Function-based identification of mammalian enhancers using site-specific integration.
Nat Methods
; 11(5): 566-71, 2014 May.
Article
in En
| MEDLINE
| ID: mdl-24658141
ABSTRACT
The accurate and comprehensive identification of functional regulatory sequences in mammalian genomes remains a major challenge. Here we describe site-specific integration fluorescence-activated cell sorting followed by sequencing (SIF-seq), an unbiased, medium-throughput functional assay for the discovery of distant-acting enhancers. Targeted single-copy genomic integration into pluripotent cells, reporter assays and flow cytometry are coupled with high-throughput DNA sequencing to enable parallel screening of large numbers of DNA sequences. By functionally interrogating >500 kilobases (kb) of mouse and human sequence in mouse embryonic stem cells for enhancer activity we identified enhancers at pluripotency loci including NANOG. In in vitro-differentiated cardiomyocytes and neural progenitor cells, we identified cardiac enhancers and neuronal enhancers, respectively. SIF-seq is a powerful and flexible method for de novo functional identification of mammalian enhancers in a potentially wide variety of cell types.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Enhancer Elements, Genetic
/
Myocytes, Cardiac
/
Embryonic Stem Cells
/
Neural Stem Cells
Type of study:
Diagnostic_studies
Limits:
Animals
/
Humans
Language:
En
Journal:
Nat Methods
Journal subject:
TECNICAS E PROCEDIMENTOS DE LABORATORIO
Year:
2014
Type:
Article
Affiliation country:
United States