Transfection of HPV11 genome DNA into human keratinocyte cell line HaCaT

• DOI: 10.3760/cma.j.issn.0412-4030.2009.02.006
• Publication Date: 2009-02-15
• Authors: Jianbo Wu; Xinyu Li; Jiarun Zheng
• Keywords: HaCaT; Lipofectamine; Agarose gel electrophoresis

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 amplified. Then the extracted, purified digested with BamH Ⅰ enzyme to release viral from bacterial vector. After recovering low-melting point agarose gel electrophoresis, self-circulated T4 DNA ligase. The religated cotransfected pTK-neo into HaCaT using Lipofectamine reagent. G418 for 2 3 weeks, clonal pooled cultures were expanded analyzed. Fluorescent quantitative PCR (FQ-PCR) nested reverse transcriptase (nRT-PCR) applied detect spliced E1^E4 mRNA expression in transfected cells. Results cotransfection of two-week selection,G418-resistant cell colonies morphological features indistinguishable normal keratinocytes. As shown FQ-PCR, present G418-selected average load capacity 15.9±16.8 copies/cell primary culture cells 23.9±1.1 third passage cells; there no statistical difference between two passages (t=-0.822, P>0.05). nRT-PCR targeting transcript produced a specific 628-bp fragment, which electrophoresis. Conclusions Our data indicate successfully introduced DNA-positive selection. Moreover, is still cells.

Key words: Human papillomavirus 11; Genes; Transfection