Stable modulation of gene expression in deregulated human neoplasias

The aim of this project is to establish stable cell systems (for example cells of a MCL-/ B-CLL-origin) which harbour the ability to modulate the expression of single genes deregulated in malignancies resulting from 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 which is able to counteract both gene inactivation and overexpression on a single and site-specific genomic basis without changing the other remaining genomic aberrations.



Fig. 1: RMCE (Recombination Mediated Cassette Exchange) mittels Cre-lox Technologie.


The resulting functional effects of these modulations permit an insight into the biological relevance of the specific genes and places emphasis on the modulated genes, for example in 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 for the gene-complementation or gene knock-down strategy. Genomic deletions are replaced by stable transfection of adequate genomic BAC-clones, which include the native promoter/enhancer sequences of the relevant genes (Fig. 2), resulting in the restoration of wild type-regulation.



Fig. 2: MCL cell line Granta-519 after RMCE Genomic integration of BAC-clone into a MCL-cell line mediated by RMC. Detected by FISH-assays: green: control-probe on 10p; red: INK4 BAC-clone.

To counteract the overexpression of amplified genes the RNAi-technology is used, mediating its gene knock-down effect using a stably integrated vector-system.
In total, his system represents a stable modulation and correction strategy for gene expression, regardless of inactivated, deleted or over expressed genes. Due to the specific Cre/lox-recombination system and the defined genetic background, the observed expression- and phenotypical alterations point toward the physiological role of the relevant gene within the deregulated cell systems.


Cooperation Partner:
Prof. Dr. Hartmut Doehner, Division of Internal Medicine III, University of Ulm.