Alpha 1-antitrypsin Matrix Attachment Region Enhances RNA PolymeraseⅡ-dependent Transcription / 中国生物工程杂志

ABSTRACT

Eukaryotic DNA element called Matrix Attachment Regions (MARs) can function on regulating the structure and activity of chromosome. Traditional quantitation in vitro and indirect functional analysis can not always reflect MAR-involved physiological state. In order to study transcription regulation and make a try in methodism,? 1-antitrypsin MAR (?1-AT MAR) is cloned and incorporated into pEGFP-C1 vector. Non-MAR-containing and MAR-containing plasmids were then transfected into HEK-293 cells with LipofectamineTM 2000 respectively. Positive cell clones were assayed after 20 days of selection by G418. Semiquantitative RT-PCR and fluorescence microscope analysis show that this MAR has a positive effect on modulating nearby gene expression. Further, co-localization with newly CMV promoter and RNA polymeraseⅡ(RNAPⅡ) was detected by chromatin immunoprecipitation (ChIP), The PCR result demonstrates that more RNAPⅡwas recruited to the CMV promoter to initiate transcription in presence of MAR. ChIP can be used to confirm the MAR-mediated transcriptional activation and provide more reliable information than RT-PCR in real time. The technology is also providing a platform for our research in gene expression regulation.