Serial analysis of gene expression identifies metallothionein-II as major neuroprotective gene in mouse focal cerebral ischemia.

We applied serial analysis of gene expression (SAGE) to study differentially expressed genes in mouse brain 14 hr after the induction of focal cerebral ischemia. Analysis of >60,000 transcripts revealed 83 upregulated and 94 downregulated transcripts (more than or equal to eightfold). Reproducibility was demonstrated by performing SAGE in duplicate on the same starting material. Metallothionein-II (MT-II) was the most significantly upregulated transcript in the ischemic hemisphere. MT-I and MT-II are assumed to be induced by metals, glucocorticoids, and inflammatory signals in a coordinated manner, yet their function remains elusive. Upregulation of both MT-I and MT-II was confirmed by Northern blotting. MT-I and MT-II mRNA expression increased as early as 2 hr after 2 hr of transient ischemia, with a maximum after 16 hr. Western blotting and immunohistochemistry revealed MT-I/-II upregulation in the ischemic hemisphere, whereas double labeling demonstrated the colocalization of MT with markers for astrocytes as well as for monocytes/macrophages. MT-I- and MT-II-deficient mice developed approximately threefold larger infarcts than wild-type mice and a significantly worse neurological outcome. For the first time we make available a comprehensive data set on brain ischemic gene expression and underscore the important protective role of metallothioneins in ischemic damage of the brain. Our results demonstrate the usefulness of SAGE to screen functionally relevant genes and the power of knock-out models in linking function to expression data generated by high throughput techniques.

Improved protocol for SAGE tag-to-gene allocation.

Serial analysis of gene expression (SAGE) is a powerful method for large-scale analysis of gene expression patterns. SAGE yields digital information on transcript abundance by the use of short sequence fragments (tags). Because SAGE does not require a priori knowledge of the expressed genes in the starting material, SAGE can be used for gene discovery. Unfortunately, correct tag-to-gene discovery. Unfortunately, correct tag-to-gene allocation after SAGE remains problematic when the short sequence of the tag corresponds to more than one gene in the reference database or when novel yet uncloned genes were detected. To overcome this problem, we developed an improved protocol for the proper identification of tag-corresponding genes. It relies on the isolation of 3'-terminal cDNA restriction fragments by the use of paramagnetic streptavidin beads and the ligation of linkers before the amplification step. Our protocol benefits from additional information encoded in each SAGE tag: its location 3'-terminal to the last NlaIII restriction site in the cDNA. In contrast to previously described protocols, stringent PCR conditions can be applied because of the length of the specific primers, which are composed of linker- and tag-specific sequences. Additionally, we demonstrate that our protocol yields quantitative information that can be used for further expression analysis of specific SAGE tags.