Establishment and characterization of P19 embryonal carcinoma cells harboring an inducible Sty gene.
|Title:||Establishment and characterization of P19 embryonal carcinoma cells harboring an inducible Sty gene.|
|Authors:||Choi, Kyung Bok.|
|Abstract:||In order to establish Myc-Sty inducible clones, I generated a bio-cistronic Lac repressor expression construct (PCJ2LacIN) and subsequently transfected it along with pPOP $\beta$-gal into P19 EC cells. One of the clones (CJ2-8) showed 35 fold induction in $\beta$-gal activity between un-induced and induced cells. Subsequently, I also generated an inducible Sty expression vector (pPOPMycSty) containing two Lac operator sequences which is to be bound by Lac repressor to regulate the expression of the target genes at the downstream of the operator sequences. Following the co-transfection of pPOPMycSty, a construct containing PGK-1 promoter and its intragenic region, B17 and PGKNeo and subsequent drug selection, most of the drug resistant colonies turned out to be positive for Myc-Sty although its expression level and inducibility were somewhat variable among the clones. Myc-Sty from the inducible clone #9 is catalytically active, can dimerize with GST-Sty in vitro, phosphorylated on Tyr in vivo, localized in the nucleus and furthermore, immunofluorescence result showed that it phosphorylates and redistribute SR proteins in the nucleus resulting in affecting alternative splicing. Western analysis result using $\alpha$-SR monoclonal antibody (mAb104) recognizing the phospho-epitope common to every SR protein suggested that Sty may selectively phosphorylate SRp30 and SRp55, which were among the proteins found to be interacting with Sty by yeast two hybrid system. Subsequently, double immunofluorescence both for Myc-Sty and EC cell surface antigen (SSEA-1) or neuronal differentiation markers (A60 and HNK-1) indicated that Myc-Sty alone does not seem to give rise to the differentiation process.|
|Collection||Thèses, 1910 - 2010 // Theses, 1910 - 2010|