Abstract
Thousands of new vertebrate genes have been discovered and genetic systems are needed to address their functions at the cellular level. The chicken B cell line DT40 allows efficient gene disruptions due to its high homologous recombination activity. However, cloning the gene of interest is often cumbersome, since relatively few chicken cDNA sequences are present in the public databases. In addition, the accumulation of multiple mutations within the same cell clone is limited by the consumption of one drug-resistance marker for each transfection. Here, we present the DT40 web site (http://genetics.hpi.uni-hamburg.de/dt40.html), which includes a comprehensive database of chicken bursal ESTs to identify disruption candidate genes and recyclable marker cassettes based on the loxP system. These freely available resources greatly facilitate the analysis of genes and genetic networks.
INTRODUCTION
The precise modification of chromosomal sequences by targeted integration of artificial constructs provides a powerful approach to determine the function of genes and regulatory elements within a living cell. In vertebrates, heterozygous mutations are most often introduced into murine embryonic stem cells which are then used to breed homozygous knock-out strains of mice (1).
If the mutant phenotype is measurable in cell culture, the production of homozygous mutant cell clones by step-wise disruption of both alleles can be a valid alternative to experiments with whole animals. The chicken B cell line DT40 has been popular in the study of cell autonomous (2–4) and B cell specific processes (5,6) due to high ratios of targeted to random integration of transfected constructs (7).
Although the identification of targeted integration events is straightforward, a common inconvenience of the DT40 system used to be the necessity to isolate the chicken cDNA of the target gene either by cross-hybridization or by inverse PCR. Another shortcoming has been the need of a new selectable marker for each transfection. While this is not a concern for the disruption of a single gene, the flexibility to freely accumulate multiple mutations is highly desirable for the analysis of genetic pathways.
THE BURSAL EST DATABASE
To allow the electronic retrieval of chicken cDNA sequences of interest, a large database of ESTs from bursal lymphocytes is imbedded into the DT40 web site (http://genetics.hpi.uni-hamburg.de/dt40Est.html). The first version of the database included about 7000 sequences derived from a bursal cDNA library named dkfz426 (8). In the meantime another 3000 sequences from this library have been added to the database. The dkfz426 library seems to faithfully reflect the gene expression profile of chicken bursal lymphocytes and has yielded sequences of thousands of new chicken orthologs of mammalian genes. However, dkfz426 was not normalized and contained only a low percentage of full-length gene coding inserts.
Another bursal B cell cDNA library was therefore synthesized using the biotinylated cap trapper method (9,10). This new normalized library named riken1 is of excellent quality with a high percentage of full-length gene inserts. More than 5000 ESTs of riken1 have been incorporated to the Bursal EST database and further sequences are added a rate of about 300 sequences per week.
All bursal ESTs are automatically annotated based on the results of BLAST searches against the public databases. In addition to the sequences and the BLAST reports, the PHRED scores as well as information about possible sequence cluster and polymorphisms are accessible at the web site. The software developed for the creation of the database and the Internet presentation is freely available under the open source license (http://genetics.hpi.uni-hamburg.de/FOUNTAIN.html).
It is hoped that the Bursal EST database will eventually comprise all genes expressed in bursal B cells and the DT40 cell line. Based on the searches for members of DNA repair pathways it seems that the database already represents orthologs of most of the housekeeping repair genes known from mammalian species. Likewise most orthologs of the known B cell specific transcription factors can be found. Since a high percentage of the ESTs from the riken1 library include the 5′ end, it is possible to determine the full-length reading frame of these ESTs by simple sequence extension.
RECYCLABLE MARKER CASSETTES
To allow recycling of the resistance markers, new marker cassettes have been designed in which the drug resistance genes are flanked by mutant LoxP sites (11). These new marker cassettes can be efficiently excised by the CRE recombinase (12) after targeted integration into the chromosome. Genomic instability after marker excision should be minimal, as the new LoxP sites resulting from the excision will be resistant to further Cre mediated cleavage. The new marker cassettes are freely distributed as part of the DT40 web site (http://genetics.hpi.uni-hamburg.de/dt40Reagents.html).
Drug resistance markers for knock-out experiments in DT40 were previously distributed as BamHI fragments and it was recommended that the cassettes are cloned into BamHI or BglII sites between the upstream and downstream arms of the target gene. Since the new marker cassettes are also available as BamHI fragments, it is possible to convert previously designed knock-out constructs by a simple cloning step. All knock-out constructs based on this principle can be combined with each other in the same cell, if the transfection is followed by the excision of the marker.
Acknowledgments
ACKNOWLEDGEMENTS
This research was supported by grant Bu 631/2-1 from the Deutsche Forschungsgemeinschaft (DFG) and from the EU Framework V programs ‘Chicken Image’ and ‘Genetics in a cell line’ and by a Research Grant for the RIKEN Genome Exploration Research Project from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government.
REFERENCES
- 1.Ledermann B. (2000) Embryonic stem cells and gene targeting. Exp. Physiol., 85, 603–613. [PubMed] [Google Scholar]
- 2.Nakayama T. and Takami,Y. (2001) Participation of histones and histone-modifying enzymes in cell functions through alterations in chromatin structure. J. Biochem. (Tokyo), 129, 491–499. [DOI] [PubMed] [Google Scholar]
- 3.Um M., Yamauchi,J., Kato,S. and Manley,J.L. (2001) Heterozygous disruption of the TATA-binding protein gene in DT40 cells causes reduced cdc25B phosphatase expression and delayed mitosis. Mol. Cell. Biol., 21, 2435–2448. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Takata M., Sasaki,M.S., Tachiiri,S., Fukushima,T., Sonoda,E., Schild,D., Thompson,L.H. and Takeda,S. (2001) Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs. Mol. Cell. Biol., 21, 2858–2866. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Bezzubova O., Silbergleit,A., Takeda,S. and Buerstedde,J.M. (1997) Reduced X-ray resistance and homologous recombination frequencies in a RAD54–/– mutant of the chicken DT40 cell line. Cell, 89, 185–193. [DOI] [PubMed] [Google Scholar]
- 6.Kurosaki T. (1999) Genetic analysis of B cell antigen receptor signaling. Annu. Rev. Immunol., 17, 555–592. [DOI] [PubMed] [Google Scholar]
- 7.Buerstedde J.M. and Takeda,S. (1991) Increased ratio of targeted to random integration after transfection of chicken B cell lines. Cell, 67, 179–188. [DOI] [PubMed] [Google Scholar]
- 8.Abdrakhmanov I., Lodygin,D., Geroth,P., Arakawa,H., Law,A., Plachy,J., Korn,B. and Buerstedde,J.M. (2000) A large database of chicken bursal ESTs as a resource for the analysis of vertebrate gene function. Genome Res., 10, 2062–2069. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Carninci P. and Hayashizaki,Y. (1999) High-efficiency full-length cDNA cloning. Methods Enzymol., 303, 19–44. [DOI] [PubMed] [Google Scholar]
- 10.Carninci P., Shibata,Y., Hayatsu,N., Sugahara,Y., Shibata,K., Itoh,M. Konno,H., Okazaki,Y. and Hayashizaki,Y. (2000) Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes. Genome Res., 10, 1617–1630. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Araki K., Araki,M. and Yamamura,K. (1997) Targeted integration of DNA using mutant lox sites in embryonic stem cells. Nucleic Acids Res., 25, 868–872. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Verrou C., Zhang,Y., Zurn,C., Schamel,W.W. and Reth,M. (1999) Comparison of the tamoxifen regulated chimeric Cre recombinases MerCreMer and CreMer. Biol. Chem., 380, 1435–1438. [DOI] [PubMed] [Google Scholar]