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The role of the Raf-1 protein in mouse development was investigated by generating a targeted disruption of the raf-1 gene. The pTC4.Raf-1 targeting vector was constructed using raf-1 genomic DNA 5' and 3' to the kinase domain. This was designed such that homologous recombination with the wild type raf-1 gene resulted in the deletion of the kinase domain. Four ES clones carrying integrations of raf-1 were used to generate chimaeric mice and -/- raf-1 homozygotes were obtained. Embryos-/-for raf-1 were isolated up to day E10.5. Two phenotypes were observed: 1) Developmental arrest at day E8.5-E9, 2) Vascular and somatogesis irregularities with cranial defects. The -/- embryos were also smaller. Expression patterns of raf-1 and A-raf- were analysed in adult tissues and embryos by a combination of in situ hybridisation, immunohistochemistry and a reporter transgenic mouse for A-raf. Embryonic expression analysis of raf-1 showed a general level of expression with elevated levels in the developing embryo, vascular system, tail bud, otic pit and hindbrain. Comparison of the expression of A-raf and raf-1 showed that raf-1 was ubiquitously expressed but had a higher level of expression spatially in development. A-raf was more localised to cell types with higher metabolic requirements. Generation of an ES cell -/- for raf-1 by two rounds of gene targeting in in vitro demonstrated that Raf-1 was not required for ES cell 'self renewal'. The results indicate that there is a requirement for raf-1 protooncogene during development in the mouse. |
