Transgenic mouse lines expressing the 3xFLAG-dCas9 protein for enChIP analysis.
Toshitsugu FujitaFusako KitauraAsami OjiNaoki TanigawaMiyuki YunoMasahito IkawaIchiro TaniuchiHodaka FujiiPublished in: Genes to cells : devoted to molecular & cellular mechanisms (2018)
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.
Keyphrases
- dna binding
- transcription factor
- crispr cas
- genome editing
- genome wide
- gene expression
- induced apoptosis
- copy number
- dna damage
- type diabetes
- oxidative stress
- metabolic syndrome
- adipose tissue
- dna methylation
- escherichia coli
- cell proliferation
- small molecule
- binding protein
- mass spectrometry
- nk cells
- wild type
- high fat diet induced