Designer nuclease-mediated generation of knockout THP1 cells

15Citations
Citations of this article
51Readers
Mendeley users who have this article in their library.
Get full text

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

Cite

CITATION STYLE

APA

Schmidt, T., Schmid-Burgk, J. L., Ebert, T. S., Gaidt, M. M., & Hornung, V. (2016). Designer nuclease-mediated generation of knockout THP1 cells. In Methods in Molecular Biology (Vol. 1338, pp. 261–272). Humana Press Inc. https://doi.org/10.1007/978-1-4939-2932-0_19

Register to see more suggestions

Mendeley helps you to discover research relevant for your work.

Already have an account?

Save time finding and organizing research with Mendeley

Sign up for free