Construction of recombinant lentiviral vectors and production of viruses

The rat-derived PEG3 promoter (−101 to +190 bp) was amplified by polymerase chain reaction (PCR) using rat genomic DNA as the template with the following primer pair: forward, 5’-TCCGGTGAATTCGCCACCATGACGACCGCGT-3’; and reverse, 5’-GCAGATCCTTACTAGTATCGATGGATC-3’. The human FTH1 gene (accession number {"type":"entrez-nucleotide","attrs":{"text":"BC000857","term_id":"12654092","term_text":"BC000857"}}BC000857) was amplified by PCR with the following primer pair: forward, 5’-AACCGTCAGATCGCACCGGTGCCACCATGACGACCGCGTCCACCTC-3’; and reverse, 5’-TCCTTGTAGTCCATGAATTCGCTTTCATTATCACTGTCTC-3’. The PEG3 promoter and FTH1 were inserted into the lentiviral vector pHBLV-CMV-MCS-3flag-EF1-ZSgreen-puro by using two ClaI sites (2180 and 2942) to generate the recombinant lentiviral vector pHBLV-PEG3-FTH1-3flag-EF1-ZSgreen-puro (LV-PEG3-FTH1); FTH1 alone was inserted into the lentiviral vector pHBLV-CMV-MCS-3flag-EF1-ZSgreen-puro by using a Xba and BamHI double-digestion system to generate pHBLV-CMV-FTH1-3flag-EF1-ZSgreen-puro (LV-CMV-FTH1) as the positive control. FTH1 gene and PEG3 promoter cDNA sequences were verified by PCR and DNA sequencing. Plasmids of each group were validated by agarose gel electrophoresis and DNA sequencing. Lentiviruses were generated by cotransfecting LV-CMV-FTH1 or LV-PEG3-FTH1 together with pSPAX2 and pMD2G into 293T packaging cells (Invitrogen, Carlsbad, CA, USA). Fresh medium containing 10% FBS was added 6 h after transfection, and then, the viral medium was collected at 48 and 72 h. Cell debris was removed by centrifugation at 4°C and 2000×g for 10 min. Viruses were enriched by centrifugation at 4°C and 82,700×g for 120 min and stored at −80°C.