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

  title={Production of gene-targeted sheep by nuclear transfer from cultured somatic cells},
  author={Kenneth J. McCreath and J. Howcroft and Keith Henry Stockman Campbell and Alan Colman and Angelika Schnieke and Alexander J. Kind},
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… 
New frontiers in gene targeting and cloning: success, application and challenges in domestic animals and human embryonic stem cells.
Improvements in the efficiency of somatic cell gene targeting and a greater understanding of the reprogramming events that occur during NT are required for the routine application of what is currently an inefficient process.
Genetic modification of sheep by nuclear transfer with gene-targeted somatic cells.
Protocols for culturing primary sheep fibroblasts, introducing and selecting targeted modifications into them and then using these modified cells in nuclear transfer experiments are described.
Generation of transgenic livestock by somatic cell nuclear transfer
This review summarizes the major advantages and disadvantages of currently available techniques to produce genetically modified livestock with special emphasis on the benefits of using somatic cell nuclear transfer (SCNT) to create geneticallymodified livestock for agriculture and biomedical applications.
Evaluation of gene targeting by homologous recombination in ovine somatic cells
Targeting of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in ovine somatic cells using homologous recombination of targeting constructs with extensive (>11 kb) homology suggests that at least for this locus, it is an extremely inefficient process.
Gene targeting in livestock.
This review provides data that illustrate the difficulty in targeting non-expressed genes and discusses some of the practical issues associated with providing targeted nuclear donor cells that are competent for nuclear transfer.
Challenges and Prospects for Targeted Transgenesis in Livestock
Until recently the only ways of making transgenic livestock were by pro-nuclear injection or, more controversially, by sperm mediated DNA transfer. Both techniques usually result in multiple copies
Gene targeting in primary fetal fibroblasts from sheep and pig.
Targeted deletion at the GGTA1 (alpha 1,3-galactosyl transferase) and PrP (prion protein) loci in primary fibroblasts from livestock are described and growth characteristics of the primary cell cultures are considered, since these are key to determining success.
Cell-Mediated Transgenesis in Rabbits: Chimeric and Nuclear Transfer Animals1
The first live born ES-derived rabbit chimera is reported, the first live cloned rabbits from genetically manipulated MSCs, and reactivation of fluorescence reporter gene expression in reconstructed embryos was investigated as a means of identifying viable embryos in vitro but was not a reliable predictor.
The present review summarizes the methods by which transgenes can be introduced into zygotes of domestic animals and concludes that the most reliable method for transgenesis which bypasses mosaics is SCNT.
25th ANNIVERSARY OF CLONING BY SOMATIC CELL NUCLEAR TRANSFER Generation of genetically engineered livestock using somatic cell nuclear transfer
Development of somatic cell nuclear transfer has bypassed the need in livestock ESCs and revolutionized the field of livestock transgenesis by offering the first cell-based platform for precise genetic manipulation in farm animals.


Cloned transgenic calves produced from nonquiescent fetal fibroblasts.
The life-span of near senescent fibroblasts could be extended by nuclear transfer, as indicated by population doublings in fibroblast lines derived from a 40-day-old fetal clone.
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 and produced viable animals by nuclear transfer.
Sheep cloned by nuclear transfer from a cultured cell line
This is the first report, to the authors' knowledge, of live mammalian offspring following nuclear transfer from an established cell line, and 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.
Selective ablation of differentiated cells permits isolation of embryonic stem cell lines from murine embryos with a non–permissive genetic background
A strategy to continuously remove differentiated cells by drug selection is described, which generates germline competent ES lines from genotypes that are non-permissive in the absence of selection.
Homologous Recombination Based Gene Therapy
  • L. Lai, Y. Lien
  • Biology, Medicine
    Nephron Experimental Nephrology
  • 1999
Homologous recombination based gene therapy has the potential to develop into a powerful therapeutic modality for genetic diseases and probably can be used for treating dominantly inherited diseases such as polycystic kidney disease.
Therapeutic gene targeting
A brief review of the challenges of gene therapy by gene targeting is provided, followed by a critical overview of recent developments in gene targeting techniques, and in the understanding of the underlying processes of homologous and nonhomologous recombination.
Gene targeting and somatic cell genetics--a rebirth or a coming of age?
Transgenic Livestock as Bioreactors: Stable Expression of Human Alpha-1-Antitrypsin by a Flock of Sheep
Although the inheritance of the same transgene in mice was reminiscent of the situation in sheep, stable expression was observed in only one of four lines studied, and the importance of these observations to the use of transgenic livestock as bioreactors for the production of human proteins is discussed.
Lymphoid hypoplasia and somatic cloning