Biolistic transfection and morphological analysis of cultured sympathetic neurons.
We have developed a protocol for introducing foreign genes into postmitotic peripheral neurons and their subsequent analysis by computer-assisted morphometry. The application of the biolistic approach results in transfection of approximately 10% of dissociated sympathetic neurons. Gold particles coated with plasmid DNA are propelled into sympathetic neuronal nuclei using a hand-held 'gene gun'. Transfection efficiency is optimized by choosing a microcarrier loading quantity of 0.7 mg of gold per experiment and a DNA loading ratio of 2 microg DNA per mg of gold. The gene gun is modified to minimize pressure-induced cell damage. Neurons are transfected with plasmids encoding enhanced green fluorescent protein (EGFP) or red fluorescent protein (DsRed), which serve as suitable markers for the cell body, axonal processes and growth cones including lamellipodia and filopodia. A detailed routine for quantitative morphometric image analysis using METAMORPH software is described. The protocol is demonstrated by comparing the effects of nerve growth factor (NGF) and dibutyryl cyclic adenosine monophosphate (dbcAMP) on neurite outgrowth of transfected sympathetic neurons plated on glass floor dishes.
Klimaschewski
Lars
L
Institute of Anatomy and Histology, University of Innsbruck, Muellerstrasse 59, A-6010 Innsbruck, Austria. lars.klimaschewski@uibk.ac.at
Nindl
Werner
W
Pimpl
Michael
M
Waltinger
Peter
P
Pfaller
Kristian
K
D000818:Animals; D001369:Axons / Q000648:ultrastructure; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D004247:DNA / Q000235:genetics; D003712:Dendrites / Q000648:ultrastructure; D006046:Gold; D049452:Green Fluorescent Proteins; D007091:Image Processing, Computer-Assisted; D008164:Luminescent Proteins; D008856:Microscopy, Fluorescence; D009414:Nerve Growth Factors / Q000494:pharmacology; D009474:Neurons / Q000648:ultrastructure*; D051381:Rats; D013564:Sympathetic Nervous System / Q000033:anatomy & histology* / Q000166:cytology / Q000648:ultrastructure; D014162:Transfection / Q000379:methods*; D016212:Transforming Growth Factor beta
C525821:Bmp7 protein, rat; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins; D008164:Luminescent Proteins; D009414:Nerve Growth Factors; D016212:Transforming Growth Factor beta; D049452:Green Fluorescent Proteins; D006046:Gold; D004247:DNA; D000242:Cyclic AMP