Interplay in lipoplexes between type of pDNA promoter and lipid composition determines transfection efficiency of human growth hormone in NIH3T3 cells in culture
M. Kerner et al., Interplay in lipoplexes between type of pDNA promoter and lipid composition determines transfection efficiency of human growth hormone in NIH3T3 cells in culture, BBA-MOL C B, 1532(1-2), 2001, pp. 128-136
Citations number
36
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS
This study was aimed to investigate if and to what extent there is an inter
play between lipoplex physicochemical properties and plasmid promoter type
affecting transfection efficiency in vitro. To reduce the number of variabl
es 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 properti
es 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 us
ed for lipoplex preparation (large unilamellar vesicles, LUV, versus multil
amellar vesicles, MLV), and DNA (-)/cationic lipid(+) charge ratio, all con
taining the same human growth hormone but differing in their promoter enhan
cer region. Two of the promoters were of viral origin: (a) SV40 promoter (s
imian virus early promoter) and (b) CMV promoter (cytomegalovirus early pro
moter); two were of mammalian cell origin: (c) PABP promoter (human poly(A)
-binding protein promoter) and (d) S16 promoter (mouse ribosomal protein (r
p) S16 promoter). Transfection studies showed that, irrespective of promote
r type, large (greater than or equal to 500 nm) MLV were superior to simila
r 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 D
OTAP/cholesterol). The optimal DNA(-)/DOTAP(+) charge ratio for all types o
f lipoplexes used was 0.2 or 0.5 (namely, when the lipoplexes were positive
ly charged). Scoring the six best lipoplex formulations (out of 128 studied
) revealed the following order: pCMV (DOTAP/ DOPE)>> pSV (DOTAP/DOPE) = pCM
V(DOTAP/cholesterol) = pS16 (100% DOTAP) = pS16 DOTAP/DOPE >> pCMV (DOTAP/D
OPC). The lack of trivial consistency in the transfection efficiency score,
the pattern of transfection efficiency, and statistical analysis of the da
ta suggest that there is cross-talk between promoter type and lipoplex lipi
d composition, which may be related to the way the promoter is associated w
ith the lipids. (C) 2001 Published by Elsevier Science B.V.