Abstract
Dictyostelium discoideum is an exceptionally powerful eukaryotic model to study many aspects of growth, development, and fundamental cellular processes. Its small-sized, haploid genome allows highly efficient targeted homologous recombination for gene disruption and knock-in epitope tagging. We previously described a robust system for the generation of multiple gene mutations in Dictyostelium by recycling the Blasticidin S selectable marker after transient expression of the Cre recombinase. We have now further optimized the system for higher efficiency and, additionally, coupled it to both, knock-out and knock-in gene targeting, allowing the characterization of multiple and cooperative gene functions in a single cell line.
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Acknowledgements
We thank Alexander Junemann and Moritz Winterhoff for thoroughly testing plasmid pTX-NLS-Cre and Drs. Katrin Koch and Ralf Gräf for their TAP-tag plasmid. This research was supported by Deutsche Forschungsgemeinschaft (Fa 330/4-2, Fa 330/5-1), the Intramural Research Program of the National Institutes of Health, the National Institute of Diabetes and Digestive and Kidney Diseases, and the WellcomeTrust/NIH Program Studentship to J.L.P.; J.L.P. is joint student with Dr. Adrian Harwood (Cardiff University) and A.R.K. (NIH). There are no conflicts or competing interests.
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Faix, J., Linkner, J., Nordholz, B., Platt, J.L., Liao, XH., Kimmel, A.R. (2013). The Application of the Cre-loxP System for Generating Multiple Knock-out and Knock-in Targeted Loci. In: Eichinger, L., Rivero, F. (eds) Dictyostelium discoideum Protocols. Methods in Molecular Biology, vol 983. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-302-2_13
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DOI: https://doi.org/10.1007/978-1-62703-302-2_13
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