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Biomedical subjects

A Colman

Publications and source records attributed to A Colman.

At least 19 recordsLinked to original sources
PubMed2001

A method for the amidation of recombinant peptides expressed as intein fusion proteins in Escherichia coli.

The increasing use of peptides as pharmaceutical agents, especially in the antiviral and anti-infective therapeutic areas, requires cost-effective production on a large scale. Many peptides need carboxy amidation for full activity or prolonged bioavailability. However, this modification is not possible in prokaryotes and must be done using recombinant enzymes or by expression in transgenic milk. Methods employing recombinant enzymes are appropriate for small-scale production, whereas transgenic milk expression is suitable for making complex disulfide-containing peptides required in large quantity. Here we describe a method for making amidated peptides using a modified self-cleaving vacuolar membrane ATPase (VMA) intein expression system. This system is suitable for making amidated peptides at a laboratory scale using readily available constructs and reagents. Further improvements are possible, such as reducing the size of the intein to improve the peptide yields (the VMA intein comprises 454 amino acids) and, if necessary, secreting the fusion protein to ensure correct N-terminal processing to the peptide. With such developments, this method could form the basis of a large-scale cost-effective system for the bulk production of amidated peptides without the use of recombinant enzymes or the need to cleave fusion proteins.

Amides↗
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

Therapeutic cloning: concepts and practicalities.

The concept of using embryonic stem (ES) cells as a source of multiple cell types for use in tissue repair has existed for approximately 20 years. Recent breakthroughs in somatic nuclear transfer and human ES cell derivation have produced a flurry of new activity in this area, with the recognition that ES cell lines that are customized and genetically identical to those of the patient are a distinct possibility. This article examines the background of and prospects for these exciting new developments.

Animals↗
PubMed1999

Therapeutic cloning: needs and prospects.

There has been widespread speculation regarding the possibility of reprogramming human somatic nuclei by transfer into oocytes. Stem cells derived from such reconstructed embryos could theoretically provide a source of autologous tissue for therapeutic transplantation. However the biological, technical and ethical hurdles which must be overcome before this can be realised are considerable. Here we review the background and prospects for such work.

Animals↗
PubMed1999

Introduction

Explore the source record for details and available documents.

Journal Article↗
PubMed1999

Dolly, Polly and other 'ollys': likely impact of cloning technology on biomedical uses of livestock.

The idea of generating transgenic livestock which secrete into their milk large quantities of proteins for therapeutic use, was pioneered in the late 1980s with the disclosure of the production of a number of transgenic sheep. One particular animal, a sheep called Tracy, produced milk where over 50% of the protein consisted of human alpha 1 anti-trypsin. Sheep-derived protein has now entered clinical trials for cystic fibrosis (UK, USA) and congenital emphysema (UK). There are many other examples where this technology is making inroads into more traditional ways of making biopharmaceuticals. However, although robust, this technology has several limitations, including an inability to allow targeted insertion/modification of the animal genome, long timelines to production flocks/herds, and the rather unpredictable expression levels seen when different transgenic founders are compared. We believe that there is now a technical solution to all of these problems. Dolly is a high profile example of a new technology comprising the generation of identical animals from cultured somatic cells. This work has many implications. In the commercial context, the real benefits of this advance will be seen when genetically engineered somatic cells are shown to be suitable nuclear donors, and particularly when the manipulations are targeted to pre-determined sites in the host cell genome. The first objective has now been achieved with the birth of Polly, a cloned sheep which contains the human gene encoding Factor IX, a protein involved in preventing haemophilia.

Animal Husbandry↗
PubMed1998

Production of therapeutic proteins in the milk of transgenic livestock.

With the advent of the Human Genome Project and associated developments in 'functional genomics', there are going to be increasing numbers of proteins identified and developed for clinical use. There are a number of production methods available, although only three, bacterial, yeast and mammalian cell culture, have produced recombinant proteins that have been approved for clinical use. Nevertheless other production systems are under development, and one, the production of human proteins in the milk of transgenic livestock, is showing great promise, with two proteins now in clinical trials. This chapter will compare and contrast the various competing technologies and will then concentrate on factors influencing the choice and use of the transgenic system.

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

Uncommon entrance? The recruitment of probationers to Merseyside hospitals 1919-1938.

That voluntary and municipal hospitals in inter-war Britain enjoyed different levels of prestige is well known. This article explores ways in which the status of hospitals was reflected in the recruitment of probationer nurses on Merseyside and argues that there were overlapping hierarchies between and within the two sectors.

Education, Nursing↗
PubMed1996

Selective ablation of differentiated cells permits isolation of embryonic stem cell lines from murine embryos with a non-permissive genetic background.

Embryonic stem (ES) cells enable the engineering of precise modifications to the mouse genome by gene targeting. Although there are reports of cultured cell contributions to chimaeras in golden hamster, rat and pig, definitive ES cell lines which contribute to the germline have not been demonstrated in any species but mouse. Among mouse strains, genetic background strongly affects the efficiency of ES isolation, and almost all ES lines in use are derived from strain 129 (refs 1,4,5) or, less commonly, C57BL/6 (refs 6-8). The CBA strain is refractory to ES isolation and there are no published reports of CBA-derived ES lines. Hence, CBA mice may provide a convenient model of ES isolation in other species. In ES derivation it is critical that the primary explant be cultured for a sufficient time to allow multiplication of ES cell progenitors, yet without allowing extensive differentiation. Thus, differences in ES derivation between mouse strains may reflect differences in the control of ES progenitor cells by other lineages within the embryo. Here we describe a strategy to continuously remove differentiated cells by drug selection, which generates germline competent ES lines from genotypes that are non-permissive in the absence of selection.

Animals↗
PubMed1996

Production of proteins in the milk of transgenic livestock: problems, solutions, and successes.

The milk of livestock can be modified dramatically by introducing foreign DNA into the germline. Exclusive expression of this DNA is ensured by the presence of regulatory sequences from mammary gland-specific genes. In sheep > 50% of the protein in milk can be encoded by a transgene and it appears that the foreign protein is additional to the normal complement of proteins. However, many technical hurdles (DNA configuration, low efficiency of transgenesis, and transgene stability) still prevent the routine use of this technology. In addition, such milk products have not yet received regulatory approval. These difficulties are not insurmountable. Transgenic methods can also be used to study the molecular basis of milk biogenesis. The effect on milk production in mice with all endogenous alpha-lactalbumin genes removed and in mice in which the murine genes were then replaced with human homologues is described. alpha-Lactalbumin, and consequently lactose, is essential for normal milk formation, and the human gene is expressed more efficiently than the murine gene.

Animals↗