A matrilin-1 gén transzkripciós szabályozásának vizsgálata transzgenikus egerekben

Role of Sox trio, Nfi and Hmgb1 factors in the unique transcriptional regulatory mechanisms of the matrilin-1 gene

“…Nucleotide sequences of Ine and Pe1 described previously [88], and that of Dpe1 is presented later in Fig. 5A.…”

“…Furthermore, the 2-kb long Matn1 promoter also includes two conserved distal promoter elements, distal promoter element 1 (Dpe1) and distal promoter element 2 (Dpe2) ( Fig.2A). In transient expression assay, multiple copies of the Dpe1 element robustly increased the short promoter activity in CEC culture [88]. Eight copies of Dpe1 fused to the short promoter directed zonal transgene expression of high level in distal structures of the developing skeletal elements in transgenic mice [88].…”

“…CEC and CEF cultures were transfected with 2 µg reporters, while HDM, LDM, and COS-7 cultures were transfected with 5 µg reporters using the Ca-phosphate coprecipitation method as described previously [86,88,89]. Cotransfection with pRL-TK (Promega) served as an internal control to correct for transfection efficiency, but parallel plates were also transfected with Cotransfection assays were performed with 2 µg or 5 µg AC8Luc reporter using effector plasmids pcDNA5'UT-FLAG-L-Sox5 (pFSox5), pcDNA5'UT-FLAG-Sox6 (pFSox6), and pCDMA-SOX9 (pSOX9) or pcDNA5'UT-FLAG-SOX9 (pFSOX9) [52], plasmids expressing…”

“…5A. Double-stranded oligonucleotides were synthesized for the Dpe1 element and Hmgb1 were expressed and purified, and crude cell extracts were made as described [88]. 20-30 fmol end-labeled DNA probes were incubated either with 0.6-3.2 μg purified GSTfused Hmgb1, SOX9, L-Sox5 or 3 μg crude CEC or CEF cell extracts in the presence of 100-500 ng poly(dG-dC)·(dG-dC) and separated on pre-run 5% or 6.6% PAGE.…”

“…3) [86] and Pe1 and Ine were demonstrated to interact with SOX9 in vitro [86,88,89]. Therefore, we hypothesized that Sox factor-mediated interactions may play important role in the chondroblast developmental stage-dependent regulation of Matn1.…”