Functional analysis of mammalian genes in vivo is primarily achieved through analysing knockout mice. Now that the sequencing of several mammalian genomes has been completed, understanding functions of all the genes represents the next major challenge in the post-genome era. Generation of knockout mutant mice has currently been achieved by many research groups but only by making individual knockouts, one by one. New technological advances and the refinements of existing technologies are critical for genome-wide targeted mutagenesis in the mouse. We describe here new recombineering reagents and protocols that enable recombineering to be carried out in a 96-well format. Consequently, we are able to construct 96 conditional knockout targeting vectors simultaneously. Our new recombineering system makes it a reality to generate large numbers of precisely engineered DNA constructs for functional genomics studies.

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In children, the majority of brain tumors arise in the cerebellum. Medulloblastomas, the most common of these, are believed to originate from the granule cell lineage. We have recently identified a mammalian gene, capicua (Cic), the ortholog of a Drosophila gene implicated in c-erbB (Egfr) signaling, which is predominantly expressed during mouse granule cell development. Its expression in medulloblastoma is therefore of particular interest. In the present study the expression of human CIC in medulloblastoma was analyzed. In silico SAGE analysis demonstrated that medulloblastomas exhibited the highest level of CIC expression and expression was most common in tumors of the CNS in general. RT-PCR and in situ hybridization verified the expression of CIC in tumor cells, although the level of expression varied between different medulloblastoma subtypes. The expression of CIC did not correlate with other markers, such as neurofilament, GFAP and Mib-1. In postnatally developing cerebellum, in silico analysis and in situ hybridization both indicated a strong correlation between Cic expression and the maturation profile of cerebellar granule cell precursors. Expression of CIC is therefore a feature shared between immature granule cells and the tumors derived from them. Cic has been implicated as a mediator of ErbB signaling and this pathway has been associated with a poor prognosis for medulloblastomas. Therefore, further analysis of the role of Cic is likely to provide valuable insight into the biology of these tumors. Additionally, study of genes such as CIC should provide objective criteria by which, in combination with other markers and clinical data, to categorize these tumors into subgroups that might allow better allocation into specific treatment regimes.