Blood—Brain Barrier Genomics

Abstract

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, IkappaB, 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.

Blood—Brain Barrier Genomics and Cloning of a Novel Organic Anion Transporter

“…First, the SSH procedure allows for the rapid cloning of partial cDNAs corresponding to BBB-selective genes (Figure 1), and the availability of the brain capillary cDNA library allows for cloning of the full-length cDNAs corresponding to the BBB-selective gene. Second, one such gene, originally designated BSAT1 (Li et al, 2001a), is highly expressed at the BBB, and is not expressed in major rat peripheral tissues ( Figure 2A). Third, unlike another BBB-selective gene, LAT1, which is expressed in rat glial tumors, the BSAT1 gene is not expressed in rat glial cells either in cell culture or as rat brain tumors ( Figure 2B).…”

“…Suppression subtractive hybridization was performed using the PCR-Select cDNA subtraction kit as reported previously (Li et al, 2001a). The SSH procedure was performed using rat brain capillary mRNA to produce tester cDNA and rat liver/kidney mRNA to produce driver cDNA.…”

“…cDNA size was normalized by digestion with RsaI, and specific oligodeoxynucleotide adaptors were ligated to the RsaIdigested tester cDNA. After hybridization of tester and driver cDNAs, the rat brain capillary-specific sequences were amplified by polymerase chain reaction (PCR) (Li et al, 2001a). The PCR products were cloned into the pCR2.1 vector, and INVaF 0 super competent cells were transformed to form a subtracted rat brain capillary cDNA library (Li et al, 2001a).…”

“…After hybridization of tester and driver cDNAs, the rat brain capillary-specific sequences were amplified by polymerase chain reaction (PCR) (Li et al, 2001a). The PCR products were cloned into the pCR2.1 vector, and INVaF 0 super competent cells were transformed to form a subtracted rat brain capillary cDNA library (Li et al, 2001a). For the differential hybridization, randomly selected colonies were blotted onto GeneScreen Plus membranes using a 96-well dot-blot apparatus (Bio-Rad).…”

“…Antisense and sense RNA probes were generated using SalI and XbaI linearized pSPT19-BSAT1 plasmid and T7 or SP6 polymerases, respectively, in the presence of digoxigenin-11-uridine triphosphate (DIG-11-UTP). Freshly prepared rat brain capillaries were cytocentrifuged onto glass slides, and the capillaries were fixed with 10% formalin and stored at À701C (Li et al, 2001a). The slides were hybridized with 12 ng digoxigenin-labeled probes in 30 mL of hybridization solution overnight at 421C, and control sections were hybridized with equal amount of sense cRNA as reported previously (Li et al, 2001a).…”