Subtractive cloning identifies tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) increased gene expression following focal stroke

Abstract

Background and Purpose - Differential gene expression has been reported following the onset of focal stroke. To identify de novo expression of ischemia-induced genes, we applied subtractive cDNA library strategy to identify the genes that are selectively upregulated by focal stroke. Methods - Spontaneously hypertensive rats were subjected to permanent occlusion of the middle cerebral artery (MCAO). mRNAs prepared from ischemic and nonischemic cortex 2 and 12 hours after MCAO were subtracted, and a subtractive cDNA library was constructed. A cDNA that encodes for tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) was identified in the subtractive cDNA library. The temporal expression of cortical TIMP-1 mRNA was further characterized in ischemic cortex subjected to permanent or temporary (160-minute) MCAO. Results - A panel of genes isolated from the subtractive cDNA library was subjected to Southern analysis to confirm ischemia-induced gene expression. TIMP-1 demonstrated robust induction after ischemic injury. Time-course studies revealed that TIMP-1 mRNA was induced threefold over controls at 12 hours (P