Construction And In Vitro Expression Study Of Neuron-specific Vector With Cre Recombinase

Conditional gene knockout mediated by Cre-loxP system is one of the most important technologies for study of gene function in vivo.It circumvented some limitations including fetal lethal led to by conventional knockout technology. To explore finely the gene function in brain,this technology was used in nervous system to made it possible to limit the targeted gene in one subregion of the brain or one period of development. It is important to choose a specific promoter to control Cre recombinase expression in certain region of brain and certain time of development during the conditional gene knockout in nervous system. Previous sdudy indicated that CaMKⅡαgene can express in a highly temporal and spacial manner [1-4],therefore CaMKⅡαpromoter is suitable to generate the transgenic mouse for forbrain-specific expression of Cre recombinase.Two fragments of CaMKⅡα5'flanking sequences were cloned and identified.Two neuron-specific vectors with Cre recombinase were constructed with them and the transcription activity of the vectors were studied. This stuty lays the foundation for the generation of the mice expressing Cre in forebrain neuron and the conditional ADAM10 gene knockout mice, which will helpfully understand the pathogenesis of Alzheimer's disease.5.26kb and 8.1kb fragments of CaMKⅡα5'flanking regulation region were obtained from genomic DNAs of C57BL/6J mice by PCR. These fragments were subcloned and partially sequenced,then ligated to the upstream of Cre gene in the plasmid pCre-IRES-EGFP respectively to construct neuron-specific vectors with Cre recombinase;i.e.pCaMKⅡα-Cre-EGFPⅠ(pCCEⅠ)和pCaMKⅡα-Cre-EGFPⅡ(pCCEⅡ). Primary culture of hippocampal and cortical neurons from C57BL/6J mice(P2,P4w) were performed.To study tissue-specific expression of pCCEⅠand pCCEⅡ, pCCEⅠ,pCCEⅡand pCre-EGFP were separately transfected into neuron,neuroblastoma-glioma cell NG108-15 and human breast carcinoma cell ZR-75-30 mediated by LiPide2000(control group: pCre-EGFP;test group:pCCEⅠor pCCEⅡ).Result: Restriction endonuclease digestion confirmed that the restriction site of the two fragments corresponded with those of the reported sequences from the mouse line C57BL/6J.Sequencing results indicated that the determined sequences are the same as those of the reported sequences from the mouse line C57BL/6J.Only the sites at -940(A→C) and -1.4kb(A→G) were changed . -900bp sequences full of cis-acting elements were the same as those reported[5]. 5.26kb and 8.1kb fragments were exactly inserted at upstream of the Cre recombinase gene respectively. The transgenic vectors were completely constructed. A large number of neurons from newborn mice were obtained (≧106/cm3 per 35mm plate).The neuronal morphology from P4w mice was normal,however, a small number of cells were observed(<300 cells per 35mm plate).After transfection:In NG108-15 cells,geen fluorescence was detected in group pCCEⅠ(low level) and group pCre, while not in group pCCEⅡ. In ZR-75-30 cells, no geen fluorescence was detected in group pCCEⅠor group pCCEⅡ, while normal geen fluorescence was visualized in group pCre. In the neurons of newborn mice, no geen fluorescence was detected in group pCCEⅠor group pCCEⅡ,while weak geen fluorescence was detected in group pCre.In the neurons of P4w mice, because of a few neurons and the high mortality caused by transfection, no geen fluorescence were detected in any of the groups.In conclusion,the neuron-specific vectors(pCCEⅠand pCCEⅡ) for expressing Cre recombinase were successfully constructed and identified. Hippocampal and cortical neurons from C57BL/6J mice(P2,P4w) were cultured. The fact that pCCEⅠcan express geen fluorescence in neuroblastoma-glioma cell NG108-15 demonstrated that 5.26kb fragment containing CamKIIαpromoter in pCCEⅠhas transcription activity. pCCEⅠcan't express geen fluorescence in human breast carcinoma cell ZR-75-30, comfirming the neuronal specificity of gene expression. The result that pCCEⅠcan't express in the neurons from P2 mice supported the temporal control of gene expression. The transcription activity of 8.1kb fragment including almost all the regulation region of CamKIIαgene remains to study in vivo.In brief, these results showed that the neuron-specific vector with Cre recombinase(pCCEⅠ) has transcription activity and that the gene expression is spatially and temporally controlled,indicating the feasibility that the transgenic mice carrying CaMKIIαpromoter - Cre gene will be generated with pCCEⅠor pCCEⅡ.