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Jean-Marie Buerstedde GSF Research Center for Environment and Health Institute for Molecular Radiobiology Ingolstädter Landstr. 1 85764 Neuherberg Germany.

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Presentation on theme: "Jean-Marie Buerstedde GSF Research Center for Environment and Health Institute for Molecular Radiobiology Ingolstädter Landstr. 1 85764 Neuherberg Germany."— Presentation transcript:

1 Jean-Marie Buerstedde GSF Research Center for Environment and Health Institute for Molecular Radiobiology Ingolstädter Landstr. 1 85764 Neuherberg Germany E-mail: buersted@gsf.de

2 Genetics in the time of genomics Full genome sequence of model organisms including the human Complete gene catalog (about 30 000 - 40 000 human genes) Clarify the function of the discovered genes for development, cell biology and disease Identify targets for the next generation of medical drugs and therapies New Resources Challenges

3 Approaches to gene analysis Screen for mutants with interesting properties Identification of the responsible genes Artificial disruption of candidate genes Analysis of the mutant phenotype Traditional genetics Reverse Genetics

4 Gene disruption by targeted integration target gene wild-type chromosome mutant chromosome knock-out construct

5 Reverse Genetics Organism versus Cell line Needed for the study of development, cancer and complex diseases Experiments in vertebrates are expensive and involve animal suffering Measurement of the mutant phenotypes are limited to cell culture Experiments are simpler and animal free Organism Cell line

6 The DT40 cell line as a genetic model Easy gene disruption by targeted integration Stable mutant phenotypes Multiple genes can be disrupted Conditional gene expression systems Established read-out assays Advantages Mutant phenotype must be measurable in cell culture Requirement

7 Targeted integration in DT40 0123 5 6 7 8 4 9101112 E -actin locus Targeted -actin locus HindIII Probe Targeting construct puc18 HindIII neoR

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9 Objectives of the Framework V consortium Somatic cell genetics as an alternative to animal experimentation Resources for gene identification and disruption in DT40 (gene catalog, marker recycle, microarray) Improvements of cell culture systems for drug developments and biopharmaceutical manufactoring

10 Partners of the consortium William Brown, Nottingham University Centromere function Jean-Marie Buerstedde, GSF Recombination Olli Lassila, Turku University Lymphoid transcription factors Berndt Müller, Aberdeen University RNA metabolism Martin Fussenegger, Cistronics Zürich Protein Engineering and Production

11 The RAD51 gene is essential Hours Human Rad51 expression in a RAD51 -/- clone Cell Proliferation 10 1 10 2 10 3 10 4 06121824303642485660

12 RAD54 -/- mutants are radiosensitive Colony survival 10 1 10 2 10 3 10 4 Cl18 +/+ Cl18 +/- Cl18.1 -/- Cl18.2 -/- Cl18.1 R 0 2 4 6 8 10 Dose in Gray

13 V segments rearranged light chain gene Immunoglobulin gene conversion

14 Assay for immunoglobulin gene conversion frameshift frameshift repair by gene conversion sIgM(-) cell sIgM(+) cell

15 0.12% 6.24% 0.37% 0.14% 9.43% events in the sIgM(+) gates The AID gene is required for immunoglobulin gene conversion

16 The Future of Somatic Cell Genetics Greatest potential for the analysis of cell biology processes Recessive mutant screens possible using the new RNA interference technique Without alternative for the systematic analysis of human genes Refinement and reduction of animal experiments through improved knowledge of gene functions


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