POL Scientific / JBM / Volume 3 / Issue 2 / DOI: 10.14440/jbm.2016.110
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ARTICLE

A comparison of strategies for immortalizing mouse embryonic fibroblasts

Melissa M. St. Amand1,2 John A. Hanover2 Joseph Shiloach1
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1 Biotechnology Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 14-A Room 176, Bethesda, MD 20892, USA
2 Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 8 Room B127, Bethesda, MD 20892, USA
Published: 23 May 2016
© 2016 by the author. Licensee POL Scientific, USA. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
Abstract

The genetically amenable mouse model has led to a large collection of genetically defined lines from which mouse embryonic fibroblasts (MEFs) have been derived. Despite their widespread use, MEFs are time consuming to generate and have a limited lifespan. Immortalizing primary MEFs with the desired genetic manipulations greatly reduces culture maintenance time, enables the generation of near limitless amounts of protein lysate, and facilitates biological replicates during experimentation. In this work, we have evaluated several approaches for MEF immortalization. When cultivated at 3% O2, some primary MEF lines could be proliferated for > 40 passages with a median doubling rate of 45 ± 55 h (n = 8). However, serial passaging at 3% Oachieved spontaneous immortalization with varying success. If cultures seemed to be reaching their Hayflick limit when cultivated at 3% O2, supplementing the culture media with 5 µM ROCK inhibitor Y-27632 helped to extend proliferation and achieve spontaneous immortalization. MEFs immortalized via SV40 Ta infection reliably produced cell lines with a median doubling rate of 25 ± 9 h (n = 9) and viability greater than 90%. In addition to a discussion of the characteristics of cell lines generated with various immortalization strategies, pros and cons of each strategy are included as are recommendations for generating immortalized MEFs.

Keywords
mouse embryonic fibroblast
immortalization
ROCK inhibitor
SV40 Ta
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Journal of Biological Methods, Electronic ISSN: 2326-9901 Print ISSN: TBA, Published by POL Scientific