中国生物工程杂志

Eukaryotic DNA element called Matrix Attachment Regions (MARs) can function on regulating the structure and activity of chromosome. In order to study in vivo chromatin accessibility and transcription regulation involved MAR, here, we show that alpha 1-antitrypsin MAR (α1-AT MAR) is cloned from human blood and incorporated in pEGFP-C1 vector. non-MAR-containing and MAR-containing plasmids were transfected into HEK-293 cells with LipofectamineTM 2000, respectively. Pools of clones were assayed after 20 days of selection of G418. Semiquantitative RT-PCR and fluorescence microscope analysis show that this MAR has a positive effect on modulate nearby gene expression. Then, chromatin immunoprecipitation (ChIP) where it co-localizes with newly CMV promoter and RNA polymeraseⅡ(RNAPⅡ) is detected by PCR, The result demonstrates that more RNAPⅡwas recruited to the CMV promoter in presence of MAR. ChIP can be used to confirm the MAR-mediated transcriptional activation and provide more dynamic information than RT-PCR in real time. The technology is also providing a platform for our research in gene expression regulation.