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 Neuroscience
Unit Number:  Appendix 1F
DOI:  10.1002/0471142301.nsa01fs10
Online Posting Date:  May, 2001
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Abstract

The development of high-efficiency methods for the introduction of functional genetic material into eukaryotic cells using cationic lipid-based transfection reagents has accelerated biology research in studies of gene expression, control of cell growth, and cell lineage. In this unit, DNA transfection is described for adherent mammalian cells (both cells and primary cultures) along with an alternate procedure for enhanced transfection. A protocol is also described for transfection of suspension cells (lymphoid, myeloid and leukemic-derived cells). In addition, transfection of RNA into adherent mammalian cells and DNA transfection into insect cells are presented. Importantly, a detailed procedure is described for optimization of reagents and transfection conditions.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Cationic Lipid–Mediated Transfection of Adherent Mammalian Cells with DNA
  • Alternate Protocol: Enhanced Cationic Lipid–Mediated Transfection of Adherent Mammalian Cells with DNA
  • Basic Protocol 2: Cationic Lipid-Mediated Transfection of the Adherent, RAT, Pheochromocytoma Cell Line, PC12
  • Basic Protocol 3: Cationic Lipid-Mediated Transfection of the Adherent, Human, Neuroblastoma Cell Line, BE(2)C
  • Basic Protocol 4: Cationic Lipid–Mediated Transfection of Adherent Sf9 and Sf21 Insect Cells with Baculovirus DNA
  • Support Protocol: 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, appendix 2A)
  • Dilution medium: cell culture medium without serum 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 cpmb unit 2.1B or Goldsborough et al., 1998), cesium chloride density gradient, or alkaline lysis (see cpmb units 1.6 & 1.7 and appendix 1A in this manual)
  • Cationic lipid reagent (see Table A.1F.1)
  • Polystyrene or polypropylene tubes
  • Additional reagents and equipment for trypsinization and counting of cells (appendix 3B), Xgal staining (unit 3.16), and selection of stable transformants (see cpmb unit 9.5 and appendix 1A in this manual).

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

 Additional Materials (also see Basic Protocol 1)
  • Cationic lipid reagent and enhancer (e.g., LipofectAMINE PLUS, including LipofectAMINE and PLUS reagents; Life Technologies)

Basic Protocol 2: Cationic Lipid-Mediated Transfection of the Adherent, RAT, Pheochromocytoma Cell Line, PC12

 Materials
  • Rat pheochromocytoma cell line: PC12 (ATCC #CRL 1721) cultured in Dulbecco's modified Eagle medium (DMEM, high glucose [4500 mg/liter d-glucose], with l-glutamine, and phenol red) supplemented with 0.1 mM non-essential amino acids (NEAA), 10% heat-inactivated horse serum and 5% fetal bovine serum (FBS)
  • Cationic lipid reagent (e.g., LipofectAMINE 2000 Reagent, trademark of Life Technologies; also see Table A.1F.1)
  • Poly-lysine– (Sigma) or poly-Dlysine– (Becton Dickinson) pre-coated 24-well plates
  • Additional reagents and equipment for trypsinization and counting of cells (appendix 3B).

Basic Protocol 3: Cationic Lipid-Mediated Transfection of the Adherent, Human, Neuroblastoma Cell Line, BE(2)C

 Materials
  • Human neuroblastoma BE(2)C cells (ATCC #CRL 2268) cultured in 1:1 mixture of DMEM/F12 supplemented with 0.1 mM non-essential amino acids and 10% FBS
  • 1:1 mixture of supplemented DMEM/F12 culture medium with and without 20% FBS
  • Cationic lipid reagent (e.g., LipofectAMINE 2000 Reagent, Life Technologies; also see Table A.1F.1)
  • Poly-lysine– (Sigma) or poly-d-lysine– (Becton Dickinson) pre-coated 24-well plates
  • Additional reagents and equipment for trypsinization and counting of cells (appendix 3B).

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 4.18)
  • Insect medium (cpmb unit 16.9; e.g., Sf-900 II SFM, Life Technologies) with and without serum and antibiotics
  • Baculovirus DNA: purified DNA or bacmid DNA miniprep (cpmb units 16.9 & 16.10 and appendix 1A in this manual; Anderson et al., 1995)
  • Cationic lipid reagent (Table A.1F.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 (see cpmb unit 16.10 and appendix 1A in this manual)

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

 Additional Materials (also see Basic Protocol 1 and Alternate Protocol)
  • 24-well tissue culture plates
  • 96-well round-bottom plates (sterile, with lid)
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Figures

  •  FigureFigure A.1F.1 Diagram of cationic lipid–mediated transfection procedure. Steps and can be performed by two methods that differ in whether or not complexes are added to dry cells; see protocol steps for details.
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  •  FigureFigure A.1F.2 Diagram of enhanced cationic lipid–mediated transfection procedure.
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  •  FigureFigure A.1F.3 A sample matrix for fine tuning (optimizing) transfection reagent efficiencies using cationic lipid reagents.
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  •  FigureFigure A.1F.4 Results of fine tuning or optimizing conditions for transfection. Before transfection, 293 H cells were plated at 2 × 105/well in a 24-well plate precoated with poly-D-lysine. The following day, cells were transfected with pCMV×SPORTgal DNA using LipofectAMINE 2000 as described (see Support Protocol). One day post transfection, cells were fixed and stained with Xgal. (A) Amounts of DNA and lipid reagent used. (B) Results of Xgal staining.
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  •  FigureFigure A.1F.5 Stable transfection of NIH 3T3 cells. Cells were plated at 6 × 104 cells/well in 24-well plates. The day after plating, cells were transfected with LipofectAMINE PLUS complexed with pSV2neo DNA. The following day, cells were passaged at a total dilution of 1/150. Cells were exposed to 0.6 mg/ml geneticin antibiotic from day 3 to day 13, and were then washed once with PBS and stained with 0.2% toluidine blue in PBS with 10% formalin.
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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.
    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.
    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. Focus18.2:43-44.
    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.
    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
    Felgner et al., 1987. See above.

The original description of cationic lipid transfection.

    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.

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