Development of a high efficient promoter finding method based on transient transfection.

Abstract

In metazoan genome, the mechanism of gene expression regulation between transcriptional regulatory elements and their target gene is spatiotemporal. Active promoters possess many specific chromosomal features, such as hypersensitive to DNaseI and enrichment of specific histone modifications. In this article, we proposed a novel method which possesses a high efficiency to find promoters in vitro. A promoter-trap library was constructed with totally 706 random mouse genomic DNA fragment clones, and 260 promoter-active fragments of the library were screened by transient transfection into 4T1 cells. To demonstrate the accuracy of this promoter finding method, 13 fragments with promoter activities were randomly selected for published DNase-seq and ChIP-seq data analysis, downstream transcripts prediction and expression confirmation. qRT-PCR results showed that six predicted transcription units were successfully amplified in different mouse tissues/cells or in reconstituted mouse mammary tumors. Our results indicate that this promoter finding method can successfully detect the promoter-active fragments and their downstream transcripts.