The use of in situ hybridization to study the transgene pathway following cellular transfection with cationic phosphonolipids.
Gene therapy is a promising field of research and biotechnological development. Considering their safety and non-immunogenicity, cationic lipids are widely used for gene transfer in vitro and show promise for in vivo gene transfer applications. However, a better understanding of the mechanisms by which transfection occurs and the limiting steps in cellular transfer of foreign DNA are critical for significant improvements of gene transfer. In this work, we have traced the plasmid DNA into human hematopoietic cell line (K562) using the in situ hybridization method in order to define the main difficulties in transfection and to design new agents better adapted to cellular constraints. In this hematopoietic cell line, after showing the efficiency of our synthetic vectors and optimizing their formulation, we observed that only 5 h after transfection the nucleus to cytoplasm signal ratio was three to one, whereas at 24 h it was one to one. In parallel, the level of the reporter protein strongly increased between these times. Those results emphasize the rapidity of transfection and lead one to imagine chemical modifications adjusted to the environment.
Montier
Tristan
T
Equipe Mixte INSERM-Université Bretagne Occidentale 01-15, Institut des Sciences et Synergies de la Santé, University Teaching Hospital, EFS-Région Bretagne rue Félix Le Dantec, BP 454, 29275 Brest cedex, France.
Cavalier
Annie
A
Delépine
Pascal
P
Guillaume
Christine
C
Clément
Jean-Claude
JC
Yaouanc
Jean-Jacques
JJ
Morel
Gérard
G
Thomas
Daniel
D
Férec
Claude
C
D019921:Biotinylation; D002412:Cations / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism / Q000648:ultrastructure; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods; D020014:K562 Cells; D007700:Kinetics; D008163:Luminescent Measurements; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010743:Phospholipids / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013997:Time Factors; D014162:Transfection / Q000379:methods; D019076:Transgenes / Q000235:genetics*
C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; D002412:Cations; D010714:Phosphatidylethanolamines; D010743:Phospholipids; C028299:phosphonolipids; D004247:DNA