Preparation, Culture, and Immortalization of Mouse Embryonic Fibroblasts

Jianming Xu1

1 Baylor College of Medicine, Houston, Texas
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 28.1
DOI:  10.1002/0471142727.mb2801s70
Online Posting Date:  May, 2005
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Abstract

Genetically manipulated transgenic and gene‐targeted mouse models are indispensible tools used in defining the role of genes in development and organ function. Similarly, cells isolated from these mice bear the same genetic alterations and therefore can be extremely useful for studying the molecular and cellular mechanisms and regulatory gene networks involving the mutated gene under well defined culture conditions. In this unit, detailed protocols regarding isolation of mouse embryonic fibroblasts (MEFs) from mouse embryos, culture of MEFs, and immortalization of MEFs are described. These protocols should be particularly useful to those investigators who intend to establish primarily cultured MEFs and to immortalize primary MEFs into stable cell cultures with a permanent growth feature from wild‐type and mutant mouse embryos.

Keywords: mouse embryonic fibroblasts; MEFs; transgenic mouse models; gene‐targeted mouse models; development

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of Mouse Embryo Fibroblasts from Mouse Embryos
  • Basic Protocol 2: Culture and Use of Primary Mouse Embryo Fibroblasts
  • Basic Protocol 3: Immortalization of Mouse Embryo Fibroblasts by Serial Passages
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of Mouse Embryo Fibroblasts from Mouse Embryos

  Materials
  • 2 to 3 male and 4 to 6 female mice (8 to 18 weeks of age)
  • 2.5% Avertin (see recipe)
  • 70% ethanol
  • Phosphate‐buffered saline, pH 7.2 (PBS; appendix 22), sterilized by autoclaving
  • 0.25% trypsin‐EDTA solution (Invitrogen, cat. no. 25300‐056)
  • MEF culture medium (see recipe)
  • Mouse ear tags (National Band and Tag)
  • Cages (11 in. wide × 7 in. long × 7 in. tall) with food and water dishes
  • Appropriate mouse food
  • Dissecting instruments, autoclaved:
    • Regular and fine dissection scissors
    • Two pairs autoclaved watchmaker's no. 5 forceps
    • Blunt fine forceps
  • Powder‐free latex gloves
  • 15‐ and 50‐ml sterile screw‐cap plastic conical tubes
  • 10‐cm tissue culture dishes or 75‐cm2 tissue culture flasks

Basic Protocol 2: Culture and Use of Primary Mouse Embryo Fibroblasts

  Materials
  • MEFs prepared from mouse embryos (see protocol 1)
  • Phosphate‐buffered saline, pH 7.2 (PBS; appendix 22), sterilized by autoclaving
  • MEF culture medium (see recipe)
  • 0.05% trypsin‐EDTA solution (Invitrogen, cat. no. 25300‐054)
  • MEF freezing medium (see recipe)
  • Inverted phase‐contrast microscope (suitable for examining the cell culture)
  • 0.75‐ or 1‐ml cryovials
  • Centrifuge and rotor with adaptors for 15‐ and 50‐ml plastic centrifuge tubes
  • Liquid nitrogen tank with canes for holding cryovials
  • 10‐cm tissue culture dishes or 75‐cm2 tissue culture flasks
  • Additional reagents and equipment for mammalian cell culture and trypsinization of cells ( appendix 3F)

Basic Protocol 3: Immortalization of Mouse Embryo Fibroblasts by Serial Passages

  Materials
  • MEFs growing in culture (see protocol 2)
  • MEF culture medium (see recipe)
  • Phosphate‐buffered saline, pH 7.2 (PBS; appendix 22), sterilized by autoclaving
  • 25‐cm2 culture flasks
  • Additional reagents and equipment for mammalian cell culture, trypsinization of cells, and counting cells ( appendix 3F), and expansion and cryopreservation of cultured MEFs (see protocol 2)
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Figures

  •   FigureFigure 28.1.1 A representative growth curve of MEFs during serial passages. Primary MEFs were isolated from E14.5 mouse embryos with a mixed C57BL/6 and 129SvEv strain background. MEFs were cultured and transferred every 3 days as described in the protocol. The fold of cell growth was calculated by dividing the number of harvested MEFs with the number of inoculated MEFs in each passage. Data represent the average of two samples.

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Literature Cited

Literature Cited
   Aaronson, S.A. and Todaro, G.J. 1968. Development of 3T3‐like lines from Balb‐c mouse embryo cultures: Transformation susceptibility to SV40. J. Cell. Physiol. 72:141‐148.
   Abbondanzo, S.J., Gadi, I., and Stewart, C.L. 1993. Derivation of embryonic stem cell lines. Methods Enzymol. 225:803‐855.
   Hogan, B., Beddington, R., Costantini, F., and Lacy, E. 1994. Manipulating the Mouse Embryo: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Kuang, S.Q., Liao, L., Zhang, H., Pereira, F.A., Yuan, Y., DeMayo, F.J., Ko, L., and Xu, J. 2002. Deletion of the cancer‐amplified coactivator AIB3 results in defective placentation and embryonic lethality. J. Biol. Chem. 277:45356‐45360.
   Puigserver, P., Adelmant, G., Wu, Z., Fan, M., Xu, J., O'Malley, B. W., and Spiegelman, B.M. 1999. Activation of PPARgamma coactivator‐1 through transcription factor docking. Science 286:1368‐1371.
   Todaro, G.J., and Green, H. 1963. Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J. Cell Biol. 17:299‐313.
   Todaro, G.J., and Green, H. 1966. Cell growth and the initiation of transformation by SV40. Proc. Natl. Acad. Sci. U.S.A. 55:302‐308.
   Wu, R. C., Qin, J., Yi, P., Wong, J., Tsai, S.Y., Tsai, M.J., O'Malley, B.W. 2004. Selective phosphorylations of the SRC‐3/AIB1 coactivator integrate genomic reponses to multiple cellular signaling pathways. Mol. Cell 15:937‐949.
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