Abstract
Recent developments in the field of designer nucleases allow the efficient and specific manipulation of genomic architectures in eukaryotic cell lines. To this end, it has become possible to introduce DNA double strand breaks (DSBs) at user-defined genomic loci. If located in critical coding regions of genes, thus induced DSBs can lead to insertions or deletions (indels) that result in frameshift mutations and thereby the knockout of the target gene. In this chapter, we describe a step-by-step workflow for establishing knockout cell clones of the difficult-to-transfect suspension cell line THP1. The here described protocol encompasses electroporation, cell cloning, and a deep sequencing-based genotyping step that allows the in-parallel analysis of 96 cell clones per gene of interest. Furthermore, we describe the use of the analysis tool OutKnocker that allows rapid identification of cell clones with all-allelic frameshift mutations.
Tobias Schmidt and Jonathan L. Schmid-Burgk contributed equally to this work
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Acknowledgement
This work is financially supported by SFB 670, SFB 704, ERC starting grant, the ImmunoSensation cluster of excellence to V.H., and by the Studienstiftung des deutschen Volkes (J.L.S.-B.)
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Schmidt, T., Schmid-Burgk, J.L., Ebert, T.S., Gaidt, M.M., Hornung, V. (2016). Designer Nuclease-Mediated Generation of Knockout THP1 Cells. In: Kühn, R., Wurst, W., Wefers, B. (eds) TALENs. Methods in Molecular Biology, vol 1338. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2932-0_19
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DOI: https://doi.org/10.1007/978-1-4939-2932-0_19
Publisher Name: Humana Press, New York, NY
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