Specific Expression In Brain Research Articles

Specific expression in brain of exogenous gene by transpassing blood-brain barrier after intravenous injection

To study the expression of exogenous LacZ gene in brain via a delivery of OX26-pGFAP-IL. pCMV-Liposome, OX26-pCMV-IL, OX26-pGFAP-IL and blank liposome were injected into rats via femoral vein. At 24 h post-injection, the method of Q-PCR was adopted to calculate the relative quantities of LacZ gene mRNA in brain and peripheral organs. At 48 h post-injection, the protein expression of LacZ gene was detected by the activity of beta-galactosidase and the method of histochemical stain. The result of Q-PCR showed that, at 24 h post-injection, the relative quantities of LacZ mRNA in OX26-pCMV-IL group (49.2 x 10(-6)) and OX26-pGFAP-IL group (44.9 x 10(-6)) were significantly higher than pCMV-liposome and blank liposome groups (P < 0.05). In peripheral organs, the relative quantity of LacZ mRNA in OX26-pCMV-IL group were significantly higher than that in OX26-pGFAP-IL group (P < 0.05). At 48 h post-injection, the activity of beta-galactosidase in OX26-pCMV-IL (0.67 pg/mg) and OX26-pGFAP-IL groups (0.92 pg/mg) were significantly higher than pCMV-liposome and blank liposome groups (P < 0.05). There was no significant difference between OX26-pCMV-IL group and OX26-pGFAP-IL group in terms of the expression of beta-galactosidase. The result of histochemical stain showed that OX26-pGFAP-IL achieved a specifically positive expression in brain and had a decreased expression in peripheral organs. OX26-pGFAP-IL injected via femoral vein can cross the brain-blood barrier and achieve a specific expression in brain under the control of GFAP promoter. OX26-pGFAP-IL decreases the non-specific expression in peripheral organs and it may be used as an non-viral gene therapy for intra-cranial diseases.

Characterization of tissue expression and full-length coding sequence of a novel human gene mapping at 3q12.1 and transcribed in oligodendrocytes

Macro-array differential hybridization of a collection of 5058 human gene transcripts represented in an IMAGE infant brain cDNA library has led to the identification of transcripts displaying preferential or specific expression in brain (Genome Res. 9 (1999) 195; http://idefix.upr420.vjf.cnrs.fr/IMAGE). Most of these genes correspond to as yet undescribed functions. Detailed characterization of the expression, sequence, and genome assignment of one of these genes named C3orf4, is reported here. The full-length sequence of the transcript was obtained by 5′ extension RT-PCR. The gene transcript (2.8 kb) encodes a 253 amino acid long protein, with four transmembrane domains. The position of the C3orf4 gene was determined at 3q12.1 thanks to the draft sequence of the human genome. It is composed of five exons spanning more than 7 kb. No TATAA box but a CpG island was found upstream of the beginning of the gene. Northern blot analysis and in situ hybridization revealed a predominant expression in myelinated structures such as corpus callosum and spinal cord. RT-PCR showed expression of the C3orf4 gene in rat optic nerve and cultured oligodendrocytes, the myelinating cells of the central nervous system, but not in astrocytes. This work supports further investigations aimed at determining the role of the C3orf4 gene in myelinating cells.

Identification and characterization of a putative C. elegans potassium channel gene (Ce-slo-2) distantly related to Ca(2+)-activated K(+) channels.

Two putative homologues of large conductance Ca(2+)-activated K(+) channel alpha-subunit gene (slowpoke or slo) were revealed by C. elegans genome sequencing. One of the two genes, F08B12.3 (Ce-slo-2), shows a relatively low amino acid sequence similarity to other Slo sequences and lacks key functional motifs, which are important for calcium and voltage sensing. However, its overall structure and regions of homology, which are conserved in all Slo proteins, suggest that Ce-SLO-2 should belong to the Slo channel family. We have cloned a full-length cDNA of the Ce-slo-2, which encodes a protein containing six putative transmembrane segments with a K(+)-selective pore and a large C-terminal cytosolic domain. Green fluorescent protein (GFP) and whole-mount immunostaining analyses revealed that Ce-slo-2 is specifically expressed in neuronal cells at the nerve ring, at the ventral nerve cord of the mid-body, and at the tail region. We have also identified a putative human counterpart of Ce-slo-2 from a human brain EST database, which shows a stretch of highly conserved amino acid residues. Northern blot and mRNA dot blot analyses revealed a strong and specific expression in brain and skeletal muscle. Taken together, our data suggest that Ce-slo-2 may constitute an evolutionarily conserved gene encoding a potassium channel that has specific functions in neuronal cells.

Specific Expression in Brain and Liver Driven by the Hemopexin Promoter in Transgenic Mice

Transgenic mice harboring the human hemopexin promoter sequences linked to the lacZ reporter gene were generated and analyzed for temporal and spatial distribution of β-galactosidase. Upstream sequences spanning from −1800, −700 and −500 bp to the transcription start point direct regulated β-galactosidase expression specifically to the liver and to the brain of transgenic mice. These results suggest that the 500 bp DNA fragment flanking the 5′end of the human hemopexin gene contains the cis-acting elements required for tissue and developmental stage-specific expressionin vivoand provide evidence for a new extrahepatic site of expression of the hemopexin gene.

Functional Dissection of the Brain-specific Rat Aldolase C Gene Promoter in Transgenic Mice: ESSENTIAL ROLE OF TWO GC-RICH BOXES AND AN HNF3 BINDING SITE

The aldolase C gene product is a glycolytic isoenzyme specifically detected in brain. We have previously defined a short 115-base pair promoter fragment able to confer on a reporter chloramphenicol acetyltransferase (CAT) gene a specific expression in brain of transgenic mice. In this promoter fragment, two GC-rich regions (A/A' and B boxes) were detected by in vitro DNase1 footprinting experiments with brain, fibroblast, or liver nuclear extracts. Both A/A' and B boxes, sharing structural homology, are able to interact with Sp1, Krox20/Krox24 factors and with other proteins (Thomas, M., Makeh, I., Briand, P., Kahn, A., and Skala, H. (1993) Eur. J. Biochem. 218, 143-151). In this paper, we describe a new ubiquitous factor termed Ub able to bind the A/A' box. We also delimit a third element (box C) binding a hepatocyte-enriched protein displaced by a hepatocyte nuclear factor 3-specific oligonucleotide. The functional involvement of each binding site in brain-specific transcription of the aldolase C gene has been tested in transgenic mice carrying different mutant promoters cloned in front of the CAT gene. A promoter containing only box C was totally inactive, suggesting an essential role of the region containing A/A' and B boxes. However, mutations or deletions of either the A/A' or the B box have no significant effect on the CAT gene expression. We therefore hypothesize that the A/A' and B sites may be functionally redundant. Indeed, constructs harboring only one of these two boxes (A/A' or B) linked to the C box displayed a brain-specific CAT activity similar to that obtained with the wild-type promoter. Furthermore, a transgene with disruption of the C box, keeping intact the A/A' and B boxes, was totally inactive, suggesting a crucial role of the hepatocyte nuclear factor 3 binding site in activation of the aldolase C gene.

Purification and molecular cloning of a novel acidic calmodulin binding protein from rat brain

An acidic protein was identified among the insoluble proteins of a growth cone-enriched fraction, and this protein was purified from the Triton- and high NaCl-insoluble fraction of newborn rat brain using several column chromatographies after solubilization at an alkaline condition. The purified protein showed a Ca(2+)-dependent calmodulin binding activity. This protein showed an anomalous behavior in SDS-polyacrylamide gel electrophoresis as that observed in case of GAP-43 (neuromodulin, F1, pp46, p57, B-50) and MARCKS (p87, p80), namely shifts of apparent molecular weights under different acrylamide concentrations. Its physicochemical characteristics, such as heat stability, acidic isoelectric point, and solubility in a 2.5% perchloric acid solution, also resemble the properties of these proteins. cDNA cloning of this protein showed that the NH2-terminal 50-amino acid sequence was almost identical to CAP-23, a previously reported chicken protein of unknown function. Since the COOH-terminal half-regions of these proteins are less similar, the entire sequences of these proteins have 65% homology (52% identity), suggesting that these proteins belong to a family. Immunoblotting of several tissue extracts using a monoclonal antibody against this protein showed its specific expression in brain.