Nuclear transfer offers a cell-based route for producing precise genetic modifications in a range of animal species. Using sheep, we report reproducible targeted gene deletion at two independent loci in fetal fibro-blasts. Vital regions were deleted from the alpha(1,3)galactosyl transferase (GGTA1) gene, which may account for the hyperacute rejection of xenografted organs, and from the prion protein (PrP) gene, which is directly associated with spongiform encephalopathies in humans and animals. Reconstructed embryos were prepared using cultures of targeted or nontargeted donor cells. Eight pregnancies were maintained to term and four PrP-/+ lambs were born. Although three of these perished soon after birth, one survived for 12 days. These data show that lambs carrying targeted gene deletions can be generated by nuclear transfer.

Nuclear transfer offers a new cell-based route for introducing precise genetic modifications in a range of animal species. However, significant challenges, such as establishment of somatic gene targeting techniques, must be overcome before the technology can be applied routinely. In this report, we describe targeted deletion at the GGTA1 (alpha 1,3-galactosyl transferase) and PrP (prion protein) loci in primary fibroblasts from livestock. We place particular emphasis on the growth characteristics of the primary cell cultures, since these are key to determining success.

Human fibrillin, a major component of the extracellular matrix, exists as two highly homologous forms (fibrillin-1 and -2). Several modules of fibrillin are homologous to TGF-beta1 binding protein. Two of these modules, D25 (the 25th module of fibrillin-1 and -2 D segment) and D12 (the 12th module of fibrillin-2 D segment) contain the cell adhesion motif arginyl-glycyl-aspartyl (RGD). The ability of RGD to mediate adhesion to D25-1 and D12-2 was investigated using bacterially expressed fusion proteins. Human skin fibroblasts and murine L-cells were used in microassays of cell attachment and cell spreading on fibrillin fusion-protein substrata. Dose-dependent experiments and competitive inhibition by soluble RGD-containing peptides demonstrated that D25-1 and D12-2 mediate RGD-dependent cell adhesion. These results provide evidence for a cell adhesion function of fibrillin-2. Inhibition with anti-integrin antibodies showed that alpha(v) and beta3 integrins mediate adhesion to D25-1, while alpha3, alpha(v) and beta1 are involved in adhesion to D12-2. Binding of different receptors may elicit distinct cell signalling supporting the hypothesis that fibrillin-1 and fibrillin-2 have distinct roles.

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.

Until recently genetically modified livestock could only be generated by pronuclear injection. The discovery that animals can be cloned by nuclear transfer from cultured somatic cells means that it will now be possible to achieve gene targeting in these species. We discuss current developments in NT, the prospects and technical challenges for introducing targeted changes into the germline by this route, and the types of application for which this new technology will be used.