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. 2006 May 9;94(11):1663–1671. doi: 10.1038/sj.bjc.6603148

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Copyright © 2006 Cancer Research UK

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Generation of stable dermcidin transfectants. (A) Dermcidin PCR. Lane 1 – Untransfected HuH7 cDNA. Lane 2 – Empty vector transfected HuH7 cDNA. Lane 3 – pcDNA3.1+PIF transfected HuH7 cDNA. Lane 4 – Blank. Lane 5 – pcDNA3.1+PIF plasmid. Cells were cultured under standard conditions and stably transfected using Fugene with G418 selection. RNA was extracted using Trizol, DNAse treated, reverse transcribed and amplified by PCR using primers to the coding region of the PIF cDNA. RNA from the HuH7 cell line was positive for PIF message only after transfection with pcDNA3.1+PIF. (B) Dermcidin PCR. Lane 1 – Empty vector transfected HuH7 cDNA. Lane 2 – pcDNA3.1+PIF transfected HuH7 cDNA. Lane 3 – pcDNA3.1+N32Q transfected HuH7 cDNA. Lane 4 – pcDNA3.1+N44Q transfected HuH7 cDNA. Lane 5 – pcDNA3.1+N32QN44Q transfected HuH7 cDNA. Lane 6 – untransfected HuH7 cDNA. Lane 7 – Positive control (CF-PAC cDNA). Lane 8 – Negative control (no cDNA). All transfected cells were strongly positive for dermcidin expression. Untransfected cells, which had been maintained in culture for the same period of time, became weakly positive for expression. (C) Immunoblot for neomycin phosphotransferase from transfected cells. Lane 1 – Empty vector transfected HuH7 lysate. Lane 2 – pcDNA3.1+PIF transfected HuH7 lysate. Lane 3 – pcDNA3.1+N32Q transfected HuH7 lysate. Lane 4 – pcDNA3.1+N44Q transfected HuH7 lysate. Lane 5 – pcDNA3.1+N32QN44Q transfected HuH7 lysate. Lane 6 – untransfected HuH7 lysate. Membranes were probed with a monoclonal antibody to neomycin phosphotransferase. All transfected cells demonstrated expression from the pcDNA3.1+ CMV-driven promoter. No expression was observed in untransfected cells.