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 Not surprisingly new genome resources are changing the use of genetics for biological research. Most prominent about the new resources are the full genome sequences of model organisms and complete gene catalogs. Most of the newly discovered genes are not defined by mutants and often even their structure does not provide clues to their function (so-called orphan genes). The challenge is therefore to clarify their function by genetic approaches. These studies will enormously contribute to our understanding of all biological proccesses and should lead to new medical drugs and therapies.

Genetics in the time of genomics New Resources Full genome sequence of model organisms including the human Complete gene catalog (about 30 000 - 40 000 human genes) Challenges Not surprisingly new genome resources are changing the use of genetics for biological research. Most prominent about the new resources are the full genome sequences of model organisms and complete gene catalogs. Most of the newly discovered genes are not defined by mutants and often even their structure does not provide clues to their function (so-called orphan genes). The challenge is therefore to clarify their function by genetic approaches. These studies will enormously contribute to our understanding of all biological proccesses and should lead to new medical drugs and therapies. Clarify the function of the discovered genes for development, cell biology and disease Identify targets for the next generation of medical drugs and therapies

Approaches to gene analysis Traditional genetics Screen for mutants with interesting properties Identification of the responsible genes Reverse Genetics - Traditional genetic approaches try to identify mutants with interesting properties (phenotypes) in a population of animals. - In the second step the responsible genes are identified and their structures are compared to the genes of normal animals - Reverse genetic approaches introduce mutations in candidate genes with suspected functions. - In the second step the properties of the resulting mutants are analyzed. Both approaches have advantages and disadvantages. The most difficult step in traditional genetics is often the identification of the causative gene mutations. Reverse genetics has the disadvantage that it is difficult to predict the properties of the engineered mutants. Artificial disruption of candidate genes Analysis of the mutant phenotype

Gene disruption by targeted integration wild-type chromosome target gene knock-out construct The chicken B-cell line DT40 integrates transfected gene constructs at high ratios targeted into its endogenous gene loci. This is shown here by Southern blot analysis for a construct derived from the b-actin locus. Lane 1 contains the DNA of the untransfected control. Targeted integration leads to neighboring double bands like seen in lane 2. Nine of the 11 randomly picked neomycin resistant transfectants (lanes 2 - 12) show evidence for the targeted integration of the transfected construct into the b-actin locus. High ratios of targeted to random integration are also seen for many other loci after transfection of DT40. This high homologous recombination acitivity seems to be specific to chicken B-cell lines and may be related to immunoglobulin gene conversion. mutant chromosome

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 Cell line Reverse genetic studies are usually done in a whole organism. So-called gene knock-out mice are frequently generated and analyzed. However it is also possible to do reverse genetics in a cell line. - Studies using knock-out mice are ideal for the analysis of developmental processes and the generation of animal reflecting human diseases (disease models) - The production of the mutant mice is however expensive and has led to large increases in animal experimentation - The limitation of reverse genetics in a cell line is that the mutants properties have to be measured using cell culture assays - The experiments are often simpler and cheaper than the mouse experiments Experiments are simpler and animal free Measurement of the mutant phenotypes are limited to cell culture

The DT40 cell line as a genetic model Advantages Easy gene disruption by targeted integration Stable mutant phenotypes Established read-out assays Multiple genes can be disrupted Conditional gene expression systems - The possibility to easily disrupt genes by targeted integration makes DT40 a useful model for vertebrate gene analysis. Other advantages are: - stable mutant phenotypes - established read-out assays for mutant analysis - the possibility to disruption multiple gene using marker recycling - the availability of conditional gene expression systems - For the study to be successful it is however required that the mutant phenotype manifests itself in cell culture Requirement Mutant phenotype must be measurable in cell culture

Targeted integration in DT40 1 2 3 4 5 6 7 8 9 10 11 12 E Probe HindIII HindIII The chicken B-cell line DT40 integrates transfected gene constructs at high ratios targeted into its endogenous gene loci. This is shown here by Southern blot analysis for a construct derived from the b-actin locus. Lane 1 contains the DNA of the untransfected control. Targeted integration leads to neighboring double bands like seen in lane 2. Nine of the 11 randomly picked neomycin resistant transfectants (lanes 2 - 12) show evidence for the targeted integration of the transfected construct into the b-actin locus. High ratios of targeted to random integration are also seen for many other loci after transfection of DT40. This high homologous recombination acitivity seems to be specific to chicken B-cell lines and may be related to immunoglobulin gene conversion. b-actin locus Targeting construct HindIII puc18 neoR HindIII HindIII Targeted b-actin locus neoR

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) An European Consortium with the name "Somatic cell genetics" was started in Framework V to exploit the potential of the DT40 genetic model for the systematic analysis of vertebrate gene function. The main objectives are: - Establish somatic cell genetics as an alternative to animal experimentation -Provide generic resources facilitating gene identification and disruption in DT40 - Promote research in the fields of gene regulation, recombination, and chromosome structure -Advance aspects of the DT40 model which might be commercially exploited Improvements of cell culture systems for drug developments and biopharmaceutical manufactoring

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

The RAD51 gene is essential Human Rad51 expression in a RAD51-/- clone 6 12 18 24 30 36 42 48 56 60 Hours Cell Proliferation 104 103 Although the yeast RAD51 gene is not essential, attempts to obtain a RAD51-/- DT40 mutant were not successful. Both chicken RAD51 gene copies could however be disrupted in a clone which conditionally expresses the human RAD51 cDNA. Shutdown of the human RAD51 expression in this mutant leads to rapid depletion of the Rad51 protein as shown by Western blotting and a simultaneous decrease in cell viability. This experiment shows that DT40 can be used to determine whether genes are essential for the survival of vertebrate cells. 102 101

RAD54-/- mutants are radiosensitive Colony survival 104 103 102 101 RAD54 -/- DT40 mutants show a phenotypes similar to yeast mutants of the RAD52 pathway. As shown by a colony survival assay the homozygous clones are highly x-ray sensitive compared to wild-type and heterozygous clones. The defect can be fully complemented by the transfection of the chicken RAD54 cDNA. This provided the first evidence that double-strand break repair mediated by the RAD52 pathway contributes to the repair of radiation damage in vertebrate cells. 2 4 6 8 10 Dose in Gray Cl18+/+ Cl18+/- Cl18.1-/- Cl18.2-/- Cl18.1R

Immunoglobulin gene conversion y V segments rearranged light chain gene V(D)J recombination contributes little to the immunoglobulin repertoire in chicken B-cells, since it is limited to the rearrangement of single functional V- and J-segment. The chicken immunoglobulin gene diversity is created by homologous recombination in form of gene conversion. During this process blocks of pseudo V-gene segments are incorporated into the rearranged VJ gene. Immunoglobulin gene conversion also plays an important role of the repertoire development in a number of mammalian species like rabbits and cows.

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

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

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