nucleus to an enucleated oocyte or zygote allows for the production of genetically identical individuals. This has advantages for research (that is, as biological controls) and commercial applications (that is, multiplication of genetically valuable livestock). However, the number of offspring that can be produced from a single embryo is limited both by the number of blastomeres (embryos at the 32-64-cell stage are the most widely used in farm animal species) and the limited efficiency of the nuclear transfer procedure. The ability to produce live offspring by nuclear transfer from cells that can be propagated and maintained in culture offers many advantages, including the production of many identical offspring over an extended period (since cultured cells can be frozen and stored indefinitely) and the ability to modify genetically or to select populations of cells of specific genotypes or phenotypes before embryo reconstruction. This objective has been achieved with the production of lambs using nuclei from cultured cells established from embryonic, fetal and adult material. In addition, lambs transgenic for human factor IX have been produced from fetal fibroblasts transfected and selected in culture.

Animals↗
PubMed1998

Embryonic and somatic cell cloning.

Revolutionary opportunities in biology, medicine and agriculture arise from the observation that offspring are obtained after nuclear transfer if somatic donor cells are induced to become quiescent. Exploitation of many of these opportunities will depend upon optimizing procedures for nuclear transfer. This may come about through an understanding of the means by which factors in the oocyte cytoplasm act upon the DNA of the transferred nucleus to regulate gene expression. Similarly, research will extend the procedure to other species. This technology may be used for embryo production, the introduction of genetic change and the derivation of cells needed to treat human diseases. Groups of genetically identical animals will be used in research to control genetic variation and to allow transfer of cells between individuals. In agriculture, production of a small number of clones will separate genetic and environmental effects, whereas production of larger numbers of offspring will disseminate genetic improvement from nucleus herds. Precise genetic modification will be achieved by site specific recombination in the donor cells before nuclear transfer. In all mammals it will become possible to define the role of any gene product and to analyse the mechanisms that regulate gene expression. Medical uses of these techniques will include the production of proteins needed to treat disease and the supply of organs such as hearts, livers and kidneys from pigs. As genome mapping projects identify loci associated with traits of commercial importance in agriculture then gene targeting will be used to study this effect. Finally, cells capable of differentiation into any of the tissues of a patient will provide treatment for diseases reflecting damage to a specific cell population that neither repairs nor replaces itself.

Animals↗
PubMed1998

Maintenance of bovine oocytes in meiotic arrest and subsequent development In vitro: A comparative evaluation of antral follicle culture with other methods.

The frequency of development of bovine embryos produced by maturation, fertilization, and culture in vitro is lower than that observed in vivo. One factor that may affect both the frequency of development and the quality of the embryos produced is the developmental competence of the oocyte. In current in vitro production systems, oocyte maturation, characterized by the resumption of meiosis, occurs after oocyte aspiration from the follicle. The developmental competence of individual oocytes may be improved by inducing maturation after culturing under conditions that inhibit the resumption of meiosis. In order to test this hypothesis, a system has been established in which intact antral follicles (3-8 mm in diameter) are cultured in vitro. During this period the oocytes are maintained at the germinal vesicle (GV) stage under the inhibitory effects of the follicle. Culture of intact antral follicles was compared with two other "physiological" methods for the maintenance of GV arrest: oocytes were cultured attached to a small part of the follicle wall or within hemisections of follicles. It was found that 96.8% of oocytes recovered from intact antral follicles-as compared to 24.6% attached to a small part of the follicle wall and 62.7% within hemisections of follicles-were maintained at the GV stage after 24-h culture. The effects on GV arrest and subsequent maturation of the oocytes were evaluated after longer periods of antral follicle culture (2, 4, and 7 days). As the culture period increased, the number of GV-arrested oocytes decreased; the maximum percentage of GV arrest was observed after 24-h culture. The majority of these oocytes matured to metaphase II. A comparison of blastocyst production was made after fertilization and subsequent development of oocytes obtained following follicle culture and of control oocytes aspirated directly from antral follicles. The cleavage rate and percentage of blastocyst production in these two groups were 54.6 +/- 13.9%, 48.4 +/- 8.4% and 68.6 +/- 8.6%, 32.8 +/- 10.8%, respectively. Statistical analysis showed significant differences in both cleavage rate and blastocyst production between these two groups. Total cell numbers in the control group were 144.6 +/- 7.28 and 152.0 +/- 25.8 after follicle culture. It is concluded that culture of intact antral follicles for 24 h is an alternative method for the maintenance of bovine oocytes in meiotic arrest and that these oocytes acquire a greater developmental competence in vitro.

Animals↗
PubMed1997

Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts.

Ovine primary fetal fibroblasts were cotransfected with a neomycin resistance marker gene (neo) and a human coagulation factor IX genomic construct designed for expression of the encoded protein in sheep milk. Two cloned transfectants and a population of neomycin (G418)-resistant cells were used as donors for nuclear transfer to enucleated oocytes. Six transgenic lambs were liveborn: Three produced from cloned cells contained factor IX and neo transgenes, whereas three produced from the uncloned population contained the marker gene only. Somatic cells can therefore be subjected to genetic manipulation in vitro and produce viable animals by nuclear transfer. Production of transgenic sheep by nuclear transfer requires fewer than half the animals needed for pronuclear microinjection.

Animals↗
PubMed1997

Viable offspring derived from fetal and adult mammalian cells.

Fertilization of mammalian eggs is followed by successive cell divisions and progressive differentiation, first into the early embryo and subsequently into all of the cell types that make up the adult animal. Transfer of a single nucleus at a specific stage of development, to an enucleated unfertilized egg, provided an opportunity to investigate whether cellular differentiation to that stage involved irreversible genetic modification. The first offspring to develop from a differentiated cell were born after nuclear transfer from an embryo-derived cell line that had been induced to become quiescent. Using the same procedure, we now report the birth of live lambs from three new cell populations established from adult mammary gland, fetus and embryo. The fact that a lamb was derived from an adult cell confirms that differentiation of that cell did not involve the irreversible modification of genetic material required for development to term. The birth of lambs from differentiated fetal and adult cells also reinforces previous speculation that by inducing donor cells to become quiescent it will be possible to obtain normal development from a wide variety of differentiated cells.

Animal Husbandry↗
PubMed1996

Sheep cloned by nuclear transfer from a cultured cell line.

Nuclear transfer has been used in mammals as both a valuable tool in embryological studies and as a method for the multiplication of 'elite' embryos. Offspring have only been reported when early embryos, or embryo-derived cells during primary culture, were used as nuclear donors. Here we provide the first report, to our knowledge, of live mammalian offspring following nuclear transfer from an established cell line. Lambs were born after cells derived from sheep embryos, which had been cultured for 6 to 13 passages, were induced to quiesce by serum starvation before transfer of their nuclei into enucleated oocytes. Induction of quiescence in the donor cells may modify the donor chromatin structure to help nuclear reprogramming and allow development. This approach will provide the same powerful opportunities for analysis and modification of gene function in livestock species that are available in the mouse through the use of embryonic stem cells.

Animals↗
PubMed1996

Cell cycle co-ordination in embryo cloning by nuclear transfer.

Exciting new opportunities in embryo cloning have been made possible by recent studies on the interaction of the donor nucleus with the recipient cytoplasm after embryo reconstruction. This article reviews information regarding the co-ordination of nuclear and cytoplasmic events during embryo reconstruction, in particular the direct and indirect effects of maturation/ meiosis/mitosis-promoting factor (MPF), upon the transferred nucleus. This will be discussed in relation to DNA replication, the maintenance of correct ploidy, the occurrence of chromosomal abnormalities and development of reconstructed embryos. Although this review is primarily concerned with the reconstruction of mammalian embryos, specific examples from amphibians will also be cited.

Animals↗