In vitro translation of papillomavirus authentic and codon-modified L1 capsid gene mRNAs in mouse keratinocyte cell-free lysate

Zhao, Kong-Nan (2014). In vitro translation of papillomavirus authentic and codon-modified L1 capsid gene mRNAs in mouse keratinocyte cell-free lysate. In Kirill Alexandrov and Wayne A. Johnston (Ed.), Cell-free protein synthesis: methods and protocols (pp. 205-218) New York, NY, United States: Humana Press. doi:10.1007/978-1-62703-782-2_13


Author Zhao, Kong-Nan
Title of chapter In vitro translation of papillomavirus authentic and codon-modified L1 capsid gene mRNAs in mouse keratinocyte cell-free lysate
Title of book Cell-free protein synthesis: methods and protocols
Place of Publication New York, NY, United States
Publisher Humana Press
Publication Year 2014
Sub-type Research book chapter (original research)
DOI 10.1007/978-1-62703-782-2_13
Series Methods in Molecular Biology
ISBN 9781627037815
9781627037822
ISSN 1064-3745
1940-6029
Editor Kirill Alexandrov
Wayne A. Johnston
Volume number 1118
Chapter number 13
Start page 205
End page 218
Total pages 14
Total chapters 21
Language eng
Abstract/Summary 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.
Keyword Primary mouse keratinocytes
Cell-free lysate
Codon usage
Aminoacyl-tRNAs
Papillomavirus L1 gene mRNAs
In vitro translation
Q-Index Code BX
Q-Index Status Confirmed Code
Institutional Status UQ

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