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Jean-Marie Buerstedde
GSF Research Center for Environment and Health
Institute for Molecular Radiobiology
Ingolstädter Landstr. 1
85764 Neuherberg
Germany
E-mail: [email protected]
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 genesfor development, cell biology and disease
Identify targets for the next generation of medical drugs and therapies
New Resources
Challenges
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
Gene disruption by targeted integration
target gene
wild-type chromosome
mutant chromosome
knock-out construct
Reverse Genetics Organism versus Cell
line
Needed for the study of development, cancer and complex diseases
Experiments in vertebrates are expensiveand involve animal suffering
Measurement of the mutant phenotypesare limited to cell culture
Experiments are simpler and animal free
Organism
Cell line
The DT40 cell line as a genetic model
Easy gene disruption by targeted integrationStable 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
Targeted integration in DT400 1 2 3 5 6 7 84 9 10 11 12
E
-actin locus
Targeted -actin locus
HindIII HindIIIProbe
Targeting constructpuc18
HindIII
HindIII HindIII
neoR
neoR
Objectives of the Framework V consortium
Somatic cell genetics as an alternative to animal experimentation
Resources for gene identification anddisruption in DT40 (gene catalog, marker recycle, microarray)
Improvements of cell culture systems for drug developments and biopharmaceuticalmanufactoring
Partners of the consortium
William Brown, Nottingham UniversityCentromere function
Jean-Marie Buerstedde, GSF Recombination
Olli Lassila, Turku UniversityLymphoid transcription factors
Berndt Müller, Aberdeen UniversityRNA metabolism
Martin Fussenegger, Cistronics ZürichProtein Engineering and Production
The RAD51 gene is essential
Hours
Human Rad51 expression in a RAD51-/- clone
Cell Proliferation
101
102
103
104
0 6 12 18 24 30 36 42 48 56 60
RAD54-/- mutants are radiosensitiveColony survival
101
102
103
104
Cl18+/+ Cl18+/- Cl18.1-/- Cl18.2-/- Cl18.1R
0 2 4 6 8 10Dose in Gray
V segments rearranged light chain gene
Immunoglobulin gene conversion
Assay for immunoglobulin gene
conversionframeshift
frameshift repairby gene conversion
sIgM(-) cell
sIgM(+) cell
0.12%
6.24%
0.37% 0.14%
9.43%
events in the sIgM(+) gates
The AID gene is required for immunoglobulin gene
conversion
The Future of Somatic Cell Genetics
Greatest potential for the analysis of cell biology processes
Recessive mutant screens possibleusing the new RNA interference technique
Without alternative for the systematic analysis of human genes
Refinement and reduction of animal experimentsthrough improved knowledge of gene functions
