High-efficiency transfection of primary human keratinocytes with positively charged lipopolyamine:DNA complexes.
The ability to introduce DNA into mammalian cells has provided a powerful means to examine the regulation of gene expression and the function of gene products. However, the most commonly used techniques for DNA transfection are not always suitable for primary cells. Primary human keratinocytes are particularly stringent in their growth requirements and are also very refractory to transfection, rendering transient gene expression studies difficult. We have investigated the ability of several polycationic lipids to promote DNA uptake into human epidermal keratinocytes, as monitored with the bacterial beta-galactosidase reporter gene. We report that the cationic lipopolyamine dipalmitoyl phosphatidylethanolamine spermine as well as another procedure using Polybrene can achieve a 20% to 30% transfection efficiency, superior to any other agent tested on these cells. Gene transfer was accomplished by a 3-h exposure of monolayer cells to DNA complexes formed with either reagent by simple mixing in a serum-free medium, followed by a brief osmotic shock with glycerol. Neither DNA carrier showed any toxicity at the effective concentrations nor interfered with cell attachment, growth or differentiation. The use of a fully biodegradable lipopolyamine as DNA carrier should make it possible to extend this transfection method to gene transfer for in vivo therapeutic applications.
Staedel
C
C
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, New York.
Remy
J S
JS
Hua
Z
Z
Broker
T R
TR
Chow
L T
LT
Behr
J P
JP
D004247:DNA / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology / Q000502:physiology; D006801:Humans; D007231:Infant, Newborn; D015603:Keratinocytes / Q000502:physiology*; D007700:Kinetics; D008297:Male; D010714:Phosphatidylethanolamines / Q000235:genetics / Q000494:pharmacology; D011073:Polyamines / Q000378:metabolism*; D013096:Spermine / Q000031:analogs & derivatives / Q000494:pharmacology / Q000502:physiology; D014162:Transfection / Q000379:methods
D010714:Phosphatidylethanolamines; D011073:Polyamines; C061134:dioctadecylamidoglycylspermine; C061135:1,2-dipalmitoylphosphatidylethanolamidospermine; D013096:Spermine; D004247:DNA; D005998:Glycine