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

Area of Science:

• Genomics
• Molecular Biology
• Gene Regulation

Background:

• Gene expression regulation in metazoans is spatiotemporal, involving transcriptional regulatory elements and target genes.
• Active promoters exhibit specific chromosomal features like DNaseI hypersensitivity and histone modification enrichment.

Purpose of the Study:

• To propose and validate a novel, high-efficiency in vitro method for identifying gene promoters.
• To screen promoter-active fragments and confirm their downstream transcriptional activity.

Main Methods:

• Construction of a promoter-trap library with 706 random mouse genomic DNA fragments.
• Screening of 260 promoter-active fragments via transient transfection into 4T1 cells.
• Validation using DNase-seq, ChIP-seq data, transcript prediction, and qRT-PCR analysis of 13 selected fragments.

Main Results:

• Successfully identified 260 promoter-active fragments from the library.
• Confirmed promoter activity and downstream transcript expression for six predicted transcription units via qRT-PCR.
• Demonstrated successful amplification in various mouse tissues, cells, and reconstituted mammary tumors.

Conclusions:

• The developed promoter-trap method is highly efficient for detecting promoter-active DNA fragments in vitro.
• The method accurately identifies functional promoters and their associated downstream transcripts.