Production of chimaeric mice containing embryonic stem (ES) cells carrying a homoeobox Hox 1.1 allele mutated by homologous recombination.

SEVERAL mouse gene families related to Drosophila developmental control genes and containing a homoeobox, a paired box or a finger domain, have been cloned and structurally analysed. On the basis of structural similarities to the Drosophila genes and of their spatially and temporally restricted expression patterns during mouse embryogenesis, it has been proposed that these mammalian genes also are involved in the control of develop-ment1–4. To elucidate the function of homoeobox genes by genetic means, mouse mutants must be generated. We have developed a technique for mutagenesis in vivo and have used it to mutate the homoeobox Hox 1.1 gene. In vivo mutagenesis was achieved through homologous recombination between an endogenous Hox 1.1 allele and a microinjected mutated gene in pluripotent embryonic stem (ES) cells5–9. Mutant cells were identified by means of the polymerase chain reaction (PCR) 10 and mutant clones were used to generate chimaeric mice. Because the homologous recombi-nation event is formally a gene conversion event and no selection is required to screen for cells carrying the mutated allele, in vivo mutagenesis allows specific alterations in the target sequence to be made without the introduction of any other sequences

Molecular Signals for Glial Activation: Pro- and Anti-Inflammatory Cytokines in the Injured Brain

Injury to the central nervous system leads to cellular changes not only in the affected neurons but also in adjacent glial cells. This neuroglial activation is a consistent feature in almost all forms of brain pathology and appears to reflect an evolutionarily-conserved program which plays an important role for the repair of the injured nervous system. Recent work in mice that are genetically-deficient for different cytokines (M-CSF, IL-6, TNF-alpha, TGF-beta 1) has begun to shed light on the molecular signals that regulate this cellular response. Here, the availability of cytokine-deficient animals with reduced or abolished neuroglial activation provides a direct approach to determine the function of the different components of the cellular response leading to repair and regeneration following neural trauma

Endogenous transforming growth factor ß1 suppresses inflammation and promotes survival in adult CNS.

Transforming growth factor beta 1 (TGF beta 1) is a pleiotropic cytokine with potent neurotrophic and immunosuppressive properties that is upregulated after injury, but also expressed in the normal nervous system. In the current study, we examined the regulation of TGF beta 1 and the effects of TGF beta 1 deletion on cellular response in the uninjured adult brain and in the injured and regenerating facial motor nucleus. To avoid lethal autoimmune inflammation within 3 weeks after birth in TGF beta 1-deficient mice, this study was performed on a T- and B-cell-deficient RAG2-/- background. Compared with wild-type siblings, homozygous deletion of TGF beta 1 resulted in an extensive inflammatory response in otherwise uninjured brain parenchyma. Astrocytes increased in GFAP and CD44 immunoreactivity; microglia showed proliferative activity, expression of phagocytosis-associated markers [alpha X beta 2, B7.2,and MHC1 (major histocompatibility complex type 1)], and reduced branching. Ultrastructural analysis revealed focal blockade of axonal transport, perinodal damming of axonal organelles, focal demyelination, and myelin debris in granule-rich, phagocytic microglia. After facial axotomy, absence of TGF beta 1 led to a fourfold increase in neuronal cell death (52 vs 13%), decreased central axonal sprouting, and significant delay in functional recovery. It also interfered with the microglial response, resulting in a diminished expression of early activation markers [ICAM1 ( intercellular adhesion molecule 1), alpha 6 beta 1, and alpha M beta 2] and reduced proliferation. In line with axonal and glial findings in the otherwise uninjured CNS, absence of endogenous TGF beta 1 also caused an similar to 10% reduction in the number of normal motoneurons, pointing to an ongoing and potent trophic role of this anti-inflammatory cytokine in the normal as well as in the injured brain

Disruption of the murine IL-4 gene blocks Th2 cytokine responses

Murine T-helper clones are classified into two distinct subsets (Th1 and Th2) on the basis of their patterns of lymphokine secretion. Th1 clones secrete interleukin-2 (IL-2), tumour necrosis factor-beta (TNF-beta) and interferon-gamma (IFN-gamma), whereas Th2 clones secrete IL-4, IL-5 and IL-10 (ref. 1). These subsets are reciprocally regulated by IL-4, IL-10 and IFN-gamma and differentially promote antibody or delayed-type hypersensitivity responses. To evaluate whether IL-4 is required for mounting Th2 responses, we generated IL-4-mutant mice (IL-4-/-) and assessed the cytokine secretion pattern of T cells both from naive and Nippostrongylus brasiliensis infected mice. CD4+ T cells from naive IL-4-/- mice failed to produce Th2-derived cytokines after in vitro stimulation. The levels of Th2 cytokines IL-5, IL-9 and IL-10 from CD4+ T cells obtained after nematode infection were significantly reduced. The reduced IL-5 production in IL-4-/- mice correlated with reduced helminth-induced eosinophilia, which has been shown to be dependent on IL-5 in vivo. We conclude that IL-4 is required for the generation of the Th2-derived cytokines and that immune responses dependent on these cytokines are impaired