A combined method of laser capture microdissection and X-Gal histology to analyze gene expression in c-Fos-specific neurons.
c-Fos is a member of the activator protein 1 family that regulates transcription of target genes. c-Fos is transiently induced in specific regions of the brain after a variety of external stimuli including learning and memory formation. Analysis of gene expression in c-Fos-expressing cells of the brain may help identify target genes that play important roles in synaptic strength or neuronal morphology. In the present study, we developed a combined method of laser capture microdissection and 5-bromo-4-chloro-3-indoly-beta-D-galactopyranosidase (X-Gal) histology to analyze gene expression in stimulus-induced c-Fos-positive cells. Using transgenic mice carrying a c-fos-lacZ fusion gene, c-Fos-positive cells were easily identified by measuring of beta-galactosidase (beta-Gal) activity. To establish the fidelity of the reporter transgene, the time course of endogenous c-Fos and the c-fos-lacZ transgene expression in the amygdala induced by LiCl administration was investigated by immunohistochemistry and X-Gal staining. LiCl increased the numbers of c-Fos- and beta-Gal-positive cells in the central and basolateral amygdala of the transgenic mice. To ensure that RNA was preserved in X-Gal stained tissue sections, different fixations were examined, with the conclusion that ethanol fixation was best for both RNA preservation and X-Gal staining quality. Finally, in combining X-Gal staining, single-cell LCM and RT-PCR, we confirmed mRNA expression of endogenous c-fos and beta-actin genes in LiCl-induced beta-Gal-positive cells in the CeA, cortex and hippocampus. Combining LCM and transgenic reporter genes provides a powerful tool with which to investigate tissue- or cell-specific gene expression.
Kwon
Bumsup
B
Department of Biological Science and Program in Neuroscience, Florida State University, Tallahassee, FL 32306, USA.
Houpt
Thomas A
TA
D000818:Animals; D001921:Brain / Q000378:metabolism; D005697:Galactosides; D015870:Gene Expression; D016762:Genes, fos; D005821:Genetic Techniques; D006652:Histological Techniques / Q000379:methods; D007211:Indoles; D007763:Lac Operon; D007834:Lasers; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D042282:Microdissection / Q000379:methods; D009474:Neurons / Q000378:metabolism*; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D001616:beta-Galactosidase / Q000378:metabolism
D005697:Galactosides; D007211:Indoles; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D012313:RNA; D001616:beta-Galactosidase; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside