Rat Blood–Brain Barrier Genomics. II

The tissue-specific gene expression at the brain microvasculature, which forms the blood–brain barrier (BBB) in vivo, can be elucidated with a brain vascular genomics program, which starts with the isolation of gene products derived from purified brain microvessels. Genes commonly expressed in peripheral organs are subtracted with the suppression subtractive hybridization method using driver cDNA produced from a pool of rat liver/kidney–derived poly A+RNA. From a screen of 480 clones in the subtracted tester cDNA library, 156 clones were sequenced. The clones fell into 3 groups: known genes (51%), rat expressed sequence tags (31%), and novel rat genes not found in databases (18%). The known genes could be categorized into families of common function including vascular remodeling, signal transduction, transcription factors, biologic transport, vascular amyloid, hemostasis, myelin, lipids, secretion, cytoskeleton, and junctional complexes. Brain vascular genomics, or BBB genomics, allows for an accelerated discovery of the gene families that are differentially expressed at the microvasculature in brain.

Blood—Brain Barrier Genomics

The blood–brain barrier (BBB) is formed by the brain microvascular endothelium, and the unique transport properties of the BBB are derived from tissue-specific gene expression within this cell. The current studies developed a gene microarray approach specific for the BBB by purifying the initial mRNA from isolated rat brain capillaries to generate tester cDNA. A polymerase chain reaction–based subtraction cloning method, suppression subtractive hybridization (SSH), was used, and the BBB cDNA was subtracted with driver cDNA produced from mRNA isolated from rat liver and kidney. Screening 5% of the subtracted tester cDNA resulted in identification of 50 gene products and more than 80% of those were selectively expressed at the BBB; these included novel gene sequences not found in existing databases, ESTs, and known genes that were not known to be selectively expressed at the BBB. Genes in the latter category include tissue plasminogen activator, insulin-like growth factor-2, PC-3 gene product, myelin basic protein, regulator of G protein signaling 5, utrophin, IκB, connexin-45, the class I major histocompatibility complex, the rat homologue of the transcription factors hbrm or EZH1, and organic anion transporting polypeptide type 2. Knowledge of tissue-specific gene expression at the BBB could lead to new targets for brain drug delivery and could elucidate mechanisms of brain pathology at the microvascular level.