Abstract
Reverse genetics is gaining importance in the field of modern biological sciences. Gene disruption and the use of siRNAs are the favored techniques for current research. Many researchers, however, are aware that the data from siRNA experiments are frequently inconsistent and that epistatic analysis of multiple genes using siRNAs is barely feasible. In recognition of the drawbacks of the siRNA technique, many researchers, especially in the field of DNA repair, are now introducing multiple genetic disruption techniques using the chicken DT40 cell line into their research. Thus, recent publications increasingly include data utilizing DT40 cells. In this chapter, we describe the current standard methods of multiple genetic manipulation in DT40 cells. We place a particular emphasis on describing the basic concepts and theoretical background of the genetic manipulation of DT40 cells for researchers who are new to such techniques.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Adamson B, Smogorzewska A, Sigoillot FD et al (2012) A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response. Nat Cell Biol 14(3):318–328
Jackson AL, Linsley PS (2010) Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application. Nat Rev Drug Discov 9(1):57–67
Singh S, Narang AS, Mahato RI (2011) Subcellular fate and off-target effects of siRNA, shRNA, and miRNA. Pharmacuet Res 28(12):2996–3015
Baba TW, Giroir BP, Humphries EH (1985) Cell lines derived from avian lymphomas exhibit two distinct phenotypes. Virology 144(1):139–151
Buerstedde JM, Takeda S (1991) Increased ratio of targeted to random integration after transfection of chicken B cell lines. Cell 67(1):179–188
Buerstedde J-M, Takeda S (2006) Reviews and protocols in DT40 research, vol 40, Subcellular biochemistry. Springer, New York, NY
Ishiai M, Uchida E, Takata M (2012) Establishment of the DNA repair-defective mutants in DT40 cells. Methods Mol Biol 920:39–49
Kitao H, Hirano S, Takata M (2011) Evaluation of homologous recombinational repair in chicken B lymphoma cell line, DT40. Methods Mol Biol 745:293–309
Yamazoe M, Sonoda E, Hochegger H et al (2004) Reverse genetic studies of the DNA damage response in the chicken B lymphocyte line DT40. DNA Repair 3(8–9):1175–1185
Sonoda E, Morrison C, Yamashita YM et al (2001) Reverse genetic studies of homologous DNA recombination using the chicken B-lymphocyte line, DT40. Phil Trans Roy Soc Lond B Biol Sci 356(1405):111–117
Iiizumi S, Nomura Y, So S et al (2006) Simple one-week method to construct gene-targeting vectors: application to production of human knockout cell lines. Biotechniques 41(3):311–316
Arakawa H, Lodygin D, Buerstedde JM (2001) Mutant loxP vectors for selectable marker recycle and conditional knock-outs. BMC Biotech 1:7
Chang H, Delany ME (2004) Karyotype stability of the DT40 chicken B cell line: macrochromosome variation and cytogenetic mosaicism. Chromosome Res 12(3):299–307
Nakai A, Ishikawa T (2001) Cell cycle transition under stress conditions controlled by vertebrate heat shock factors. EMBO J 20(11):2885–2895
Arakawa H, Moldovan GL, Saribasak H et al (2006) A role for PCNA ubiquitination in immunoglobulin hypermutation. PLoS Biol 4(11):e366
Zhang Y, Wienands J, Zurn C et al (1998) Induction of the antigen receptor expression on B lymphocytes results in rapid competence for signaling of SLP-65 and Syk. EMBO J 17(24):7304–7310
Fujimori A, Tachiiri S, Sonoda E et al (2001) Rad52 partially substitutes for the Rad51 paralog XRCC3 in maintaining chromosomal integrity in vertebrate cells. EMBO J 20(19):5513–5520
Acknowledgments
We thank Dr. Masamichi Ishiai (Kyoto Univ.) for critical reading of the manuscript. This work was supported in part by Grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan (to AM and MT) and by Mochida Memorial Foundation for Medical and Pharmaceutical Research (to AM).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Motegi, A., Takata, M. (2014). Multiple Genetic Manipulations of DT40 Cell Line. In: Storici, F. (eds) Gene Correction. Methods in Molecular Biology, vol 1114. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-761-7_3
Download citation
DOI: https://doi.org/10.1007/978-1-62703-761-7_3
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-760-0
Online ISBN: 978-1-62703-761-7
eBook Packages: Springer Protocols