4.2. Cloning of IL1RN into the pBabe-Puro Plasmid Vector and Ectopic Expression in T24 Cells

A PCR product encoding the IL1RN gene was amplified from RT4 cDNA using the Phusion High-Fidelity PCR Kit (ThermoFischer Scientific, Waltham, MA, USA). The cDNA was generated by reverse transcription (RT) using 1 μg total RNA from RT4 cell cells with the GoScript^TM Reverse Transcription System (Promega, Fitchburg, WI, USA) in a 20 µL reaction mixture. The PCR reaction was conducted with 5 µL of the RT reaction and the cloning primers (forward primer: 5′-CGATGCGGATCCGAGGCCCTCCCCATGGCTTTAG-3′ and reverse primer 5′-CGATGCGTCGACGGCAGTACTACTCGTCCTCC-3′), which included BamHI and SalI restriction enzyme recognition sites, respectively. Cycling conditions: initial denaturation at 98 °C for 30 s, 25 cycles of 98 °C for 10 s, 55 °C for 30 s, followed by a final extension at 72 °C for 10 min. The PCR product was run on an agarose gel, followed by gel purification with the QIAquick^® Gel Extraction Kit (QIAGEN, Hilden, Germany). Both the expression vector pBabe-Puro and the purified PCR product were digested with the restriction enzymes BamHI-HF^® and SalI-HF^® (New England Biolabs, Ipswich, MA, USA) and ligated using Quick Ligase (New England Biolabs, Ipswich, MA, USA). The ligation mix was transformed into Stbl 3 chemically competent Escherichia coli (ThermoFischer Scientific, Waltham, MA, USA) and the plasmid DNA was isolated from a single bacterial colony with the PureYield™ Plasmid Miniprep System (Promega, Fitchburg, MA, USA). The cloning of IL1RN was validated by Sanger sequencing using pBABE-5′ or pBABE-3′ sequencing primers (Eurofins, Ebersberg, Germany). Plasmid DNA was amplified with the NucleoBond^® Xtra Midi/Maxi Kit (Macherey-Nagel, Düren, Germany). For virus production, HEK293 cells were cotransfected with the IL1RN expression vector, the pCMV-Gag-Pol and pCMV-VSV-G plasmids. The virus was harvested after 48 h, filtered twice through a 0.45 µm filter (Sarstedt, Nürnbrecht, Germany), and used to infect T24 cells. The infected target cells were selected with 1 μg/mL Puromycin (ThermoFischer Scientific, Waltham, MA, USA) for 7 days and permanent selection was continued with 0.5 μg/mL Puromycin.