Liposome‐Mediated Transfection

Michael Whitt1, Linda Buonocore2, John K. Rose2

1 University of Tennessee, Memphis, Tennessee, 2 Yale University School of Medicine, New Haven, Connecticut
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.16
DOI:  10.1002/0471142735.im1016s03
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Using liposomes to deliver DNA into different eukaryotic cell types results in higher efficiency and greater reproducibility than other transfection methods. In this unit, the describes a transient expression system while an involves stable transformation and expression of DNA integrated into the genome of the transfected cell. In both protocols, plasmid DNA derived from either crude (miniprep) or purified (through CsCl) preparations is mixed with a liposome suspension comprised of cationic lipids and applied to monolayer cell cultures.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Transient Expression Using Liposomes
  • Alternate Protocol 1: Stable Transformation Using Liposomes
  • Commentary
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Transient Expression Using Liposomes

  Materials
  • Exponentially growing eukaryotic cells (Table 10.16.1)
  • Plasmid DNA ( miniprep or CsCl purified; Table 10.16.1)
  • Complete DMEM, serum free (complete DMEM‐SF), and complete DMEM‐10 and ‐20 (or other appropriate growth medium; appendix 22)
  • Liposome suspension (Table 10.16.1; Lipofectin or TransfectACE; GIBCO/BRL #8292SA and 8301SA, respectively)
  • 6‐well, 35‐mm tissue culture dishes
  • Polystyrene tubes (Falcon #2058 or Corning #25310)

Alternate Protocol 1: Stable Transformation Using Liposomes

  Additional Materials
  • Selective medium (unit 10.17)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Buonocore, L. and Rose, J.K. 1990. Prevention of HIV‐1 glycoprotein transport by soluble CD4 retained in the endoplasmic reticulum. Nature (Lond.) 345:625‐628.
   Crise, B., Buonocore, L., and Rose, J.K.. 1990. CD4 is retained in the endoplasmic reticulum by the human immunodeficiency virus type 1 glycoprotein precursor. J. Virol. 64:5585‐5593.
   Elroy‐Stein, O. and Moss, B. 1990. Cytoplasmic expression system based on constitustem based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 83:8122‐8126.
   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.
   Shaw, A.S., Amrein, K.E., Hammond, C., Stern, D.F., Sefton, B.M., and Rose, J.K. 1989. The lck protein kinase interacts with the cytoplasmic tail of stem based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 83:8122‐8126.
   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.
   Shaw, A.S., Amrein, K.E., Hammond, C., Stern, D.F., Sefton, B.M., and Rose, J.K. 1989. The lck protein kinase interacts with the cytoplasmic tail of the CD4 glycoprotein through its unique amino‐terminal domain. Cell 59:627‐636.
   Whitt, M.A., Chong, L., and Rose, J.K. 1989. Glycoprotein cytoplasmic domain sequences required for rescue of a vesicular stomatitis virus glycoprotein mutant. J. Virol. 63:3569‐3578.
   Whitt, M.A., Buonocore, L., Rose, J.K., Ciccarone, V., and Gebeyehu, G. 1990. TransfectACE reagent promotes transient transfection frequencies greater than 90%. Focus 13:8‐12.
Key References
   Felgner et al., 1987. See above.
  These two references present comparative studies on various liposome suspensions and their ability to promote transfection in a wide range of mammalian cell types.
   Rose et al., 1991. See above.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library