Transfection of HPV11 genome DNA into human keratinocyte cell line HaCaT / 中华皮肤科杂志

ABSTRACT

Objective To explore if keratinocytes that stably maintain HPV11 genome can be obtained by transfection and selection methods. Methods Escherichia coil containing pBR322.HPV11 plasmid was cultured and amplified. Then the plasmid was extracted, purified and digested with BamH Ⅰ enzyme to release viral genome from the bacterial vector. After recovering from the low-melting point agarose gel by electrophoresis, the genome was self-circulated with T4 DNA ligase. The religated DNA was cotransfected with pTK-neo DNA into HaCaT keratinocytes using Lipofectamine reagent. After selection with G418 for 2 to 3 weeks, clonal and pooled cultures were expanded and analyzed. Fluorescent quantitative PCR (FQ-PCR) and nested reverse transcriptase PCR (nRT-PCR) were applied to detect HPV11 DNA and spliced HPV11 E1