Deletion of the alpha(1,3)galactosyl transferase (GGTA1) gene and the prion protein (PrP) gene in sheep.

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.

BCR-ABL accelerates C2-ceramide-induced apoptosis.

In patients with chronic myeloid leukemia (CML), the neoplastic (BCR-ABL+) progenitor cells are characterized by an increased proliferative activity. Whether these cells are also resistant to apoptosis and if so, under what conditions remains controversial. We now show that highly purified populations of very primitive neoplastic progenitor cells obtained directly from CML patients survive and proliferate in vitro for several weeks in the absence of any added growth factors (except insulin). In contrast, purified primary normal progenitors maintained under the same conditions die rapidly. Nevertheless, both primary CML cells and BCR-ABL+ BAF3 cells show the same dose-dependent sensitivity to TNF-alpha or ceramide-induced apoptosis as their respective normal counterparts. In fact, time course studies demonstrated an even faster onset of apoptosis in ceramide-treated BCR-ABL+ BAF3 cells as compared to normal controls. BCR-ABL+ cells treated with ceramide also showed a rapid and sequential increase in the tyrosine phosphorylation of p210(BCR-ABL), p46-56SHC and p120Cbl. These findings suggest growth factor deprivation and treatment with TNF-alpha or ceramide trigger different initial events both of which can lead to apoptosis in factor-dependent hematopoietic cells. However, in the first case, activation of apoptosis is blocked by the basal activity of p210(BCR-ABL), whereas in the second, the presence of p210(BCR-ABL) appears to accelerate the onset of apoptosis by a mechanism that may involve an activation of its kinase function.

Gene targeting in primary fetal fibroblasts from sheep and pig.

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.

Optimisation of a functional mycobacterial growth-inhibition assay to improve its suitability for infant TB vaccine studies.

The development of vaccines against tuberculosis continues to be hindered by the lack of correlates of protection. Immunity to Mycobacterium tuberculosis (M.tb) infection relies predominantly on cell mediated response, which is routinely measured using a read-out of host cytokine profiles. However, to date none of the cytokine profiles have been found to predict protection. A number of functional in vitro approaches that measure growth of mycobacteria pre- and post-vaccination as a potential functional surrogate marker for vaccine take have been developed. The use of a reporter-gene tagged BCG-lux assay measuring the viability of mycobacteria in whole blood samples has previously been described by our group to assess vaccine immunogenicity. Since only very small blood samples are usually available in paediatric studies, we now report a modification of the BCG-lux assay to reduce the volume required and make it more field-friendly. Our results show that a 2-fold reduction in blood volume made no significant difference to bacterial growth ratios, used as the main read-out. These results confirm the suitability of the BCG-lux assay for functional studies of vaccine immunogenicity and immunopathogenesis in young children and could play a role in late-phase TB vaccine trials of novel candidates.

FOXP3 gene expression in a tuberculosis case contact study.

Regulatory T lymphocytes (T(regs)) that express FOXP3 are involved in the beneficial attenuation of immunopathology, but are also implicated in down-regulation of protective responses to infection. Their role in tuberculosis (TB) is unknown. We classified 1272 healthy TB contacts according to their tuberculin skin test (TST) and interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) results and 128 TB cases, and studied the expression of FOXP3 and interleukin (IL)-10 in blood samples. Compared to the uninfected contact group (TST(-), ELISPOT(-)), we observed higher levels of FOXP3 mRNA in blood from TB patients (< 0.001), but IL-10 expression was slightly lower (P = 0.04). In contrast, FOXP3 expression levels were significantly lower (P = 0.001) in the recently infected contacts (TST(+), ELISPOT(+)) but there was no difference for IL-10 (P = 0.74). We hypothesize that during early/subclinical TB, most of which will become latent, FOXP3(+) T(regs) may be sequestered in the lungs, but when TB becomes progressive, FOXP3 reappears at increased levels in the periphery. While these findings do not reveal the role, beneficial or harmful, of T(regs) in TB, they emphasize the probable importance of these cells.

Gene targeting in livestock: a preview.

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.

TGF-beta1 binding protein-like modules of fibrillin-1 and -2 mediate integrin-dependent cell adhesion.

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.