Interplay in lipoplexes between type of pDNA promoter and lipid composition determines transfection efficiency of human growth hormone in NIH3T3 cells in culture

Miryam Kerner, Oded Meyuhas, Danielle Hirsch-Lerner, Laura J. Rosen, Zhang Min, Yechezkel Barenholz

Research output: Contribution to journalArticlepeer-review

Abstract

This study was aimed to investigate if and to what extent there is an interplay between lipoplex physicochemical properties and plasmid promoter type affecting transfection efficiency in vitro. To reduce the number of variables only one cell type (NIH3T3 cells), one gene (human growth hormone), one cationic lipid (DOTAP) in a plasmid > 85% in supercoiled form, and the same medium conditions were used. The variables of the physicochemical properties included presence and type of helper lipid (DOPE, DOPC, or cholesterol, all in 1:1 mole ratio with DOTAP), size and lamellarity of the liposomes used for lipoplex preparation (large unilamellar vesicles, LUV, versus multilamellar vesicles, MLV), and DNA-/cationic lipid+ charge ratio, all containing the same human growth hormone but differing in their promoter enhancer region. Two of the promoters were of viral origin: (a) SV40 promoter (simian virus early promoter) and (b) CMV promoter (cytomegalovirus early promoter); two were of mammalian cell origin: (c) PABP promoter (human poly(A)-binding protein promoter) and (d) S16 promoter (mouse ribosomal protein (rp) S16 promoter). Transfection studies showed that, irrespective of promoter type, large (≥ 500 nm) MLV were superior to ∼ 100 nm LUV; the extent of superiority was dependent on liposome lipid composition (larger for 100% DOTAP and DOTAP/DOPE than for DOTAP/DOPC and DOTAP/cholesterol). The optimal DNA-/DOTAP+ charge ratio for all types of lipoplexes used was 0.2 or 0.5 (namely, when the lipoplexes were positively charged). Scoring the six best lipoplex formulations (out of 128 studied) revealed the following order: pCMV (DOTAP/DOPE) ≫ pSV (DOTAP/DOPE) = pCMV(DOTAP/cholesterol) = pS16 (100% DOTAP) = pS16 DOTAP/DOPE ≫ pCMV (DOTAP/DOPC). The lack of trivial consistency in the transfection efficiency score, the pattern of transfection efficiency, and statistical analysis of the data suggest that there is cross-talk between promoter type and lipoplex lipid composition, which may be related to the way the promoter is associated with the lipids.

Original languageEnglish
Pages (from-to)128-136
Number of pages9
JournalBiochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
Volume1532
Issue number1-2
DOIs
StatePublished - 31 May 2001
Externally publishedYes

Keywords

  • Cationic lipid
  • Gene delivery
  • Lipofection

Fingerprint

Dive into the research topics of 'Interplay in lipoplexes between type of pDNA promoter and lipid composition determines transfection efficiency of human growth hormone in NIH3T3 cells in culture'. Together they form a unique fingerprint.

Cite this