Molecular cloning and differential expression of the cat immediate early gene c-fos.

Recently, the effect of binocular central retinal lesions on the expression of immediate early genes in the visual system of adult cats was demonstrated using in situ hybridization and immunocytochemistry [2]. The present study was undertaken to quantify cat c-fos mRNA expression differences in the cat primary visual cortex after sensory deafferentation. Prior to quantification, DNA fragments obtained using reverse transcription-polymerase chain reaction (RT-PCR) in combination with rapid amplification of complementary DNA ends (RACE) were cloned and sequenced. This provided us with the necessary sequence11Note: nucleotide sequence data reported are available in the Genbank database under the accession number AF540379. information to prepare cat-specific c-fos primers for the development of a new quantitative RT-PCR assay. We optimized a reverse transcription-competitive polymerase chain reaction (RT-cPCR) method with a heterologous DNA fragment (competitor) as external standard to quantify relative amounts of cat c-fos mRNA expression levels. Internal standardization was accomplished by quantifying, in a parallel RT-cPCR, a well-characterized housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This cat-specific RT-cPCR assay allowed us to measure c-fos mRNA expression levels in central and peripheral regions of primary visual cortex in normal and retinal lesion cats.