Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents

Pamela Hawley‐Nelson1, Valentina Ciccarone2, Meredith L. Moore3

1 Centocor, Inc., Radnor, Pennsylvania, 2 MacroGenics, Inc., Rockville, Maryland, 3 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 9.4
DOI:  10.1002/0471142727.mb0904s81
Online Posting Date:  January, 2008
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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. This unit describes how to perform transfection of adherent and suspension cells, insect cells, and RNA transfection using cationic lipid reagents. Curr. Protoc. Mol. Biol. 81:9.4.1‐9.4.17. © 2008 by John Wiley & Sons, Inc.

Keywords: transfection; luciferase; cationic lipid; cell culture; transgene; transient gene expression; stable gene expression

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: 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 Cells with Single‐Stranded RNA or siRNA
  • 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
  • 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 (e.g., complete DMEM, appendix 3F), with serum but no antibiotics
  • Plasmid DNA, purified by anion‐exchange chromatography (e.g., Qiagen Plasmid Purification Kit or unit 2.1), cesium chloride density gradient (unit 1.7), or alkaline lysis (unit 1.6)
  • Dilution medium: specialized medium for lipid‐mediated transfection (e.g., Opti‐MEM I, Invitrogen) or antibiotic‐free, serum‐free cell culture medium
  • Cationic lipid reagent (see Table 9.4.1)
  • Transfection medium: specialized medium for lipid‐mediated transfection (e.g., Opti‐MEM I, Invitrogen) or antibiotic‐free cell culture medium
  • Polystyrene or polypropylene tubes
  • Additional reagents and equipment for trypsinization and counting of cells ( appendix 3F), Xgal staining (unit 9.11), and selection of stable transformants (unit 9.5)

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

  Materials
  • Cationic liposome reagent (e.g., DMRIE‐C or Lipofectamine 2000, Invitrogen; see Table 9.4.1)
  • Dilution medium: specialized medium for lipid‐mediated transfection (e.g., Opti‐MEM I, Invitrogen) or cell culture medium (e.g., complete DMEM; appendix 3F) without serum or antibiotics
  • Plasmid DNA, purified by anion‐exchange chromatography (e.g., Qiagen Plasmid Purification Kit or unit 2.1), cesium chloride density gradient (unit 1.7), or alkaline lysis (unit 1.6)
  • Cell suspension: 1 × 106 to 1 × 107 cells/ml in cell culture medium without antibiotics
  • Tubes, cell culture plates, or flasks

Basic Protocol 3: Cationic Lipid–Mediated Transfection of Cells with Single‐Stranded RNA or siRNA

  Materials
  • Cells
  • Cell culture medium (e.g., complete DMEM; appendix 3F), with serum but no antibiotics
  • Dilution medium: specialized medium for lipid‐mediated transfection (e.g., Opti‐MEM I, Invitrogen) or cell culture medium without serum or antibiotics
  • Cationic lipid reagent (e.g., Lipofectamine 2000 or DMRIE‐C, Invitrogen; see Table 9.4.1)
  • mRNA (unit 4.5) or siRNA (unit 26.2)
  • 6‐well or 35‐mm tissue culture plate
  • 12 × 75–mm polystyrene tubes
  • Additional reagents and equipment for trypsinizing, counting, and plating cells ( appendix 3F)

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

  Materials
  • Insect cells: Sf9 or Sf21 cells (unit 16.9)
  • Insect medium (unit 16.9; e.g., Sf‐900 II SFM, Invitrogen) with and without serum and antibiotics
  • Baculovirus DNA: purified DNA or bacmid DNA miniprep (units 16.9 & 16.10; Anderson et al., 9.4)
  • Cationic lipid reagent (e.g., CellFectin, Invitrogen; Table 9.4.1)
  • 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 (unit 16.10)

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

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

Videos

Literature Cited

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.
   Caplen, N.J., Alton, E.W.F.W., Middleton, P.G., Dorin, J.R., Stevenson, B.J., Gao, X., Durham, S.R., Jeffery, P.K., Hodson, M., Coutelle, C., Huang, L., Porteous, D.J., Williamson, R., and Geddes, D.M. 1995. Liposome‐mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis.. Nat. Med. 1:39‐46.
   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.
   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.
   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.
   Nabel, G.J., Nabel, E.G., Zhi‐Yong, Y., Fox, B.A., Plautz, G.E., Gao, X., Huang, L., Shu, S., and Gordon, D., 1993. Direct gene transfer with DNA‐liposome complexes in melanoma: Expression, biologic activity and lack of toxicity in humans.. Proc. Natl. Acad. Sci. U.S.A. 90:11307‐11311.
   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.
   Unger, T.F. 1997. Transfection connection: Methods of nucleic acid delivery into eukaryotic cells. The Scientist 11:18.
   Yeoman, L.C., Danels, Y.J., and Lynch, M.J. 1992. Lipofectin enhances cellular uptake of antisense DNA while inhibiting tumor cell growth. Antisense Res. Dev. 2:51.
   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.
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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