The DT40 Cell Line as a Genetic Model to Study Vertebrate Gene Function

Jean-Marie Buerstedde
Department of Cellular Immunology
Heinrich-Pette-Institute
Martinistr. 52
D-20251 Hamburg, Germany
telephone: 0049 40 48051 290
fax: 0049 40 48051 296
email: buersted@genetics.hpi.uni-hamburg.de
prestype: Platform
presenter: Jean-Marie Buerstedde

Jean-Marie Buerstedde, Igor Abdrahmanov and Bernhard Korn

Due to the high ratios of targeted integration the chicken B cell line DT40 cell line is a popular geneticsystem to study the functions of genes by disruption. However, relatively the sequences of few chicken genes are known and the isolation of the chicken cDNA by cross-hybridization or by reverse PCR is cumbersome.

To improve this situation we have started to build a comprehensive bursal B-cell EST database which currently holds over 7000 ESTs. Sequences corresponding to interesting candidate genes can be easily identified by online BLAST or keyword searches. Since the database reflects the gene expression profile of bursal lymphocytes, it provides valuable hints as to which genes might be involved in B-cell specific processes related to immunoglobulin repertoire formation, signal transduction, transcription and apoptosis.

This large collection of chicken ESTs will also be useful for gene expression studies and comparative gene mapping within the chicken genome project. In collaboration with the Department of Olli Lassila at Turku University in Finnland we are building filter and glas slide gene arrays which could be used to quantitate gene expression profiles in wild-type and mutant DT40 cells.

This bursal EST sequencing project will continue for the next three years. To avoid redundant sequencing of highly expressed genes we have successfully normalized the bursal cDNA library by hybridization with already sequenced ESTs. Since our current bursal library has only a low percentage of full-length cDNAs, we have also produced a second bursal cDNA library containing a high percentageof full length cDNA inserts with the help of the Riken Center in Japan. This library is currently orderedin the German Genome Resource Center. Using the normalized library and the library enriched for full-length clones we are planning to add new sequences at a rate of 1000 sequences per month for the next three years. All ESTs are freely available to all researchers.

We are producing other generic resources for genetic studies in DT40. In the past, six drug resistance markers were available, but for analysis of complex pathways a higher number of mutations are needed.

We therefore constructed new markers flanked by loxP sites which are recognized by the CRE-recombinase. These markers allow the generation of unlimited number of mutations in DT40 through the reuse of the markers after CRE-mediated excision.

Details of the bursal EST sequencing project and other resources can be found via the DT40 web site maintained by our Department.



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