Jean-Marie Buerstedde GSF Research Center for Environment and Health Institute for Molecular...

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

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