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. 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 sequence(1) 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.
Van der Gucht
Estel
E
Laboratory of Neuroendocrinology and Immunological Biotechnology, Katholieke Universiteit Leuven, Naamsestraat 59, B-3000 Leuven, Belgium. estel.vandergucht@bio.kuleuven.ac.be
Massie
Ann
A
De Klerck
Bert
B
Peeters
Kristel
K
Winters
Kurt
K
Gerets
Helga H J
HH
Clerens
Stefan
S
Vandesande
Frans
F
Arckens
Lutgarde
L
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001483:Base Sequence / Q000235:genetics; D002415:Cats; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D003714:Denervation; D005786:Gene Expression Regulation / Q000235:genetics; D017781:Genes, Immediate-Early / Q000235:genetics*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D012160:Retina / Q000293:injuries* / Q000378:metabolism / Q000601:surgery; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014793:Visual Cortex / Q000166:cytology / Q000378:metabolism* / Q000601:surgery; D014795:Visual Pathways / Q000293:injuries* / Q000378:metabolism
D018076:DNA, Complementary; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases