Electroporation-based gene transfer for efficient transfection of neural precursor cells.
Transplantation of neural precursor cells (NPCs) is a potential tool to replace dysfunctional or degenerated neuronal or glial cell types in the central nervous system. Furthermore, transplantation of genetically engineered neural precursor cells might provide a strategy to target therapeutic gene products to the diseased nervous system. Here, we describe a novel and highly efficient electroporation-based transfection protocol for mitogen-expanded mouse NPCs. Transfection of NPCs with the reporter gene enhanced green fluorescent protein (EGFP) or the neural adhesion molecule L1 revealed transfection efficacies of more than 70% as estimated by the number of EGFP-positive or L1-immunoreactive cells 1 day after transfection in vitro. The percentage of EGFP- or L1-positive cells decreased with increasing time in culture. Positive cells were detectable for up to 3 weeks after transfection. When EGFP- or L1-transfected NPCs were grafted into the retina of adult wild-type or L1-deficient mice, they differentiated into glial cells some of which expressed EGFP and L1 for up to 2 and 3 weeks, respectively, the longest post-transplantation periods investigated.
Richard
Ines
I
Kopf-und Hautzentrum, Klinik und Poliklinik für Augenheilkunde, Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany.
Ader
Marius
M
Sytnyk
Vladimir
V
Dityatev
Alexander
A
Richard
Gisbert
G
Schachner
Melitta
M
Bartsch
Udo
U
D000818:Animals; D019002:CD56 Antigen / Q000235:genetics; D002452:Cell Count; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D018274:Electroporation / Q000379:methods*; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009457:Neuroglia / Q000166:cytology / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D033581:Stem Cell Transplantation / Q000379:methods*; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D014162:Transfection / Q000379:methods*; D015854:Up-Regulation / Q000235:genetics
D019002:CD56 Antigen; C501063:Ncam1 protein, mouse; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins