Development of a reproducible procedure for plasmid DNA encapsulation by red blood cell ghosts.
OBJECTIVE
The binding and encapsulation of [3H] pGL3 luciferase reporter plasmid DNA by red blood cell (RBC) ghosts, intended as a vehicle for transfection and ultimately for gene therapy, were studied using two methods for DNA compaction.


RESULTS
In the first approach, DNA was compacted through binding electrostatically to poly-L-lysine. Complexes were constructed to have a slight negative charge. Experimentally, it was found that a high percentage of binding was to the outside of the resealed RBC ghosts. An alternative approach using polyethylene glycol6000 at a final concentration of 15% (weight/volume) was used to collapse [3H] pGL3 DNA in the presence of 0.025M MgCl2. Addition of the reagents, premixed with DNA, to a pelleted suspension of RBC ghosts followed by a short incubation and then addition of 1.5 M NaCl to restore tonicity, resulted in resealing of the ghosts. Uptake of [3H] pGL3 DNA by the ghosts was approximately 20% of the input amount of DNA. Further work showed that 60-70% of the DNA was inside the resealed ghosts and largely present in the supercoiled form. At no stage was any freezing and thawing used.


CONCLUSIONS
Transfection studies have demonstrated that pGL3 DNA carrying the luciferase gene is successfully transferred from RBC ghosts to recipient HeLa cells in culture under mild fusion conditions.
Larson
Gretchen
G
Department of Pharmacology, Faculty of Health Sciences, University of Stellenbosch, Tygerberg, South Africa.
Pieterse
Anton
A
Quick
Gwynnèth
G
van der Bijl
Pieter
P
van Zyl
Johann
J
Hawtrey
Arthur
A
D004278:DNA, Superhelical / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism*; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics; D010957:Plasmids / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D011107:Polylysine / Q000737:chemistry; D011485:Protein Binding; D015203:Reproducibility of Results; D014162:Transfection / Q000379:methods*
D004278:DNA, Superhelical; D011107:Polylysine; D011092:Polyethylene Glycols; D008156:Luciferases