Stable modulation of gene expression in deregulated human neoplasias.

The aim of this project is to establish a stable cell system of a MCL-/ B-CLL-origin which harbours the ability to modulate the expression of single genes deregulated in these malignancies as a result of genomic aberrations, increased or decreased gene copy numbers. For this purpose we use the RMCE-strategy (“recombination mediated cassette exchange”, (Fig. 1), a flexible system able to counteract both gene inactivation and overexpression on a single and site-specific genomic basis without changing the other remaining genomic aberrations. The resulting functional effects of these modulations permit an insight into the biological relevance and emphasis of the modulated genes for the lymphoproliferative diseases B-CLL and MCL. First a stable selectable selection cassette is introduced into the cells to generate a defined integration locus, which is subsequently used by the gene-complementation and gene knock-down strategy. Genomic deletions were replaced by stable transfection of adaequate genomic BAC-clones including the own promoter/enhancer sequences of the relevant genes (Fig. 2), therewith wildtype-regulation is restored.

To counteract the overexpression of amplified genes the RNAi-technology is used, mediating its gene knock-down effect by a stable integrated vector-system. (Fig. see other project)
This system represents a stable modulation and correction strategy for gene expression regardless of inactivated, deleted or overexpressed genes. Due to the specific Cre/lox-recombination system and the defined genetic background the observed expression- and phenotypical alterations admit a hint for the physiological role of the relevant gene within the deregulated cell systems. Notably since the cell system, which should be modified, are cell lines of a MCL and B-CLL origin, representing the chraracteristic genomic imbalances for these human neoplasias.

This system represents a stable modulation and correction strategy for gene expression regardless of inactivated, deleted or overexpressed genes. Due to the specific Cre/lox-recombination system and the defined genetic background the observed expression- and phenotypical alterations admit a hint for the physiological role of the relevant gene within the deregulated cell systems. Notably since the cell system, which should be modified, are cell lines of a MCL and B-CLL origin, representing the chraracteristic genomic imbalances for these human neoplasias.