Mild surfection of neural cells, especially motoneurons, in primary culture and cell lines.
Of all cell types, motoneurons (MNs), are possibly the most difficult to maintain in culture, since their development and survival is conditioned by many factors that are still in the course of identification. This may also be the reason why they are difficult to transfect. We succeed to transfect these fragile cells with lipoplex [DOTAP:PC (10:1)-pGFP]-precoated coverslips. Here, we report that this original method, also termed 'surfection' does not perturbate MN development and survival while giving important transfection yield (15%). Lipofectamine 2000 and other well-known auxiliary lipids (DOPE, Chol) give lower surfection yields. The use of (DOTAP:PC)-based lipid vector also can be extended to several neural and non-neural cell lines with appreciable transfection yield such as a glial cell line (GCL) derived from rat spinal cord (65%), HeLa S3 (60%), COS-7 (30%) and HEK 293 cells (20%). The efficiency of DOTAP:PC (10:1) and Lipofectamine 2000 vectors in our surfection method are compared on standard HeLa S3 cell lines. Lipofectamine 2000 (72%) is slightly better than DOTAP:PC (10:1) (60%). However, the surfection method improved the efficiency of Lipofectamine 2000 itself (72%) as compared to the classical (62%) approach. In summary we have developed an original standard surfection protocol for both MN primary cultures and cell lines, thus simplifying laboratory practice; moreover, Lipofectamine 2000 used in this surfection method is more efficient for the cell lines than the manufacturer-recommended method. We emphasize that our method particularly spares fragile cells like MNs from injure and therefore, might be applied to other fragile cell type in primary cultures.
Rakotoarivelo
Clovis
C
Unité 583 de l'INSERM, Institut des Neurosciences de Montpellier Hôpital Saint-Eloi, 80, rue Augustin-Fliche FR-34091 Montpellier, Cedex 05, France. rakoto@montp.inserm.fr
Petite
Didier
D
de Weille
Jan
J
Lumbroso
Serge
S
Privat
Alain
A
Sultan
Charles
C
Mersel
Marcel
M
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D004247:DNA; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D008055:Lipids; D008081:Liposomes; D009046:Motor Neurons / Q000378:metabolism*; D009457:Neuroglia / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D010957:Plasmids; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection / Q000379:methods*
D008055:Lipids; C086724:Lipofectamine; D008081:Liposomes; D049452:Green Fluorescent Proteins; D004247:DNA