The Probe Preparation Of Brd2Gene And Its Morphological Research In Mouse Cerebral Cortex

Objective In order to observe Brd2gene expression in mouse cerebral cortex andunderstand its neurochemical characteristics, this study prepared two digoxigenin labeledcRNA probes of Brd2gene. Then observed expression of Brd2gene and analyses itsneurochemical characteristics in the cerebral cortex of C57BL/6mouse by the applicationof the probe.Methods At first, the experiment designed two pairs of Brd2primers and extract totalRNA from mouse brain, then the two Brd2DNA fragments were obtained through reversetranscription PCR(RT-PCR) method and they were respectively cloned into the pCRⅡ-TOPOvectors. Using the method of in vitro transcription, we prepared two highly sensitivedigoxigenin(DIG) labeled probes and finally through fluorescent in situ hybridizationexperiment, we analysis the specificity of the two probes and the effect of hybridization.Then twelve healthy adult C57BL/6mice were randomly divided into two groups. For thefirst group of mice, we perfused mice and then observed the distribution patternof Brd2mRNA in adult mouse brain by using fluorescent in situ hybridization. For the secondgroup, we perform fluorescent in situ hybridization to detect mRNA for Brd2combined withimmunofluorescence staining to reveal neurochemical features of Brd2mRNA positive cells inmouse cerebral cortex.Results （1）First of all, the experiment successfully constructed two Brd2/pCRⅡ-TOPO plasmids by the application of molecular biology techniques and obtained high titerdigoxigenin labeled cRNA probes. Then in the fluorescent in situ hybridization experiments，using two probes shows good hybridization effects. （2）Choose one of two probes for fluorescent in situ hybridization experiment, weobserved the overall distribution of Brd2gene in cerebral cortex of C57BL/6mouse. Theresults showed that Brd2gene mainly expressed in neural cells.（3）By using double staining method of fluorescent in situ hybridization combined withimmunofluorescence, we observed the distribution and coexistence of Brd2mRNA positive cellswith NeuN, GFAP, GABA, CB, CR and PV. The results showed that, Brd2positive cells weremainly distributed in NeuN positive cells, while none of the Brd2positive cells were GFAPpositive astrocytes. In the cerebral cortex of mouse, we observed the coexistence ofBrd2mRNA with GABA, CB, CR and PV respectively. The Brd2/GABA coexistence cellsaccounted for18%and52%of total Brd2mRNA and GABA positive cells respectively. TheBrd2/CB coexistence cells accounted for23%and82%of total Brd2mRNA and CB positivecells respectively. The proportion of Brd2/CR coexistence cells accounted for20%and82%of total Brd2mRNA and CR positive cells respectively. The proportion of Brd2mRNA/PVcoexistence cells accounted for13%and31%of total Brd2mRNA and PV positive cellsrespectively.Conclusions （1）The digoxigenin labeled cRNA probes that prepared in this experimentcould specifically detect expression of Brd2mRNA in the cerebral cortex of mouse.（2）The present study revealed that Brd2mRNAis mainly expressed in neural cellsof cerebral cortex. While glial cells rarely expressed Brd2mRNA.（3）In C57BL/6mouse brain, Brd2gene has different coexistence relationship withGABA, CB, CR and PV. This provided morphological evidence for further functional studies.