title: In vitro translation of papillomavirus authentic and codon-modified L1 capsid gene mRNAs in mouse keratinocyte cell-free lysate.
abstract: Keratinocytes are the major cell type in the epidermis responsible for constructing the protective barrier of mammalian skin. Primary keratinocytes cultured in vitro can mimic the in vivo differentiation process, providing an abundant source of pure keratinocytes used for investigating the regulatory mechanisms of gene expression associated with cell proliferation and differentiation. We developed a primary mouse keratinocyte cell-free lysate system for translation of papillomavirus authentic and codon-modified L1 capsid gene mRNAs in vitro. We demonstrated that the viral gene codon usage matched available aminoacyl-tRNAs to determine their translational efficiencies, which were associated with differentiation status of the keratinocytes used for preparing cell-free lysate. We revealed that a novel regulatory mechanism of gene expression is utilized by papillomavirus to direct viral capsid protein expression to the site of virion assembly in mature keratinocytes. Here, I describe the methods in details of how to establish primary mouse keratinocyte culture, to prepare cell-free lysate, to carry out in vitro translation of the viral gene mRNAs, and to detect the translated products using Western blotting analysis.
authors.affiliation: UQ Centre for Clinical Research, The University of Queensland, Herston, Brisbane, QLD, Australia.
authors.forename: Kong-Nan
authors.initials: KN
authors.lastname: Zhao
mesh_terms: D000818:Animals; D015153:Blotting, Western; D036022:Capsid Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics*; D018929:Cell Culture Techniques; D059447:Cell Cycle Checkpoints; D016923:Cell Death; D002457:Cell Extracts; D002469:Cell Separation; D002474:Cell-Free System; D002478:Cells, Cultured; D003062:Codon / Q000235:genetics*; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D027383:Papillomaviridae / Q000235:genetics*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D036022:Capsid Proteins; D002457:Cell Extracts; D003062:Codon; D012333:RNA, Messenger; D012343:RNA, Transfer