Transgene expression, but not gene delivery, is improved by adhesion-assisted lipofection of hematopoietic cells.
In contrast to adherent cells, cells growing in suspension and particularly hematopoietic cells, are notoriously difficult to transfect in vitro using nonviral approaches. In the present study, the effect of cell adhesion on gene transfer efficacy was investigated by allowing hematopoietic cells to bind to an adherent cell monolayer (ACM) before being subjected to cationic liposome-mediated DNA transfer. Human CD34 and T CD4 cell lines were cultivated on an ACM constituted of murine fibroblast NIH3T3 cells and transfected with a plasmid carrying the beta-galactosidase gene. X-gal staining showed that up to 27% of the cells expressed the transgene. In contrast, less than 0.1% of these cells were positively transfected in suspension. This adhesion-assisted lipofection (AAL) procedure was also successfully tested on blood lymphocytes, since it resulted in up to 30% of transfected human primary T lymphocytes. Flow cytometry analysis performed on T lymphocyte subsets revealed that 8 and 9%, respectively, of CD4 and CD8 cells could be transfected with a plasmid carrying the green fluorescent protein gene. Other adherent cells, such as MS5 murine stromal cells or HeLa epithelial cells, were also a compatible matrix for AAL. Moreover, the pCMV beta plasmid was present in similar amounts in the nuclei of TF1 cells transfected in suspension or with the AAL procedure. These data raise the possibility that cell matrix/hematopoietic cell interactions might govern expression of the transgene in hematopoietic cells growing usually in suspension, but not endocytosis of liposome/DNA particles and plasmid migration ot the cell nucleus.
Keller
H
H
Département d'Immunologie, Institut Pasteur, Paris, France.
Yunxu
C
C
Marit
G
G
Pla
M
M
Reiffers
J
J
Thèze
J
J
Froussard
P
P
D016475:3T3 Cells; D000818:Animals; D018952:Antigens, CD34; D015496:CD4-Positive T-Lymphocytes; D018414:CD8-Positive T-Lymphocytes; D002448:Cell Adhesion; D002460:Cell Line; D018920:Coculture Techniques; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006412:Hematopoietic Stem Cells / Q000201:enzymology / Q000502:physiology*; D006801:Humans; D008081:Liposomes; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D013601:T-Lymphocytes / Q000201:enzymology; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics
D018952:Antigens, CD34; D008081:Liposomes; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase