Pgl3 basic luciferase reporter gene vector

Reporter vector coding for the Firefly Luciferase under the control of the Il9 promoter encompassing nucleotides −1201 to +52 bp was cloned into the promoterless pGL3 Basic luciferase reporter gene vector (Promega)^{28 (link)}. Irf4 promoter encompassing nucleotides −1562 to +122 bp was cloned into pGL3 Basic luciferase reporter gene vector. Reporter assays were carried out as described previously^{7 (link)}. Briefly, 293 T cells were transfected with 0.4 μg of the reporter vector coding for the Firefly Luciferase under the control of the Il9 or Irf4 promoter and with 0.8 μg of the Foxo1 vector. Cells were cultured for 48 hours before harvesting and the relative Il9 or Irf4 promoter activity was measured using Promega kit in accordance with the manufacturer’s instructions.

A series of luciferase reporter gene constructs was prepared in the promoter-less pGL3-basic luciferase reporter gene vector (Promega) to determine the promoter activity of the 5′-upstream region of the human TMBIM6 gene. The promoter region in the pGEM-TMBIM6 construct that contained part of the 5′-UTR and upstream flanking region (−2430/+30 from the transcription start site) was separated by KpnI and BglII digestion and sub-cloned into the pGL3-basic vector to make the reporter gene construct pGL3-TMBIM6-P1(−2430/+30). Then, five progressive 5′-deletion constructs (pGL3-TMBIM6-P2 to pGL3-TMBIM6-P6) were generated through PCR amplification by using different forward primers with the KpnI site and a common reverse primer with the BglII restriction enzyme site. Additionally, P2 specific region construct (P2ΔP3) and P2 without P6 (P2ΔP6) construct were prepared from pGL3-TMBIM6-P2 reporter construct by PCR amplification using primers specific for the indicated region. P6 region (−405/+30) was further fragmented into −405/−260, −259/−134, and −133/+30 by PCR amplification and prepared the pGL3-TMBIM6-P7, pGL3-TMBIM6-P8, and pGL3-TMBIM6-P9 respectively by cloning into luciferase reporter pGL3 basic vector. The primer sequence and enzyme sites are listed in the Table 1. The nucleotide sequences of all constructs were verified for orientation.