Identification of functional relevant and overexpressed genes in Non-Hodgkin Lymphomas via RNAi
Dr. Armin Pscherer
Fig. 1: Targeted genomic regions in B-CLL and MCL (Bentz et al. ‘t(11;14)-positive mantle cell lymphomas exhibit complex karyotypes and share similarities with B-cell chronic lymphocytic leukemia’ Genes Chromosomes and Cancer, 27, 285-294, 2000)
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This project utilizes our substantial knowledge about amplified genomic loci and overexpressed genes in the lymphoma entities B-CLL and MCL (Fig.1) to analyze potential candidate genes on a functional basis via the RNAi-technology. Since its usage in somatic mammal cells RNAi appears much more better in effectiveness, specificity and reproducibility than the antisense-technology used so far for gene „knock-down“ experiments. RNAi is a endogenous cell mechanism mediating its „knock-down“ effect on the basis of a specific mRNA degradation maintaining as long as the initiators, the genespecific siRNAs, are present in the cytoplasm of the cell (Fig.2).
Fig. 2: miRNA- and siRNA-mechnism adopted from: He L, Hannon GJ, MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet. 2004 Jul;5(7):522-31.
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This project is based on a screening approach wherby candidate genes in amplified genomic loci are functionally identified. Multiple but genespecific siRNAs are generated with Dicer-digestion of a long, genespecific dsRNA (ex vivo dicing) and these siRNAs are subsequently transfected in B-CLL or MCL cell lines, which display an amplification of the particular genomic loci and an overexpression of the gene of interest. This assay can be done in parallel (48 well plate) for up to 20 genes located within the amplified genomic region. The genespecific „knock-down“ effect of the siRNAs regulates the overexpression level of the genes downwards to „wild-typ“ expression. The transfected cells exhibiting a difference in subsequent following proliferation- or apoptosis-assays are most likely „knocked-down“ for a gene functionally relevant for B-CLL or MCL. This approach is predominantly applied for genes within amplified genomic loci not yet tested for their disease relevance, e.g. 12q13 in B-CLL and 3q26 in MCL.
Cooperation Partner:
Prof. Dr. Hartmut Doehner, Division of Internal Medicine II, University of Ulm
Funding
Since April 2004 this project is funded by a grant of the Fritz von Thyssen-Stiftung (Az.: 10.04.1.169, “Funktionelle Analyse von deregulierten und inaktivierten Genen in Non-Hodgkin-Lymphomen des B-CLL und MCL-Typs” unter dem Förderungsschwerpunkt “Molekulare Pathogenese und Modelle der Krankheitsentstehung”).
(click icon to view www.fritz-thyssen-stiftung.de)
