Abstract
Quidquid agis, prudenter agas et respice finem!—Whatever you do, do it wisely and consider the goal. In consideration of that sage advice, the chicken B-cell line DT40 is an excellent model cell system to study the function of vertebrate genes. In addition to being highly amenable to gene manipulations, the recent influx of genome and gene/protein resources allows for the straightforward selection, design, and targeting of candidate genes for knockout analysis. This chapter will give a step by step standardized protocol to creating a gene knockout mutant in DT40. With careful consideration, the methods and protocols described herein can be easily modified to allow for further gene manipulations such as creating a knockin or a conditional mutant.
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References
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.
Arakawa, H., Lodygin, D., and Buerstedde, J. M. (2001) Mutant loxP vectors for selectable marker recycle and conditional knockouts. BMC Biotechnol. 1, 7.
International Chicken Genome Sequencing Consortium (2004) Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution. Nature 432, 695–716.
International Chicken Polymorphism Map Consortium (2004) A genetic variation map for chicken with 2.8 million single-nucleotide polymorphisms. Nature 432(7018), 717–722.
Schmid, M., Nanda, I., Hoehn, H., et al. (2005) Second report on chicken genes and chromosomes 2005. Cytogenet. Genome Res. 109(4), 415–479.
Wahl, M. B., Caldwell, R. B., Kierzek, A. M., et al. (2004) Evaluation of the chicken transcriptome by SAGE of B cells and the DT40 cell line. BMC Genomics 5, 98.
Caldwell, R. B., Kierzek, A. M., Arakawa, H., et al. (2005) Full-length cDNAs from chicken bursal lymphocytes to facilitate gene function analysis. Genome Biol. 6, R6.
Deng, C. and Capecchi, M. R. (1992) Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Mol. Cell Biol. 12(8), 3365–3371.
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Caldwell, R.B., Fiedler, P., Schoetz, U., Buerstedde, JM. (2007). Gene Function Analysis Using the Chicken B-Cell Line DT40. In: Ochs, M.F. (eds) Gene Function Analysis. Methods in Molecular Biology™, vol 408. Humana Press. https://doi.org/10.1007/978-1-59745-547-3_11
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DOI: https://doi.org/10.1007/978-1-59745-547-3_11
Publisher Name: Humana Press
Print ISBN: 978-1-58829-734-1
Online ISBN: 978-1-59745-547-3
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