Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents

Pamela Hawley‐Nelson1, Valentina Ciccarone1

1 Life Technologies, Inc., Rockville, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 20.6
DOI:  10.1002/0471143030.cb2006s19
Online Posting Date:  August, 2003
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Abstract

The development of high‐efficiency methods for the introduction of functional genetic material into eukaryotic cells using cationic lipids has accelerated biological research in the studies of gene expression, control of cell growth, and cell lineage. Transfection mediated by cationic lipids is commonly used in industrial protein production as well as in some clinical gene therapy protocols. Replacing our previous unit on this topic, this new version describes how to perform transfection of adherent and suspension cells, insect cells, and RNA transfection using the cationic lipid system.

     
 
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Table of Contents

  • Basic Protocol 1: Cationic Lipid–Mediated Transfection of Adherent Mammalian Cells with DNA
  • Alternate Protocol 1: Enhanced Cationic Lipid–Mediated Transfection of Adherent Mammalian Cells with DNA
  • Basic Protocol 2: Cationic Lipid–Mediated Transfection of Suspension Cells with DNA
  • Basic Protocol 3: Cationic Lipid–Mediated Transfection of Adherent Cells with RNA
  • Basic Protocol 4: Cationic Lipid–Mediated Transfection of Adherent Sf9 and Sf21 Insect Cells with Baculovirus DNA
  • Support Protocol 1: Fine Tuning or Optimizing Conditions for Cationic Lipid Reagent Transfections
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Cationic Lipid–Mediated Transfection of Adherent Mammalian Cells with DNA

  Materials
  • Adherent cells
  • Cell culture medium with serum (e.g., complete DMEM, unit 1.2)
  • Dilution medium: serum‐free cell culture medium or specialized medium for lipid‐mediated transfection (e.g., Opti‐MEM I, Life Technologies)
  • Plasmid DNA, purified by anion‐exchange chromatography (e.g., Concert High Purity columns, Life Technologies; or see Goldsborough et al., ), cesium chloride density gradient, or alkaline lysis (unit 1.6)
  • Cationic lipid reagent (see Table 20.6.1)
  • Polystyrene or polypropylene tubes
  • Additional reagents and equipment for trypsinization and counting of cells (unit 1.1), Xgal staining, and selection of stable transformants ( appendix 3A)

Alternate Protocol 1: Enhanced Cationic Lipid–Mediated Transfection of Adherent Mammalian Cells with DNA

  • Cationic lipid reagent and enhancer (e.g., LipofectAmine Plus, including LipofectAmine and Plus reagents; Life Technologies)

Basic Protocol 2: Cationic Lipid–Mediated Transfection of Suspension Cells with DNA

  Materials
  • Dilution medium: cell culture medium without serum or specialized medium for transfection (e.g., Opti‐MEM I, Life Technologies)
  • Cationic liposome reagent (e.g., DMRIE‐C or LipofectAmine 2000, Life Technologies; also see Table 20.6.1)
  • Plasmid DNA, purified by anion‐exchange chromatography or Goldsborough et al., ), cesium chloride density gradient, or alkaline lysis
  • Cell suspension: 1 × 107 cells/ml in normal cell culture medium without serum or antibiotics
  • Cell culture medium (e.g., complete DMEM; unit 1.2)
  • Serum
  • 6‐well tissue culture plates

Basic Protocol 3: Cationic Lipid–Mediated Transfection of Adherent Cells with RNA

  Materials
  • Adherent cells
  • Cell culture medium with serum (e.g., complete DMEM; unit 1.2)
  • Dilution medium: serum‐free cell culture medium or specialized medium for transfection (e.g., Opti‐MEM I, Life Technologies)
  • Cationic lipid reagent (e.g., DMRIE‐C, Life Technologies; also see Table 20.6.1)
  • mRNA (see appendix 3A)
  • 6‐well or 35‐mm tissue culture plate
  • 12 × 75–mm polystyrene tubes
  • Additional reagents and equipment for trypsinizing, counting, and plating cells (unit 1.1)

Basic Protocol 4: Cationic Lipid–Mediated Transfection of Adherent Sf9 and Sf21 Insect Cells with Baculovirus DNA

  Materials
  • Insect cells: Sf9 or Sf21 cells
  • Insect medium (e.g., Sf‐900 II SFM, Life Technologies) with and without serum and antibiotics
  • Baculovirus DNA: purified DNA or bacmid DNA miniprep (Anderson et al., )
  • Cationic lipid reagent (Table 20.6.1)
  • 6‐well tissue culture plate
  • 27°C incubator
  • 12 × 75–mm polystyrene tubes, sterile
  • Additional reagents and equipment for culturing insect cells and harvesting baculovirus from cell supernatants ( appendix 3A)

Support Protocol 1: Fine Tuning or Optimizing Conditions for Cationic Lipid Reagent Transfections

  • 24‐well tissue culture plates
  • 96‐well round‐bottom plates (sterile, with lid)
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Figures

Videos

Literature Cited

   Anderson, D., Harris, R., Polayes, D., Ciccarone, V., Donahue, R., Gerard, G., Jessee, J., and Luckow, V. 1995. Rapid generation of recombinant baculovirus and expression of foreign genes using the Bac‐to‐Bac baculovirus expression system. Focus 17.2:53‐58.
   Behr, J.‐P., Demeneix, B., Loeffler, J.‐P., and Perez‐Nutul, J. 1989. Efficient gene transfer into mammalian primary endocrine cells with lipopolyamine‐coated DNA. Proc. Natl. Acad. Sci. U.S.A. 86:6982‐6986.
   Bennett, C.F., Chiang, M.‐Y., Chan, H., Shoemaker, J.E.E., and Mirabelli, K. 1992. Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides. Mol. Pharmacol. 41:1023.
   Brunette, E., Stribling, R., and Debs, R. 1992. Lipofection does not require the removal of serum. Nucl. Acids Res. 20:1151.
   Chiang, M.‐Y., Chan, H., Zounes, M.A., Freier, S.M., Lima, W.F., and Bennett, C.F. 1991. Antisense oligonucleotides inhibit intercellular adhesion molecule 1 expression by two distinct mechanisms. J. Biol. Chem. 266:18162‐18171.
   Ciccarone, C. and Hawley‐Nelson, P. 1995. Lipofectin transfection activity increased by protocol improvement. Focus 17:103.
   Ciccarone, V., Hawley‐Nelson, P., and Jessee, J. 1993. Cationic liposome‐mediated transfection: Effect of serum on expression and efficiency. Focus 15:80‐83.
   Ciccarone, V., Anderson, D., Lan, J., Schifferli, K., and Jessee, J. 1995. DMRIE‐C reagent for transfection of suspension cells and for RNA transfections. Focus 17.3:84‐87.
   Ciccarone, V., Chu, Y., Schifferli, K., Pichet., J.‐P., Hawley‐Nelson, P., Evans, K., Roy, L., and Bennett, S. 1999. LipofectAmine 2000 Reagent for rapid, efficient transfection of eukaryotic cells. Focus 21.2:54‐55.
   Dube, S. 1997. Transfection using LipofectAmine Plus Reagent. Focus 19.3:57.
   Felgner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P., Ringold, G.M., and Danielsen, M. 1987. Lipofection: A highly efficient, lipid‐mediated DNA‐transfection procedure. Proc. Natl. Acad. Sci. U.S.A. 84:7413‐7417.
   Goldsborough, M.D., Evans, K., Xu, L., and Young, A. 1998. High purity plasmid DNA from anion exchange chromatography. Focus 20:68‐69.
   Hawley‐Nelson, P. and Shih, P.‐J. 1995. Sensitivity of transfection efficiency to culture age. Focus 17:62.
   Hawley‐Nelson, P. and Ciccarone, V. 1996. Transient transfection efficiency of human keratinocytes in two serum‐free media. Focus 18.2:43‐44.
   Hawley‐Nelson, P., Ciccarone, V., Gebeyehu, G., Jessee, J. and Felgner, P. 1993. LipofectAmine reagent: A new, higher efficiency polycationic liposome transfection reagent. Focus 15.3:73‐79.
   Lamb, B.T., Sisodia, W.W., Lawler, A.M., Slunt, H.H., Kitt, C.A., Kearns, W.G., Pearson, P.L., Price, D.L., and Gearhart, J.D. 1993. Introduction of the 400 kilobase precursor amyloid protein gene in transgenic mice. Nature Genet. 5:22‐30.
   Life Technologies. 1999. Guide to eukaryotic transfections with cationic lipid reagents, 2nd ed. Life Technologies, Inc., Rockville, Md.
   Rose, J.K., Buonocore, L., and Whitt, M. 1991. A new cationic liposome reagent mediating nearly quantitative transfection of animal cells. BioTechniques 10:520‐525.
   Schifferli, K.P. and Ciccarone, V. 1996. Optimization of cationic lipid reagent‐mediated transfection for suspension cell lines. Focus 18:45‐47.
   Sells, M.A., Li., J., and Chernoff, J. 1995. Delivery of proteins into cells using cationic liposomes. BioTechniques 19:72‐78.
   Shih, P.J., Evans, K., Schifferli, K., Ciccarone, V., Lichaa, F., Masoud, M., Lan, J., and Hawley‐Nelson, P. 1997. High efficiency transfection with minimal optimization using the LipofectAmine Plus Reagent. Focus 19.3:52‐56.
   Tilkins, M.L., Hawley‐Nelson, P., and Battista, P. 1994. Transient transfection of endothelial cells. Focus 16.4:117‐119.
   Wagner, R.W., Matteucci, M.D., Lewis, J.G., Gutierrez, A.J., Moulds, C., and Froehler, B.C. 1993. Antisense gene inhibition by oligonucleotides containing C‐5 propyne pyrimidines. Science 260:1510.
   Yeoman, L.C., Danels, Y.J., and Lynch, M.J. 1992. Lipofec'tin enhances cellular uptake of antisense DNA while inhibiting tumor cell growth. Antisense Res. Dev. 2:51.
Key References
   Kriegler, M. 1990. Gene Transfer and Expression: A Laboratory Manual. Stockton Press, New York.
  Good general review on gene expression.
   Life Technologies, 1999. See above.
  Describes history and gives protocols and considerations for cationic lipid reagent transfections.
   Tilkins, M.L., Hawley‐Nelson, P., and Ciccarone, V. 1998. Transfection of mammalian and invertebrate cells using cationic lipids. In Cell Biology: A Laboratory Handbook, Vol. 4, 2nd ed. (J.E. Celis, ed.) pp. 145‐154. Academic Press, New York.
  Review of cationic lipid transfection procedures.
   Felgner et al., 1987. See above.
  The original description of cationic lipid transfection.
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