Identification of differentially expressed genes in rat hippocampus after transient global cerebral ischemia using subtractive cDNA cloning based on polymerase chain reaction.
OBJECTIVE
The purpose of this study is to identify new molecules that play important roles in the phenomena that occur in the hippocampus after transient global cerebral ischemia, as clues to better understanding of the mechanisms.


METHODS
A subtractive cDNA library was established by suppression subtractive hybridization of rat hippocampal tissues after transient global cerebral ischemia. With differential screening of the library, upregulated fragments were identified. The mRNA expression levels of selected genes were measured with semiquantitative reverse transcriptase polymerase chain reaction (PCR).


RESULTS
Among more than 100 isolated fragments, approximately half were determined to be identical to known sequences. The rest showed high homology to known sequences, and only 2 did not exhibit homology to any known sequences. The expression of 5 genes identified in this study increased in 24 hours after ischemia to a level twice as high as that in sham-operated controls. These included furin, prosaposin, synaptotagmin IV, heat shock protein 105, and the neutral and basic amino acid transporter (NBAT). The increases in the mRNA expression levels of the genes except NBAT, as revealed by semiquantitative reverse transcription PCR, were statistically significant at both 6 and 24 hours after ischemia.


CONCLUSIONS
Genes isolated are thought to be associated with production of proteins necessary for degeneration, neuroprotection, and reconstruction of neurons. How the expression of these genes relates to functional changes after ischemia remains to be determined. PCR-based subtractive cDNA cloning is demonstrated to be a useful tool for analyzing in vivo gene expression in animal ischemia models.
Yokota
N
N
Department of Neurosurgery, Hamamatsu University School of Medicine (Japan).
Uchijima
M
M
Nishizawa
S
S
Namba
H
H
Koide
Y
Y
D026923:Amino Acid Transport Systems, Basic; D026921:Amino Acid Transport Systems, Neutral; D000818:Animals; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics*; D004195:Disease Models, Animal; D045683:Furin; D020869:Gene Expression Profiling; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006624:Hippocampus / Q000737:chemistry / Q000378:metabolism*; D002546:Ischemic Attack, Transient / Q000235:genetics* / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D009410:Nerve Degeneration / Q000235:genetics / Q000378:metabolism; D009416:Nerve Regeneration / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D049231:Saposins; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism; D050857:Synaptotagmins
D026923:Amino Acid Transport Systems, Basic; D026921:Amino Acid Transport Systems, Neutral; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018076:DNA, Complementary; D006023:Glycoproteins; D018840:HSP70 Heat-Shock Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; C483347:Psap protein, rat; D049231:Saposins; C081009:Slc3a1 protein, rat; C494294:Syt4 protein, rat; D050857:Synaptotagmins; D013381:Subtilisins; D045683:Furin