Whilst the recruitment of phagocytic leukocytes is fundamental to the innate response against pathogenic infection, the inappropriate mobilisation of their cytotoxic potential can also lead to fatal tissue injury. To determine the contribution of Csk, a negative regulator of Src family kinases, to the regulation of phagocyte recruitment and activation in vivo, mice lacking Csk in the myeloid lineage were generated using conditional mutagenesis (cre/loxP). This Csk deficiency resulted in acute multifocal inflammation in skin and lung, accompanied by extramedullary haematopoiesis in the spleen and liver, and increased myelopoiesis in bone marrow. Animals were protected from the disease in a microbiologically controlled environment, but remained hypersensitive to LPS-induced shock. Csk-deficient granulocytes showed enhanced spontaneous and ligand-induced degranulation accompanied by hyperinduction of integrins. Hyperresponsiveness was associated with hyperadhesion and impaired migratory responses in vitro. Biochemical studies revealed spontaneous accumulation of tyrosine-phosphorylated proteins, including hyperphosphorylation of key signalling proteins including Syk and paxillin. These data support a breakdown of the activation threshold set by Csk. Thus, Csk is critical in preventing premature granulocyte recruitment through enforcing the requirement for ligand engagement while supporting the migratory capacity of activated cells through negative regulation of cell adhesion. To address the incomplete Cre mediated deletion of floxed genes in vivo, a genetic approach to elevate Cre recombinase gene expression was developed. Whilst manipulation of regulatory elements including promoter, enhancer, and untranslated regions has yielded enhanced and sustained expression in vitro, this has been difficult to achieve in vivo. Here, it is reported that construction of artificial exons through insertion of short heterologous intron sequences into the open reading frames of the Cre recombinase and enhanced green fluorescent protein results in functional expression accompanied by a 30-fold increase in transcription levels in vitro. Furthermore, green fluorescence levels were enhanced five-fold in cell lines and enhanced considerably in the rat brain after transduction with a herpes simplex virus-based vector. These data define a method of improving both the level and duration of recombinant gene expression, in addition to and independently of surrounding regulatory elements. Significantly, the method should help to increase Cre recombinase expression from weak or transiently expressed promoters thus overcoming an important limitation of Cre/loxP technology incomplete deletion. Furthermore, this method may also be applicable in gene therapy to obtain sustained and effective expression of recombinant proteins in vivo.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:644405 |
| Date | January 2007 |
| Creators | Thomas, R. M. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1445173/ |
Page generated in 0.0266 seconds