Transgenic mouse lines expressing the 3xFLAG-dCas9 protein for enChIP analysis

Genes Cells. 2018 Apr;23(4):318-325. doi: 10.1111/gtc.12573. Epub 2018 Feb 26.

Abstract

We developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology to isolate specific genomic regions while retaining their molecular interactions. In enChIP, the locus of interest is tagged with an engineered DNA-binding molecule, such as a modified form of the clustered regularly interspaced short palindromic repeats (CRISPR) system containing a guide RNA (gRNA) and a catalytically inactive form of Cas9 (dCas9). The locus is then affinity-purified to enable identification of associated molecules. In this study, we generated transgenic mice expressing 3xFLAG-tagged Streptococcus pyogenes dCas9 (3xFLAG-dCas9) and retrovirally transduced gRNA into primary CD4+ T cells from these mice for enChIP. Using this approach, we achieved high yields of enChIP at the targeted genomic region. Our novel transgenic mouse lines provide a valuable tool for enChIP analysis in primary mouse cells.

Keywords: ChIP; chromatin immunoprecipitation; dCas9; enChIP; transgenic mouse.

MeSH terms

  • Animals
  • CD4-Positive T-Lymphocytes / cytology
  • CD4-Positive T-Lymphocytes / metabolism
  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Associated Protein 9 / metabolism*
  • Cells, Cultured
  • Chromatin Immunoprecipitation / methods*
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Gene Expression Regulation
  • Genetic Engineering / methods*
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • RNA, Guide, CRISPR-Cas Systems / genetics*

Substances

  • RNA, Guide, CRISPR-Cas Systems
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes