Type: Practical with Student Seminar

Day 1-10: Retroviral gene transfer and molecular analysis of transduced cells

Date: 17.-28.10.2011

Hosts/Supervisors: Christof von Kalle, Manfred Schmidt, Ali Nowrouzi, Claudia Ball, Cynthia Bartholomä, Anna Paruzynski, Stephanie Laufs, Richard Gabriel; Translational Oncology Labs, TP4, 4th floor (contact: manfred.schmidt@nct-heidelberg.de)

Topics:

Retroviral vector based gene therapy has been becoming of age, leading to unprecedented levels of efficiency and success, but also side effects: vector integration may not be inert for the affected cell type, potentially leading to clonal dominance or, in its worst cases, to oncogenesis. As insertional mutagenesis has to be considered a ‘normal’ side effect accompanying each clinical gene transfer study, the analysis of vector integration and its influence on cellular genes represents a prerequisite of each study and associated biosafety measurements. Besides of vector biosafety testings, insertional mutagenesis has proven to be an efficient tool for the screening of genes with oncogenic potential. Moreover, viral vectors are routinely used to evaluate the function of genes in hematopoetic stem cells and solid tumor initiating cells.

Content:

• Transduction of HeLa cells with a retroviral vector expressing the marker gene eGFP.
• Enrichment of gene-marked cells by FACS analysis; subsequent separation of enriched cell fraction and cultivation (single cell colonies).
• Isolation of DNA and RNA in enriched cell fractions and single cell colonies (after 1-3 days); estimation of DNA concentration (photometrically and by semi-quantitative PCR).
• Integration site analysis by linear amplification mediated (LAM) PCR; isolation and und sequencing of identified LAM amplicons.
• Determination of genomic vector integration sites by BLAST und BLAT alignments.
• Estimation of RNA concentration and conversion into cDNA; analysis of the expression level of specific genes by RT-PCR.
• Cell culture of primary pancreatic and colon cancer initiating cells
• Differentiation and immunofluorescence of pancreatic cancer cells
• Transduction and functional analysis of colon cancer initiating cells

Day 11-15: Gene therapy for brain repair

Date: 07.-11.11.2011

Introduction: 17.10., 5 p.m.

Hosts/Supervisors: Ana Martin-Villalba, Susanne Kleber, Desirée Seib, Marcin Teodorczyk, Enric Llorens, Robert Hermann, Sabrina Laudenklos; Neurobiology of Brain Tumors Labs, TP4, 3rd floor (contact: a.martin-villalba@dkfz.de or s.kleber@dkfz.de)

Topics:

The recognition that within the tumour, there is a cell population that shares common features with their normal stem cell counterparts has opened a new window to look at neoplasia. This cancer stem cell-like population, also known as tumour-initiating cells, represents the most therapy-resistant cell population within a tumour and has therefore become a major focus in the development of future therapeutic strategies. Neural cancer stem cells (NCSCs) have been identified in GBM, medulloblastoma and ependymoma. These NCSCs share common signalling pathways for self renewal, proliferation and differentiation with normal NSCs. Understanding the pathways that are common to both normal and cancer stem cells and may be dysregulated in the latter will facilitate the discovery of new targets for gene therapy.

Content:

• Isolation of "cancer stem cells" from glioblastoma (GBM)-bearing mice
• Isolation of adult neural stem cells
• Characterization of cancer and normal stem cells by FACS-analysis
• Neurosphere Assay (Ferron et al., 2007)
• Migration Assay of CSCs
• Determination of self-renewal and differentiation capacity of CD95L-expressing stem cells