Pgl4.11 luc2p vector

In order to generate a luciferase reporter plasmid containing the promoter region of OCTN2 reference sequence, a 2,618 bp of the human OCTN2 gene, extending from −2,490 to +128 relative to the translational start site, was amplified from genomic DNA samples from the individual with a reference sequence. Then, the amplified product was inserted between the restriction sites for NheI and KpnI (Enzynomics, Daejeon, Korea) of the pGL4.11 [luc2P] vector (Promega Corporation, Madison, WI, USA). The mutant pGL4.11-OCTN2 vectors containing genetic variants of the promoter region were generated using QuikChange^® II Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) following the manufacturer’s instructions, with primers whose sequences are shown in Supplementary Table S5. The sequences of all constructs were confirmed by direct sequencing.

The 5'-flanking region of the BlVg gene was obtained using the Genome Walking Kit (Takara, China) with bumblebee genomic DNA as the template. Different lengths of the upstream regulatory region were amplified by PCR (with primers containing NheI and HindIII restriction sites) and cloned into the pGL4.11 [luc2P] vector (Promega, USA) for sequencing. The recombinant vectors were designated pGL4-Vg2.4K Luc, pGL4-Vg2.3K Luc, pGL4-Vg1.9K Luc, pGL4-Vg1.4K Luc, pGL4-Vg1K Luc, and pGL4-Vg0.9K Luc. All the plasmids were verified by automated sequencing analysis.
To analyze the functionality of the putative BrC-Z3 element within the BlVg promoter, specific mutations were introduced into pGL4-Vg1.9K Luc using overlap PCR. The core site of BrC-Z3 CRE, namely, “AAAC”, was changed to “GGGT”. The mutagenesis primers are shown in Table 1.
The open reading frames (ORFs) of BlBrC-Z (1–4) were amplified with whole body cDNA from an adult queen and cloned into the pBmFlag stable expression vector (stored in our laboratory; detailed information available in reference [38 (link)] with XhoI and KpnI sites for gene overexpression. The primers for the BlVg promoter, and BlBrC-Z (1–4) are shown in Table 1.

The 500 bp upstream of the TSPYL2 gene start site were cloned in the pGL4.11[luc2P] vector (Promega) to drive the expression of the luciferase reporter gene luc2P (Photinus pyralis). This plasmid was then used to transfect U2OS cells together with pRL-TK (encoding Renilla luciferase, Promega) and FLAG-E2F1 (GenScript) plasmids. After 48 h, cells were exposed to 20 μM etoposide for 6 h or left untreated and luciferase activity was analyzed with the Dual-Luciferase Reporter Assay System (Promega) and normalized over Renilla luciferase activity according to manufacturer protocol. Experiments were repeated three times and the mean and standard deviation were reported in the chart.

Cultured B. mori embryonic cells (BmE) maintained at 27 °C in Grace’s insect medium containing 10% fetal bovine serum (HyClone, China) were used to examine the promoter activities of fibroin genes. In brief, the promoter sequences of the fibroin genes fibH (AF226688.1), fibL (AAA27840.1) and P25 (X04226.1), with lengths of 2096 bp, 1060 bp and 1227 bp, respectively, were commercially synthesized (GenScript, China) and inserted into the empty pGL4.11[Luc2P] vector (Promega, USA). The abbreviations for the final constructs were fibH-Luc2P, fibL-Luc2P, and P25-Luc2P, respectively. Then, a mixture of 3.5 μL of transfection reagents (Roche, USA) with 1.5 μg of the fibH-Luc2P, fibL-Luc2P, or P25-Luc2P plasmid DNA was transfected into BmE cells. After 36 h of culture at 27 °C, the cells were harvested, and luciferase activity was determined using a Dual Luciferase Reporter Gene Assay Kit (Yeasen, China). pGL4.11[Luc2P] was used as a negative control. The plasmids p57S[hrA4-Gal4] (A4G4) and pGL4.11[UAS-Luc2P] (UAS-Luc2P), which we constructed based on a previously modified Gal4/UAS binary system^{23 (link),24 (link)}, were used as positive controls.