Pgl4.23 luc2 minp

The entire coding regions of mouse Pax1, mouse Pax9, human Sox5, human Sox6, and mouse Sox9 were obtained from RCAS(A)-Pax1, RCAS(A)-Pax9^{10 (link)}, pMXs-gw/hSOX5, pMXs-gw/hSOX6^{32 (link)}, and pCMV-SPORT6-Sox9 (OriGene Technologies, Rockville, USA), respectively. To construct expression plasmids, each DNA fragment was inserted into pcDNA3 or CAG-IRES-EGFP vectors. To generate N-terminal FLAG-tagged PAX1 and PAX9, each DNA fragment was amplified by PCR to add NotI sites and inserted into the pcDNA3-FLAG vector. A 359-bp UE was amplified by PCR from mouse genomic DNA with forward and reverse primers designed with BamHI and BglII sites, respectively, and then PCR products were cloned into the pCR4-TOPO vector (Thermo Fisher Scientific). Four tandem copies of UE were obtained by ligation of monomers digested with BamHI and BglII. To construct pGL4.23[luc2/minP]-1xUE-Luc or pGL4.23[luc2/minP]-4xUE-Luc, one copy or four tandem copies of UE were inserted into the pGL4.23[luc2/minP] vector (Promega, Madison, WI, USA) containing a minimal promoter and the firefly luciferase reporter gene. The 508-bp I12E or 1109-bp Nkx3.2-P were amplified by PCR from mouse genomic DNA and inserted into the pGL4.23[luc2/minP] or pGL3-Basic vector (Promega) containing the firefly luciferase reporter gene, respectively. The PCR primers for constructs are listed in Supplementary Table 2.

The full-length ICP0 promoter (−800 to +150) was previously described, and provided by the late Dr. Priscilla Schafer [35 (link)]. The respective CRM constructs were synthesized by Genscript and inserted into pGL4.23[luc2/minP] (Promega; Madison, WI, USA) at SacI and XhoI unique restriction enzyme sites. The GR-α expression construct was obtained from Dr. John Cidlowski (NIEHS, Research Triangle Park, NC, USA). The KLF15 expression plasmid was obtained from Deborah Otteson (University of Houston; Houston, TX, USA).