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Magnetic separation and antibiotics selection enable enrichment of cells with ZFN/TALEN-induced mutations.

PloS one (2013-02-27)
Hyojin Kim, Myung-Sun Kim, Gabbine Wee, Choong-il Lee, Hyongbum Kim, Jin-Soo Kim
ABSTRACT

The ability to enrich cells with targeted mutations greatly facilitates the process of using engineered nucleases, including zinc-finger nucleases and transcription activator-like effector nucleases, to construct such cells. We previously used surrogate reporters to enrich cells containing nuclease-induced mutations via flow cytometry. This method is, however, limited by the availability of flow cytometers. Furthermore, sorted cells occasionally fail to form colonies after exposure to a strong laser and hydrostatic pressure. Here we describe two different types of novel reporters that enable mutant cell enrichment without the use of flow cytometers. We designed reporters that express H-2K(k), a surface antigen, and the hygromycin resistance protein (Hygro(R)), respectively, when insertions or deletions are generated at the target sequences by the activity of engineered nucleases. After cotransfection of these reporters and the engineered nuclease-encoding plasmids, H-2K(k)- and Hygro(R)-expressing cells were isolated using magnetic separation and hygromycin treatment, respectively. We found that mutant cells were drastically enriched in the isolated cells, suggesting that these two reporters enable efficient enrichment of mutants. We propose that these two reporters will greatly facilitate the use of engineered nucleases in a wider range of biomedical research.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Hygromycin B solution from Streptomyces hygroscopicus, ≥60% (HPLC), 45-60 mg/mL in H2O
Sigma-Aldrich
Hygromycin B from Streptomyces hygroscopicus, suitable for plant cell culture, BioReagent, ≥60% (HPLC), lyophilized powder
Sigma-Aldrich
Hygromycin B from Streptomyces hygroscopicus, lyophilized powder
Sigma-Aldrich
Hygromycin B from Streptomyces hygroscopicus, powder, BioReagent, suitable for cell culture, suitable for insect cell culture