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

PLoS One. 2013;8(2):e56476. doi: 10.1371/journal.pone.0056476. Epub 2013 Feb 18.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Anti-Bacterial Agents / pharmacology*
  • Base Sequence
  • Cell Line
  • Cells, Cultured
  • Cinnamates / pharmacology
  • Drug Resistance / genetics*
  • Endonucleases / genetics
  • Endonucleases / metabolism*
  • Gene Order
  • Genes, Reporter
  • Genetic Vectors / genetics
  • H-2 Antigens / genetics
  • Humans
  • Hygromycin B / analogs & derivatives
  • Hygromycin B / pharmacology
  • Immunomagnetic Separation*
  • Mice
  • Mutation*
  • Receptors, CCR5 / genetics
  • Receptors, CCR5 / metabolism
  • Sequence Alignment
  • Zinc Fingers*

Substances

  • Anti-Bacterial Agents
  • Cinnamates
  • H-2 Antigens
  • H-2K(K) antigen
  • Receptors, CCR5
  • Hygromycin B
  • hygromycin A
  • Endonucleases

Grant support

J-SK and Hyongbum K. are supported in part by the National Research Foundation of Korea (J-SK, 2012-0001225; HK, 2012-0003376, 2012-0006735). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study.