Introduction of green fluorescent protein (GFP) into hippocampal neurons through viral infection.
Expression of green fluorescent protein (GFP), its more fluorescent mutant forms (e.g., EGFP [enhanced GFP]), or their fusion protein derivatives, affords a number of informative possibilities in cellular neuroscience. EGFP is a soluble protein and appears to be homogeneously distributed within the cytosol of neurons when expressed. Thus, it reveals the structure of the neuron, including the cell body, and axonal and dendritic arbors. It is also sufficiently bright to reveal detailed structures such as axonal boutons and dendritic spines. When expressed as a fusion protein, EGFP can provide information about the distribution characteristics of the proteins within neurons. Furthermore, during single-cell electrophysiological studies, such expression can direct the investigator to record from a cell carrying a foreign gene. In this protocol, we describe the use of the Sindbis pseudovirus expression system to deliver GFP to neurons. Sindbis is a member of the alphaviruses, which are plus-stranded RNA viruses. This protocol uses the DH(26S) strain, which preferentially infects neurons over glia (50:1). Two infection methods are given: one for dissociated hippocampal cultured neurons and one for organotypic hippocampal slices.
Malinow
Roberto
R
Hayashi
Yasunori
Y
Maletic-Savatic
Mirjana
M
Zaman
Shahid H
SH
Poncer
Jean-Christophe
JC
Shi
Song-Hai
SH
Esteban
José A
JA
Osten
Pavel
P
Seidenman
Ken
K
D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D006624:Hippocampus / Q000166:cytology*; D009474:Neurons / Q000821:virology*; D012845:Sindbis Virus / Q000235:genetics*; D013194:Staining and Labeling / Q000379:methods*; D014161:Transduction, Genetic / Q000379:methods*
D049452:Green Fluorescent Proteins