Gene transfer methods for CNS organotypic cultures: a comparison of three nonviral methods.
Organotypic slice cultures from postnatal day 12 mouse cerebellum were transfected using three nonviral methods: biolistics (gene gun), lipotransfection, and electroporation. The plasmid transferred, pHD17-25Q-GFP, encoded a fusion protein with a green fluorescent protein (GFP) component. Optimal conditions for both lipotransfection and electroporation are the same as those previously found in live animal models. Electroporation (26 +/- 6) and biolistics (34 +/- 4.4) provide a better rate of transfer than lipotransfection (15 +/- 2.2) in slice cultures and are comparable to each other. Each of the transfer methods produced positive signals in a heterogeneous population of glial and neuronal cells. These data provide a base for optimal transfection of slice cultures, allowing the development of therapeutic constructs, and support the idea that successful refinement of nonviral delivery methods for in vivo use is possible using brain slice cultures.
Murphy
R C
RC
Wadsworth Center, New York State Department of Health, Albany, New York 12201-2002, USA.
Messer
A
A
D000818:Animals; D019470:Biolistics / Q000379:methods*; D002490:Central Nervous System / Q000166:cytology*; D004305:Dose-Response Relationship, Drug; D018274:Electroporation / Q000379:methods*; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods*
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins